Yeast As A Tool in Cancer Research 2007

YEAST AS TOOL IN CANCER RESEARCH
Yeast as Tool in Cancer Research
Edited by
JOHN L.. NITISS

St. Jude Children’s Research Hospital,
Memphis, TN, U.S.A.
and
JOSEPH HEITMAN
Duke University Medical Center,
Durham, NC, U.S.A.
A C.I.P. Catalogue record for this book is available from the Library of Congress.
ISBN 978-1-4020-5962-9 (HB)

ISBN 978-1-4020-5963-6 (e-book)
Published by Springer,
P.O. Box 17, 3300 AA Dordrecht, The Netherlands.
www.springer.com
Printed on acid-free paper
All Rights Reserved

© 2007 Springer
No part of this work may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming,
recording or otherwise, without written permission from the Publisher, with the
exception of any material supplied specifically for the purpose of being entered
and executed on a computer system, for exclusive use by the purchaser of the work.
TABLE OF CONTENTS
Foreword…………………………………………………………………….ix
List of Contributors………………………………………………………….xi
Introduction………………………………………………………………....xv
Chapter 1
FROM DNA REPLICATION TO GENOME INSTABILITY
IN SCHIZOSACCHAROMYCES POMBE: PATHWAYS
TO CANCER………………………………………………………………...1
Julie M. Bailis and Susan L. Forsburg
Chapter 2
DISSECTING LAYERS OF MITOTIC REGULATION
ESSENTIAL FOR MAINTAINING GENOMIC STABILITY…………...37
Jennifer S. Searle and Yolanda Sanchez
Chapter 3
YEAST AS A TOOL IN CANCER RESEARCH:
NUCLEAR TRAFFICKING……………………………………………….75
Anita H. Corbett and Adam C. Berger
Chapter 4
STUDIES OF PROTEIN FARNESYLATION IN YEAST …………...….101
Nitika Thapar and Fuyuhiko Tamanoi
Chapter 5
FROM BREAD TO BEDSIDE: WHAT BUDDING YEAST
HAS TAUGHT US ABOUT THE IMMORTALIZATION
OF CANCER CELLS…………………………………………………..…123
Soma S. R. Banik and Christopher M. Counter
v
vi Table of Contents
Chapter 6
HSP90 CO-CHAPERONES IN SACCHAROMYCES
CEREVISIAE……………………………………………………………...141
Marija Tesic and Richard F. Gaber
Chapter 7
YEAST AS A MODEL SYSTEM FOR STUDYING
CELL CYCLE CHECKPOINTS………………………………………….179
Carmela Palermo and Nancy C. Walworth
Chapter 8
METABOLISM AND FUNCTION OF SPHINGOLIPIDS
IN SACCHAROMYCES CEREVISIAE: RELEVANCE
TO CANCER RESEARCH…………………………………………….…191
L. Ashely Cowart, Yusuf A. Hannun and Lina M. Obeid
Chapter 9
EXPLORING AND RESTORING THE p53 PATHWAY
USING THE p53 DISSOCIATOR ASSAY IN YEAST………………….211
Rainer K. Brachmann
Chapter 10
FUNCTIONAL ANALYSIS OF THE HUMAN p53 TUMOR
SUPPRESSOR AND ITS MUTANTS USING YEAST………………....233
Alberto Inga, Francesca Storici and Michael A. Resnick
Chapter 11
ABC TRANSPORTERS IN YEAST – DRUG RESISTANCE
AND STRESS RESPONSE IN A NUTSHELL………………………….289
Karl Kuchler and Christoph Schüller
Chapter 12
THE FHCRC/NCI YEAST ANTICANCER DRUG SCREEN………..…315
Susan L. Holbeck and Julian Simon
Chapter 13
YEAST AS A MODEL TO STUDY THE IMMUNOSUPPRESSIVE
AND CHEMOTHERAPEUTIC DRUG RAPAMYCIN………………....347
John R. Rohde, Sara A. Zurita-Martinez and Maria E. Cardenas
Chapter 14
USE OF YEAST AS A MODEL SYSTEM FOR IDENTIFYING
AND STUDYING ANTICANCER DRUGS…………………………..…375
Jun O. Liu and Julian A. Simon
Table of Contents vii
Chapter 15
GENETIC ANALYSIS OF CISPLATIN RESISTANCE
IN YEAST AND MAMMALS…………………………………………...393
Seiko Ishida and Ira Herskowitz
Chapter 16
USING YEAST TOOLS TO DISSECT THE ACTION
OF ANTICANCER DRUGS: MECHANISMS OF ENZYME
INHIBITION AND CELL KILLING BY AGENTS
TARGETING DNA TOPOISOMERASES………………………………409
Anna T. Rogojina, Zhengsheng Li, Karin C. Nitiss and John L. Nitiss
Index…......….…………………………………………………………….429
FOREWORD
Leland H. Hartwell
Director, Fred Hutchinson Cancer Research Center, Nobel Laureate for Medicine, 2001
Yeast has proved to be the most useful single-celled organism for

studying the fundamental aspects of cell biology. Resources are now
available for yeast that greatly simplify and empower new investigations,
like the presence of strains with each gene deleted, each protein tagged and
databases on protein–protein interactions, gene regulation, and subcellular
protein location. A powerful combination of genetics, cell biology, and
biochemistry employed by thousands of yeast researchers has unraveled the
complexities of numerous cellular processes from mitosis to secretion and
even uncovered new insights into prion diseases and the role of prions in
normal biology. These insights have proven, time and again, to foretell the
roles of proteins and pathways in human cells.
The collection of articles in this volume explores the use of yeast in
pathway analysis and drug discovery. Yeast has, of course, supplied
mankind’s most ubiquitous drug for thousands of years. In one aspect, the
role of yeast in drug discovery is much like the role of yeast in other areas of
biology. Yeast offers the power of genetics and a repetoire of resources
available in no other organism. Using yeast in the study of drug targets and
metabolism can help to make a science of what has been largely an empirical
activity. A science of drug discovery would permit rigorous answers to
important questions. What is the target of the drug? Is there more than one
target and what are the relative affinities? What is the physiological
consequence of inactivating a particular protein? Which drug in a panel is
the most specific? How many ways can a cell mutate to resistance? What is
the consequence of inhibiting two proteins? Which proteins in the cell if
inhibited would produce a desired physiological outcome? Are all the
proteins in a pathway equivalent targets? Can one identify drugs that alter
the location or interactions of proteins without affecting their activity? Each
of these questions can be rigorously answered in yeast but not in most other
ix
x Foreword
systems. Questions like these have rarely been answered in the field of drug
discovery.
A more challenging question is: Can yeast be made more applicable for
the discovery of drugs against human targets? While many human drugs are
active in yeast many are not. The lack of effect in yeast can be due to the fact
that yeast does not have the targets at all – e.g., cell surface hormone
receptors, or because the orthologous protein in yeast is sufficiently different,
or that yeast cells have redundant proteins and inhibition of one is masked by
the second. However, even if drugs against human targets are active in yeast,
the yeast orthologue is likely to be different enough to preclude optimization
of drug identification in yeast. One way to solve this problem is by
substitution of human orthologues for yeast ones. One could even substitute
human transport proteins and drug-metabolizing proteins to further optimize
the yeast system.
Moreover, our ultimate interest in drugs is to alter physiology, which is
the product not of single proteins but of pathways and networks of proteins
acting in concert. The most effective use of yeast would probably result
from substituting entire pathways of yeast proteins with their human counter-
parts. With the aid of reporters that quantitatively reveal the activity of the
pathway at different points one could enter a new era of drug discovery that
interrogates modulation of the pathway at different sites. Finally, we should
think about using the same approach for pathways that do not normally exist
in yeast – for example pathways that synthesize hormones. By constructing
the pathway in yeast de novo with appropriate reporters one could screen for
drugs that modulate hormone synthesis and easily localize the target in the
pathway. This sounds like fun. I suspect the dough has only begun to rise on
what yeast has to offer in the arena of drug discovery.
LIST OF CONTRIBUTORS
JULIE M. BAILIS ANITA H. CORBETT

The Salk Institute for Biological Department of Biochemistry
Studies and Graduate Program in
10010 North Torrey Pines Road Biochemistry
La Jolla, CA 92037 Cell and Developmental Biology
Emory University School of
SOMA S. R. BANIK Medicine
Departments of Pharmacology and 1510 Clifton Rd., NE
Cancer Biology and Radiation Atlanta, GA 30322
Oncology
Duke University Medical Center, CHRISTOPHER M. COUNTER
Box 3813 Departments of Pharmacology and
Durham, NC, USA 27710 Cancer Biology and Radiation
Oncology
ADAM C. BERGER Duke University Medical Center,
Department of Biochemistry Box 3813
and Graduate Program in Durham, NC, USA 27710
Biochemistry
Cell and Developmental Biology L. ASHELY COWART
Emory University School of Ralph H. Johnson
Medicine Veterans Administration
1510 Clifton Rd., NE and the Departments of
Atlanta, GA 30322 Medicine and Biochemistry
and Molecular Biology
RAINER K. BRACHMANN Medical University of South
Department of Medicine Carolina
University of California at Irvine Charleston, South Carolina
Irvine, CA, USA 92697
SUSAN L. FORSBURG
MARIA E. CARDENAS Molecular and Computational
Department of Molecular Genetics Biology Section
and Microbiology University of Southern California
Duke University Medical Center 835 W. 37th St., SHS 172
Durham, NC, USA 27710 Los Angeles, CA 90089-1340
xi
xii List of Contributors
RICHARD F. GABER KARL KUCHLER

Department of Biochemistry, Mqx F. Perutz Laboratories
Molecular Biology and Department of Medical Biochemistry
Cell Biology Division of Molecular Genetics
Northwestern University Medical University Vienna
Evanston, Illinois 60208 Campus Vienna Biocenter
Vienna, Austria
YUSUF A. HANNUN
Ralph H. Johnson ZHENGSHENG LI
Veterans Administration Department of Molecular
and the Departments of Pharmacology
Medicine and Biochemistry St. Jude Children’s Research
and Molecular Biology Hospital
Medical University of South Memphis, TN 38105
Carolina
Charleston, South Carolina JUN O. LIU
Departments of Pharmacology
IRA HERSKOWITZ and Neuroscience
Department of Biochemistry and Johns Hopkins School of Medicine
Biophysics Baltimore, MD 21205
University of California
JOHN L. NITISS
San Francisco, CA
Department of Molecular
Pharmacology
SUSAN L. HOLBECK
St. Jude Children’s Research
National Cancer Institute
Hospital
Developmental Therapeutics
Memphis, TN 38105
Program
Information Technology Branch, KARIN C. NITISS
Rockville, MD Department of Molecular
Pharmacology
ALBERTO INGA St. Jude Children’s Research
Laboratory of Molecular Genetics Hospital
National Institute of Memphis, TN 38105
Environmental Health Sciences
NIH, P.O. Box 12233 LINA M. OBEID
Research Triangle Park Ralph H. Johnson
NC 27709 Veterans Administration
and the Departments of
SEIKO ISHIDA Medicine and Biochemistry
Department of Biochemistry and and Molecular Biology
Biophysics Medical University of South
University of California Carolina
San Francisco, CA Charleston, South Carolina
List of Contributors xiii
CARMELA PALERMO CHRISTOPH SCHÜLLER

Department of Pharmacology Mqx F. Perutz Laboratories
UMDNJ-Robert Wood Johnson Department of Medical
Medical School and Joint Biochemistry
Graduate Program in Cellular Division of Molecular Genetics
and Molecular Pharmacology Medical University Vienna
UMDNJ-Graduate School of Campus Vienna Biocenter
Biomedical Sciences and Vienna, Austria
Rutgers
The State University of New Jersey JENNIFER S. SEARLE
675 Hoes Lane Department of Molecular Genetics,
Piscataway, NJ 08854-5635 Biochemistry and Microbiology
The University of Cincinnati
MICHAEL A. RESNICK 231 Ablert Sabin Way
Laboratory of Molecular Genetics Cincinnati, OH 45267-0524
National Institute of
Environmental Health Sciences JULIAN A. SIMON
NIH, P.O. Box 12233 Divisions of Clinical Research,
Research Triangle Park, NC 27709 Basic Sciences and Human
Biology
ANNA T. ROGOJINA Fred Hutchinson Cancer Research
Department of Molecular Center
Pharmacology Seattle, WA 98109
St. Jude Children’s Research
Hospital FRANCESCA STORICI
Memphis, TN 38105 Laboratory of Molecular Genetics
National Institute of
JOHN R. ROHDE Environmental Health Sciences
Department of Molecular Genetics NIH, P.O. Box 12233
and Microbiology Research Triangle Park, NC 27709
Duke University Medical Center
Durham, NC, USA 27710 FUYUHIKO TAMANOI
Department of Microbiology,
YOLANDA SANCHEZ Immunology & Molecular Genetics
Department of Pharmacology Genetics Jonsson Comprehensive
and Toxicology Cancer Center
Dartmouth Medical School Molecular Biology Institute
7650 Remsen University of California
Hanover, NH 03755 Los Angeles, 405 Hilgard Ave.
USA Los Angeles, CA 90095-1489
xiv List of Contributors
MARIJA TESIC NANCY C. WALWORTH

Department of Biochemistry, Department of Pharmacology
Molecular Biology and UMDNJ-Robert Wood Johnson
Cell Biology Medical School and Joint
Northwestern University Graduate Program in Cellular
Evanston, Illinois 60208 and Molecular Pharmacology
UMDNJ-Graduate School of
NITIKA THAPAR Biomedical Sciences and
Department of Microbiology, Rutgers
Immunology & Molecular The State University of New Jersey
Genetics 675 Hoes Lane
Jonsson Comprehensive Cancer Piscataway, NJ 08854-5635
Center
Molecular Biology Institute SARA A. ZURITA-MARTINEZ
University of California Department of Molecular Genetics
Los Angeles, 405 Hilgard Ave. and Microbiology
Los Angeles, CA 90095-1489 Duke University Medical Center
Durham, NC, USA 27710
INTRODUCTION
John L. Nitiss
Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis,
TN 38105
In 1970, Lee Hartwell reported a series of genetic experiments showing

that progression through the cell cycle in yeast was amenable to genetic
analysis. At about the same time, several investigators, including Michael
Resnick and Brian Cox identified the first yeast mutants that were shown to
be defective in DNA repair processes. Walt Fangman and his co-workers
were characterizing the basics of yeast chromosomes and yeast DNA
replication (even though cytogenetics was not practical, and because yeast
lack thymidine kinase, pulse labeling of DNA was not possible). Gerry Fink
was identifying the many ways a eukaryote regulates gene expression, while
Fred Sherman carried out studies on cytochrome c that illuminated
translation (and much else). Other investigators were becoming convinced
that yeast could shed light on many fundamental processes that were not
accessible in multicellular eukaryotes. Since many investigators committed
to using yeast as an experimental system, there was also considerable efforts
to increase the scope of yeast genetics by developing new genetic tools,
which became an effort to develop molecular, biochemical, and cell
biological tools. The important tools developed in yeast are too numerous to
mention (although the discovery of gene replacement by homologous
recombination surely requires note). Contemporary biologists, even those
studying “large” eukaryotes, continue to learn from yeast systems.
Despite the impressive roster of accomplishments in basic biology
obtained using yeast as a model, there are areas of importance of cancer
research where yeast has not been extensively utilized. In addition, many
investigators and clinicians working in many areas of cancer research tend
not to think of yeast as being relevant to their areas of interest. In some,
cases, yeast researchers have not made the appropriate effort to communicate
their results to the cancer research community. The goal of this book is to
highlight the contributions that yeast systems have made to in a variety of
xv
xvi Introduction
areas of cancer research. Accordingly, this volume is intentionally directed

more to workers outside the “yeast world” and toward investigators
interested in cancer. We have requested the authors to highlight areas where
yeast-based systems have made contributions not readily accessible with
other experimental systems, and to try to communicate clearly to workers
who may not be familiar with yeast.
This book is broadly organized into three sections. The first section,
including Chapters 1 through 8 highlight areas of biology that are particularly
relevant to cancer research. These include studies of DNA metabolism
(Chapters 1, 2, and 7), protein localization and trafficking (Chapters 3, 4, and
6), and cell immortalization (Chapter 5). Chapter 8, a discussion of sphingo-
lipids, is relevant both to the biology, and potentially, the development of
novel cancer treatments.
The second section, Chapters 9 and 10 describe how yeast can be used to
study human p53. These chapters highlight the ability to learn about the
function of human oncoproteins using yeast.
The third section is broadly concerned with studying anticancer drugs in
yeast. Some of the chapters discuss concerns broadly relevant to drug action
(Chapters 11 and 14), while the actions of specific anticancer drugs, such as
rapamycins, platinum compounds, and topoisomerase inhibitors are explored
in Chapters 13, 15, and 16. Finally, Chapter 12 describes one broad effort to
use yeast as a tool for drug discovery.
There are many other areas of interest not included in this volume where
yeast systems have made important contributions to cancer research. These
areas include important methodologies such as yeast two hybrid, areas of
basic biology such as the study of yeast Ras proteins and yeast kinases, and
areas of great relevance to anticancer drugs, such as yeast systems of DNA
repair. While we hope to include such topics in future volumes, we also felt
that there were other superb sources already available for topics such as a
general introduction to yeast.
This book would not have been possible without the efforts of Peggy
Vandiveer in the Word Processing Center at St. Jude Children’s Hospital.
Peggy carefully formatted all of the chapters and cheerfully and quickly
handled a huge amount of work. Thanks are also due to Jeffrey Berk and
Aman Seth in the Nitiss laboratory, who carefully checked all of the chapters
and caught many things that might have slipped through. Support for the
generation of this book was provided to JLN by the American Lebanese
Syrian Associated Charities (ALSAC).
Chapter 1
FROM DNA REPLICATION TO GENOME
INSTABILITY IN SCHIZOSACCHAROMYCES
POMBE: PATHWAYS TO CANCER
Julie M. Bailis1 and Susan L. Forsburg2

1
The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA
92037; 2Molecular and Computational Biology Section, University of Southern California,
835 W. 37th St., SHS 172, Los Angeles, CA 90089-1340
1 INTRODUCTION
The genetic integrity of cells depends on the complete, accurate

replication of each genome cell cycle. Cells are particularly susceptible to
genetic changes during the DNA synthesis (S) phase of the cell cycle,
because only one complete copy of the DNA template exists, and potentially
damaging breaks and unwinding occur as part of the replication process.
Genome instability may result in deletion or amplification of genetic
information within a chromosome, translocation of part of a chromosome to
another chromosome, or gain or loss of whole chromosomes. These changes,
in turn, can have important consequences for chromatin structure and gene
expression. In wild-type cells, DNA replication is tightly regulated and
involves multiple mechanisms that prevent amplification or loss of genetic
information. In addition, internal controls such as checkpoints delay or arrest
replication if active replication forks are blocked. Cancer cells, in contrast,
are characterized by uncontrolled proliferation and chromosome instability.
The fission yeast Schizosaccharomyces pombe provides an outstanding
model for studies of replication and chromosome dynamics, with replication
origins and centromeres that are similar to those of metazoans [46, 205].
This review will focus on DNA replication in S. pombe and its role in
maintenance of genome integrity. We will consider the choice and
1
J.L. Nitiss et al. (eds.), Yeast as Tool in Cancer Research, 1–35.
© 2007 Springer.
2 J. M. Bailis and S. L. Forsburg
organization of origins of DNA replication, the proteins that assemble at

origins and those that are responsible for actual DNA synthesis. We will also
describe regulatory mechanisms that promote origin firing, control the
timing of the replication program, and limit replication to once per cell
cycle. Then, we will discuss the cellular response to blocks to DNA
replication. Finally, we will evaluate how studies of DNA replication in
fission yeast and other model organisms have demonstrated relevance to
cancer biology, concentrating on those pathways that, when disrupted, lead
to genome instability and progression toward cancer.
1.1 DNA replication origins and the origin recognition

complex
The fission yeast genome, like that of other eukaryotes, is divided among
multiple linear chromosomes each requiring multiple origins of replication.
DNA synthesis must therefore be coordinated both between the different
chromosomes and within each individual chromosome. The identification of
specific fragments of the genome that could replicate autonomously on
plasmids provided the first step toward identifying S. pombe origins of
replication. The autonomously replicating sequence (ARS) elements in
S. pombe that have been described thus far are at least 500 bp in size and
correspond to intergenic regions of the genome [24, 53, 114, 203].
Interestingly, centromeric regions of S. pombe chromosomes appear to be
enriched for DNA fragments with ars activity [170]. In some chromosomal
regions, two or more origins are clustered close together [40, 147].
Replication initiates from discrete, defined sites within each origin region
[24, 39, 40, 53, 147, 203].
S. pombe replication origins preferentially lie in adenine–thymine (A–T)
rich regions of the genome where the local sequence of As and Ts is
asymmetric [210]. Although short consensus sequences similar to that of the
S. cerevisiae ARS element have been identified within some of the S. pombe
origins, these are not essential for origin activity [114]. Fine structure
examination of individual origins has revealed multiple short stretches of
DNA where replication initiates as well as adjacent accessory sequences that
promote origin function [29, 39, 85]. Thus, the organization of S. pombe
replication origins appears to be modular, with redundant, dispersed, and
degenerate elements contributing to efficient activity.
Different replication origins display distinct firing efficiencies [147]. It is
not known whether the choice of origins is regulated or stochastic. Within
those regions of the chromosome where origins are clustered, there may be a
hierarchy of preferential origin usage determined at least in part by local
enhancer sequences [84]. In the S. pombe genome, the number of potential
Chapter 1: Replication in Fission Yeast 3
replication origins has been estimated at 1 per 20 kb [114] to 1 per 55 kb

[203]. The recent publication of the S. pombe genome sequence [205] should
facilitate genome-wide identification of replication origins as has recently
been described for budding yeast [153, 206], as well as provide information
about origin usage under different growth conditions.
Replication initiation at origins requires the association of multiple
conserved proteins with the origin region [41, 80]. As in other eukaryotes,
the S. pombe origin recognition complex (ORC) binds to the origin DNA and
marks it as a potential site of replication initiation [27, 28, 179]. ORC is
composed of six related proteins (ORC1–6), each one of which is essential
and conserved in eukaryotic organisms [13]. In some organisms, several of
the ORC subunits may contact the origin DNA [13]. In S. pombe, ORC
binding to the origin DNA is mediated by the Orp4 subunit, which contains
multiple A–T hooks [27, 28, 90]. This motif, which is not present in the Orc4
subunit of other eukaryotes, is thought to promote protein binding within the
DNA minor groove in regions that are A–T rich [27].
Unexpectedly, S. pombe ORC can bind to multiple specific sites within a
single replication origin [28, 179]. These sites correspond to the regions of
the origin where replication initiates as determined by two-dimensional gel
analysis [179]. Thus, the position of the ORC complex within the origin may
determine the location and direction of the replication machinery. It is not
known whether S. pombe ORC also associates with nonorigin DNA, as has
been described for other eukaryotes and which may reflect the additional
role of ORC in heterochromatic silencing [13, 206].
The majority of ORC protein in S. pombe associates with chromatin,
including replication origins, throughout the cell cycle [107, 146]. The Orp2
(Orc2) subunit, originally identified as a cyclin-dependent kinase (CDK)
binding protein, contains consensus sites for CDK phosphorylation [95].
Orp2 becomes phosphorylated in a CDK-dependent manner during mitosis,
and is dephosphorylated during G1/S of the cell cycle [107, 197]. It remains
to be determined whether other ORC subunits also become modified during
the cell cycle.
2 THE PREREPLICATIVE COMPLEX

AND LICENSING OF ORIGINS
The preparation for S phase begins as early as mitosis (M phase) or G1,

when additional proteins required for DNA replication assemble sequentially
at replication origins to form the prereplicative complex (preRC). A series of
additional steps is required to convert the preRC into an initiation complex
that actively synthesizes DNA. While the proteins and general pathways
involved in this process are conserved in eukaryotes, many of the details and
order of their functions are still incomplete. Figure 1 provides a model of our
current view of the assembly and activation of proteins at replication origins.
Figure 1. Model of assembly and activation of the preRC based on information from multiple
systems. Cdc18 and Cdt1 bind to ORC at the origin and facilitate the loading of the MCM
protein complex. The preRC is activated by the actions of the CDK and Hsk1/Dfp1 kinases
(“P” indicates phosphorylation). This promotes the association of other replication factors
with the complex localized to the origin, and leads to initiation of DNA synthesis. Many of
the relevant substrates of Hsk1 and CDK have yet to be identified.
The S. pombe Cdc18 (Cdc6 in other eukaryotes) and Cdt1 proteins bind
to origin sites marked by ORC [55, 95, 141]. ORC and Cdc18 physically
interact [28], as do Cdc18 and Cdt1 [141]. ORC, Cdc18, and Cdt1 together
recruit the minichromosome maintenance (MCM) proteins to the complex
assembling at origins [79, 146]. Genetic interactions between the MCM pro-
teins and Cdc18, and the MCM proteins and ORC, have been demonstrated
in S. pombe [49, 55, 101, 102]. Although each of the preRC components is
essential for DNA replication, the different proteins carry out distinct
functions in assembly and activation of replication origins.
The Cdc18/Cdc6 and Cdt1 proteins are thought to act as a “licensing
factor” that is a critical determinant of the onset of DNA replication [81,
109, 142]. Expression of S. pombe Cdc18 and Cdt1, which is controlled by
the Cdc10 transcription factor [65, 81], is restricted to the G1/S window of
the cell cycle [81, 141]. In addition, the Cdc18 protein is regulated by
phosphorylation, which targets Cdc18 for ubiquitin-mediated proteolysis
[68, 69]. It is not known whether S. pombe Cdt1 protein levels are also
regulated. In Xenopus, Cdt1 activity is inhibited by association with another
protein, geminin [16]; however, a geminin homolog has not been described
in yeast. The cell cycle-regulated activity of Cdc18 and Cdt1 both promote
preRC assembly and prevents its reassembly until the next cell cycle [17,
80].
The MCM complex is not only a preRC component, but also a
compelling candidate for a replicative helicase [41, 80, 98, 148]. The
complex is composed of six homologous subunits (MCM2–7) with similarity
to a large family of ATPases [91]. Each subunit is conserved in other
eukaryotes, and each is essential for replication and cell viability [148, 193].
Mutation of individual S. pombe MCM genes results in defects in replication,
irreversible S-phase arrest and delocalization of the entire MCM complex
from the nucleus [34, 49, 101, 149]. The mcm mutant arrest occurs with the
bulk of replication completed and requires the DNA damage checkpoint,
suggesting that the chromosomes are damaged in mcm mutant cells [101,
102, 108, 123, 178].
Interestingly, although the MCM proteins are estimated to be at least
tenfold more abundant than the predicted number of origins, reducing the
amount of a single MCM protein results in chromosome instability and
defects in the completion of S phase [49, 102]. Replication can still initiate
with low levels of MCM protein, suggesting that the amount of MCM
protein required to initiate DNA replication is much less than that needed to
complete S phase [102]. Genetic interactions between S. pombe MCM genes
and factors involved in the elongation step of DNA replication suggest that
MCM complex function is required throughout S phase [49, 102]. This is
consistent with more direct experiments in S. cerevisiae, where specific
degradation of one of the six MCM subunits during S phase blocks further
DNA replication [94, 103]. The current model suggests that a heterohexa-
meric MCM complex with all six MCM subunits is present at replication
origins, and then travels with the replication fork [78, 148, 193].
Although all six S. pombe MCM subunits interact [1], distinct MCM
subcomplexes have been identified in vitro and in vivo, suggesting that
individual MCM proteins have different relative affinities for each other [96,
162, 163]. The Mcm4, 6, and 7 proteins are thought to form a “core” complex
that is tightly associated [96, 163]. Mcm2 associates with this core through
interactions with Mcm4 [163]. The Mcm3 and 5 proteins form a dimer that
is also loosely associated with the MCM core proteins [162], probably
through interaction with Mcm7 [101]. Similar subcomplexes of MCM
proteins have also been described in other eukaryotes [67, 160]. The core
complex of Mcm4, 6, and 7, but not the heterohexameric complex of Mcm2,
3, 4, 5, 6, and 7, demonstrates weak helicase activity in vitro in S. pombe [96]
and in human cells [67]. Curiously, point mutations of conserved residues in
each Mcm protein that are predicted to inhibit ATPase or helicase activity
display different effects in vivo in both S. pombe [49, 52] and S. cerevisiae
[160] depending on the subunit mutated. This suggests division of labor
amongst the MCM subunits, and may explain why six related proteins are
required for MCM activity.
Although MCM nuclear localization is regulated in budding yeast, MCM
proteins localize to the nucleus constitutively throughout the cell cycle in
fission yeast and in metazoans [149, 193]. In these organisms, the
association of the MCM proteins with chromatin varies: MCM proteins
localize to chromatin, including replication origins, in late M phase and
dissociate during S phase [79, 146]. Thus, regulation of MCM chromatin
binding is one mechanism of control of MCM complex function. Part of this
regulation is provided by the Cdc18 and Cdt1 loading factors, which limit
MCM binding to M/G1 of the cell cycle [80]. In some organisms, Mcm4
is also regulated by CDK-dependent phosphorylation. In Xenopus, this
promotes Mcm4 dissociation from chromatin [62, 152]; in S. cerevisiae,
Mcm4 phosphorylation leads to its exclusion from the nucleus [140].
However, CDK consensus site mutants of S. pombe Mcm4 do not display
obvious phenotypes in vivo [52].
3 REPLICATION INITIATION REQUIRES TWO

DISTINCT PROTEIN KINASE ACTIVITIES
Although several of the proteins required for DNA replication are

assembled at replication origins prior to the start of S phase, origin firing and
initiation of DNA synthesis requires the activities of two protein kinase
complexes, the CDK [17] and DDK (Dbf4-dependent) kinases [73, 110,
161]. While some substrates of these protein kinases have been identified, it
is clear that our understanding of their role is still incomplete. In fission
yeast, there is a single CDK, cdc2+, that functions as the major regulator of
cell cycle transitions including S phase onset [127]. Importantly, the ability
of S. pombe Cdc2 to functionally complement cross-species [11, 97]
suggests that the principles of function and regulation of the CDKs are
conserved in eukaryotes.
S. pombe Cdc2 associates with different cyclins in different stages of the
cell cycle. The G1/S phase transition is promoted by the assembly of Cdc2
with the B-type cyclin Cig2 [45, 125]. In mitosis, Cdc2 associates with the
Cdc13 cyclin [127]. Fission yeast CDK activity varies through the cell cycle,
and this global regulation controls the dependency of S phase on completion
of mitosis and prevents multiple rounds of replication within a single cell
cycle [21, 31, 126].
Figure 2. Control mechanisms that prevent rereplication. CDK activity is regulated so that the
preRC can only assembly once each cell cycle. CDK phosphoyrlates Drc1, which promotes
association with Rad4/Cut5 and replication initiation. CDK phosphorylation of Cdc18 and
Orc2 prevents origins from refiring.
Remarkably, the CDK kinase complex plays both positive and negative
roles in S phase progression (Figure 2). Replication initiation is positively
activated by CDK-mediated phosphorylation of Drc1, which results in the
association of Drc1 with Rad4/Cut5 [143]. Rad4/Cut5 is essential for the
initiation of DNA synthesis and also acts in the DNA damage checkpoint
[44, 156, 157]. Although the molecular role of Rad4/Cut5 is unclear, the
interaction between Rad4 and Drc1 may promote the association of the DNA
polymerases α and ε with origins [143]. The S. pombe homologs of
Rad4/Cut5 in S. cerevisiae (Dpb11) and in human cells (TopBP1) interact
with DNA polymerase as part of their replication function [112, 182]. Each
of these proteins contains BRCT motifs, which are also found in the human
DNA repair gene XRCC1 and the BRCA1 tumor suppressor [19].
CDK activity negatively regulates DNA replication by preventing origins
from refiring in a single cell cycle [17, 80]. CDK-mediated phosphorylation
of Cdc18 and its subsequent degradation prevent reassembly of the preRC
until the next cell cycle, because recruitment of preRC factors such as the
MCM proteins depends on Cdc18 [68]. Overproduction of Cdc18 or
expression of a nonphosphorylated version of Cdc18 induces re-replication,
presumably by resetting origins to the G1 state [80]. Mutation of the CDK
phosphorylation sites in Orp2 also allows re-replication [197], although the
mechanism by which CDK-mediated phosphorylation of Orp2 prevents
re-replication is not clear [95, 107, 197]. Unlike Cdc18, Orp2 appears to
remain associated with the ORC complex at the origin [107]; it is not known
whether phosphorylation of Orp2 changes its affinity for other origin-associated
proteins.
Manipulation of the Cdc2 protein kinase itself can result in re-
replication. Cells that lack mitotically active Cdc2 [21] or the mitotic cyclin
Cdc13 [61] re-replicate DNA, as do cells overproducing the CDK inhibitor
Rum1 [126]. These observations demonstrated that increased levels of CDK

kinase activity after the G1/S transition are required to prevent reinitiation of
DNA replication within a single cell cycle. Re-replication appears to mimic
a normal S phase because it requires all of the proteins that function in DNA
synthesis in a normal S phase [172]. This suggests that re-replication results
solely from a disruption of the order of the cell cycle.
CDK kinase activity is not the only control exerted over the onset of S
phase. Initiation of DNA replication also requires the activity of the Cdc7
protein kinase [161]. Hsk1, the S. pombe homolog of Cdc7 [23, 111]
associates with a subunit, Dfp1 (Dbf4 in other eukaryotes), which is required
for Hsk1 kinase activity toward its substrates [23, 180]. Although the levels
of Hsk1 protein appear to be constant in different phases of the cell cycle,
Dfp1 levels and the associated Hsk1-dependent protein kinase activity are
specifically upregulated during G1/S [22]. The Mcm2 protein is a substrate
of Hsk1/Dfp1 in vitro [22] and in vivo [181]. However, the biological effect
of Mcm2 phosphorylation remains to be determined. It is also possible that
Hsk1 has additional, as yet unidentified, substrates in vivo; in S. cerevisiae,
DNA polymerase α and another replication factor, Cdc45 (the homolog of
S. pombe Sna41), are targets of Cdc7 [82, 145, 201].
S. pombe Hsk1 has three apparent roles in the cell. First, Hsk1/Dfp1 may
activate individual origins of replication by phosphorylating components of
the preRC, as has been described for Cdc7 in S. cerevisiae [20, 37]. This role
for Hsk1 may also involve control of the temporal order of origin firing,
since the kinase is a potential target of the replication checkpoint [22, 171,
181]. Second, Hsk1 is involved in the recovery from replication blocks
such as hydroxyurea [171]. Third, Hsk1 may influence the establishment of
sisterchromatid cohesion during S phase [8, 171, 181]. The potential
mechanisms of these activities are discussed below.
4 ADDITIONAL REPLICATION FACTORS
Once the preRC is formed and activated by the CDK and Cdc7 protein
kinases, additional components of the replication machinery become
associated with the complex, which becomes the active replication fork.
Many of these proteins are essential for viability and conserved in all
eukaryotes, including Cdc23/Mcm10, Sna41/Cdc45, the single-strand DNA
binding protein replication protein A (RPA), DNA polymerase α DNA
polymerase δ DNA polymerase ε DNA, replication factor C (RFC), and
proliferating cell nuclear antigen (PCNA) [198].
S. pombe cdc23 mutants, like the corresponding mutants in other
eukaryotes, display defects in replication and demonstrate genetic
interactions with other replication mutants [7, 77, 100]. A mutant of the
S. cerevisiae homolog of cdc23+, MCM10, can be complemented by
S. pombe cdc23+, suggesting conservation of gene function [7]. S. cerevisiae
MCM10 appears necessary to recruit the MCM complex to chromatin [66],
and also functions in replication elongation [116]. In both S. cerevisiae and
S. pombe, Cdc23 associates with chromatin throughout the cell cycle [66,
101]. In Xenopus, MCM10 is dispensable for MCM complex binding to
chromatin but is required to localize Cdc45/Sna41 [204].
Sna41/Cdc45 appears to be required both for replication initiation and
elongation. The association of Sna41 with chromatin requires the Sld3
protein, which has been described in both the budding and fission yeasts [74,
136]. Sna41/Cdc45 is required to load other replication factors such as RPA,
DNA polymerase α, DNA polymerase ε, and PCNA [182]; in S. pombe,
Sna41 [124, 194] has been shown to interact with DNA polymerase α [195]
and recruit it to the MCM protein complex at replication origins [195]. A
role for Sna41/Cdc45 in origin DNA unwinding has been suggested from
experiments in Xenopus [199] and in S. cerevisiae [74]. In S. cerevisiae,
Cdc45 function has been shown to be required throughout S phase [189].
Many of the genes involved in DNA replication in S. pombe are
homologous to those described in other eukaryotes, and the enzymology of
the gene products is conserved [198]. RPA is needed to recruit the DNA
polymerases α, δ, and ε to replication origins [198]. Interestingly, DNA
polymerase α may have an additional function beyond replication initiation
and DNA synthesis (see section VIII). The eukaryotic DNA polymerase
processivity factor PCNA becomes associated with the replication fork
through the actions of the clamp loader, RFC [198]. S. pombe PCNA also
interacts with DNA polymerase δ [155] and promotes its processivity [6].
Components of S. pombe RFC and primase are involved in the replication
checkpoint as well as in DNA synthesis [56, 164].
Several proteins involved in lagging strand metabolism, such as DNA
ligase [72], the Dna2 helicase [54, 75], and the homolog of the FEN1
endonuclease, Rad2 [132] have also been characterized in S. pombe. The
dna2 mutant can be suppressed by overproduction of DNA polymerase δ,
DNA ligase or Rad2, suggesting that Dna2 has a central function in Okazaki
fragment maturation [75]. Mutants of a novel S. pombe replication factor,
cdc24+, are suppressed by overproduction of Dna2, as well as by
overproduction of PCNA or RFC [54, 184]. S. pombe cdc24+ has no known
sequence homolog in other systems [54]. Mutants of cdc24+ are defective in
the completion of S phase and display chromosome breakage, which is
not typical of replication mutants [54]. Cdc24 thus may play a role in
maintenance of chromosome integrity during S phase, perhaps through a

function in Okazaki fragment maturation.
5 TIMING OF REPLICATION ORIGIN FIRING
Coordination of multiple replication origins on multiple chromosomes

is essential to maintain stability of the genome. In wild-type cells, there is a
temporal program of origin firing: some origins fire early in S phase, others
are active in the middle of S phase, and still other replication origins do not
initiate synthesis until late in S phase. In general, heterochromatic regions
are late-replicating in most organisms [208]. However, a recent analysis of
replication origins in S. pombe [86] suggests that a centromeric origin in
S. pombe replicates early in S phase. In contrast, origins located within other
regions of heterochromatin, in particular the telomere and the rDNA,
replicate late in S phase. Although most [14, 16] of the origins analyzed in
this study [86] replicate early in S phase, it remains to be determined
whether these are representative of other S. pombe origins.
Control of the timing of origin firing has been investigated using the
drug hydroxyurea (HU) to block cells in S phase. HU treatment causes an
array of lesions in the cell, including inhibition of the enzyme ribonucleotide
reductase, which depletes the nucleotide pools and prevents further DNA
replication [80, 106, 158, 188]. For the few origins that have been analyzed
in S. pombe, HU treatment prevents the activity of normally late-firing
origins, but does not restrain early origin firing [86]. This mechanism is
checkpoint-dependent [86], as both early and late origins are active in
mutants lacking the checkpoint kinases Rad3 (a homolog of human ATM) or
Cds1 (the equivalent of human CDS1/CHK2 and budding yeast Rad53).
Checkpoint-dependent inhibition of late origin firing was previously described
in budding yeast [158, 166], and the conservation of replication timing
control between these divergent yeasts suggests that a similar mechanism
should also operate in human cells.
6 RESPONSE TO REPLICATION BLOCKS
During S phase progression, active replication forks may encounter

blocks to further synthesis. In S. pombe, HU-induced arrest has been most
often used to study the effects of blocks to replication. Cells treated with HU
undergo checkpoint-mediated arrest in S phase that depends on Cdc2 and six
“checkpoint rad” proteins [25]. Wild-type cells can recover from HU
treatment, resulting in restart of DNA replication, the completion of S phase
and subsequent progress through the cell cycle. This suggests that replication
fork structure is preserved during S phase arrest caused by HU treatment.
The checkpoint kinase Cds1 may play a role in stabilization of stalled
replication forks. S. pombe cds1 mutants arrest in HU, but are defective in
recovery from the arrest [104]. The HU-induced arrest of cds1 cells results
from the activation of the damage checkpoint kinase Chk1, suggesting that
Cds1 is needed to prevent chromosomal damage in the presence of HU
[104]. Consistent with this hypothesis, aberrant replication structures are
observed in S. pombe cds1 and rad3 mutants treated with HU [86]. Loss of
replication fork integrity has also been observed in rad53 mutants of
S. cerevisiae, suggesting a conserved mechanism of damage tolerance [106,
188].
S. cerevisiae Rad53 is phosphorylated in response to S phase damage
[150, 188] and phosphorylates components of the replication machinery to
restrain its progression [150]. Similarly, S. pombe Cds1 becomes phos-
phorrylated in response to HU treatment [104]. This in turn leads to Cds1-
dependent phosphorylation of both Hsk1 and its activator Dfp1, suggesting
that Hsk1 and Dfp1 are targets of the cellular response to replication blocks
[22, 171]. These phosphorylation events may serve to regulate the inter-
action of Hsk1/Dfp1 with proteins localized to replication origins and to
prevent the activation of origins under conditions unfavorable to the cell
[70, 110].
Although S. pombe Hsk1 and Dfp1 have an essential role in activation of
replication origins, Cds1 is dispensable during a normal S phase [104, 129].
Recently, an S-phase specific upstream activator of Cds1, Mrc1, was
identified [3, 185]; both Mrc1 and Cds1 are nonessential for cell viability [3,
129]. Future work should determine how the cell senses blocks to replication
and how this information is directed to Mrc1 and Cds1.
Recovery from HU-induced S phase arrest also requires Rqh1, the fission
yeast homolog of the Escherichia coli RecQ helicase [175]. The wild-type
function of Rqh1 during S phase is not known but may involve preventing
inappropriate recombination events when replication forks are stalled [175].
Mitotic recombination is elevated in rqh1 mutants that are treated with HU
[131, 175]. Mutants of rqh1+ also display synthetic genetic interactions with
components of the replication machinery [131, 171]. In S. cerevisiae, the
Rqh1 homolog SGS1 localizes with the Rad53 kinase and promotes its
phosphorylation in response to HU [51]. However, SGS1 mutants are
hyperrecombinant even in the absence of HU, suggesting there may be some
differences in the function between SGS1 and Rqh1 [51].
7 RECOMBINATION PROTEINS IN S PHASE
Several replication mutants display increased levels of recombination,

suggesting a link between these processes. Certain alleles of S. pombe DNA
polymerase α, DNA ligase, and rad2+ have mutator phenotypes [105]. The
increase in mutation frequency in these mutants suggests that the corres-
ponding wild-type proteins prevent genome changes and rearrangements,
which may result from recombination during S phase. Recombination is
elevated in mcm mutant cells that have been arrested in S phase [102]. In
addition, S. pombe rad2 mutants are synthetically lethal in combination with
mutants of rad50, rhp51, or rhp54 (the S. pombe homologs of RAD50,
RAD51, and RAD54), suggesting that recombination functions become
essential when Okazaki fragment metabolism is compromised [58, 130,
132]. The association of impaired replication function with increased
recombination has also been described in S. cerevisiae [36, 117] and
prokaryotes [119], suggesting this is a general feature of S phase.
Conversely, certain recombination mutants display S phase defects. In
the S. pombe rad50 mutant, S phase is delayed relative to wild type and the
cells are sensitive to HU [58]. In vertebrate cells, inactivation of the
recombination proteins Rad51 [173] or Mre11 [32] leads to DNA strand
breaks and cell lethality. These and other observations have led to the
suggestion that recombination proteins are normal components of S-phase
progression in eukaryotes that protect genome integrity [133, 135]. Thus,
replication fork stalls and starts may occur as part of normal S phase in
eukaryotes, as has been described in prokaryotes [33, 119].
There are several possible consequences of a stalled replication fork,
which may depend on its cause. Ideally, fork structure is protected and its
components remain assembled during the arrest (Figure 3). However, the
fork may lose structural integrity if this protection fails, resulting in its
collapse and the generation of DNA breaks; these breaks are likely to be
lethal to the cell if they are not repaired [33, 119].
Recombination is one mechanism that can reestablish a replication fork
from a DNA break [33, 120]. Although recombination-dependent replication
has been best characterized in prokaryotes, there is evidence that a similar
process operates in eukaryotes. In S. cerevisiae, break-induced replication
(BIR) can replicate hundreds of kilobases of DNA starting from a
chromosomal break [92]. In S. pombe, cells lacking telomerase can replicate
telomere sequences, presumably by a recombinational mechanism [137].
Importantly, replication mediated by recombination is predicted to be
independent of replication origins and origin proteins. Thus, there may be
mechanistic links between recombination and replication throughout S phase
in S. pombe and other eukaryotes, which are likely to be significant for the
maintenance of overall genome stability.
Figure 3. Model for replication restart (based on 33,119). When cells are treated with HU,
replication forks stall. If the structure of the fork can be maintained through the arrest, then
the fork may resume synthesis once HU is removed from the media. If the fork structure
cannot be maintained, the fork may collapse, generating DNA double-strand breaks.
Recombination is one mechanism that may repair DNA breaks and reestablish stalled
replication forks.
8 COORDINATION OF S PHASE EVENTS

WITH MITOSIS
The events of S phase are closely linked with the later events of the cell
cycle. As described above, maintenance of the order of the cell cycle and
alternation of S phase with mitosis preserves the genome integrity. In
addition, chromosomal processes such as sister-chromatid cohesion and
silencing are regulated coordinately with the replication of the DNA.
Intriguingly, the replication fork is the one structure that contacts all of the
DNA, once, in a single cell cycle. This positions the replication machinery
in a unique state to monitor and modify any region of the genome during
S phase.
Cohesion between newly replicated sister chromatids holds them together
from S phase until their separation during mitosis. This arrangement is
essential for the proper attachment of kinetochores to the microtubule
spindles and correct segregation of the chromatids during mitosis [139]. The
close apposition of sister chromatids throughout G2 and M phase likely also

facilitates repair of damaged DNA off the homologous template [169].
Cohesion is mediated by a conserved complex of proteins first identified in
S. cerevisiae, the cohesins [57, 118]. Homologs of the cohesins also exist in
fission yeast [15, 187, 192] and have provided important insights as to how
cohesion assembly is regulated during S phase.
The fission yeast Eso1 protein is critical for the activation of cohesion
function, although it is not required for the assembly of cohesin proteins with
chromatin [186]. Interestingly, Eso1 appears to be a fusion of two budding
yeast proteins: the cohesin activator Eco1 and the DNA damage bypass
polymerase Rad30. This homology suggests a direct link between cohesion
and DNA damage repair.
In fission yeast, cohesion sites along the chromosome have been iden-
tified and are particularly concentrated at the repeat regions of centromeres
[14, 192]. Like human centromeres, S. pombe centromeres are relatively
large (compared to S. cerevisiae centromeres) and contain heterochromatin.
Two recent studies indicate that the heterochromatin protein Swi6 (the
equivalent of mammalian HP1) is specifically required for the efficient
assembly of cohesion proteins to the centromere [14, 144]. Cohesion
association with chromatin arms occurs independently of Swi6.
Unexpectedly, the Hsk1 kinase also has a role in sister-chromatid
cohesion [171, 181]. Like Swi6, Hsk1 specifically influences the association
of the cohesin complex with centromeres [8]. Swi6 physically interacts with
both Hsk1 and its subunit Dfp1, suggesting direct recruitment of cohesin to
centromeres by these replication proteins [8]. These findings suggest a role
for Hsk1 and Dfp1 in sister-chromatid cohesion that may be separable from
their role in replication initiation.
Replication proteins also affect heterochromatin function. Within regions
of heterochromatin, including centromeres, most genes are normally
silenced. At least in budding yeast, silencing involves progression through S
phase, although perhaps not fork passage per se [50, 88, 99, 121]. As noted
above, eukaryotic ORC proteins are required for gene silencing and localize
to heterochromatin regions in addition to replication origins [13]. In
S. cerevisiae, the PCNA [209] [43], RFC, and Cdc45 proteins also contribute to
silencing [43]. In S. pombe, mutants of DNA polymerase α display defects in
silencing [138]. DNA polymerase α interacts with Swi6 and is required for
its proper localization to heterochromatin [2, 138] suggesting that disruption
in silencing can be attributed to defective heterochromatin structure. hsk1
mutants also display a defect in silencing, even though the Swi6 protein is
localized to heterochromatin [8]. This suggests that heterochromatin function
depends on the assembly of other proteins with Swi6; whether this requires a
passing replication fork or other aspects of S-phase progression remains to

be determined.
9 DNA REPLICATION IN FISSION YEAST

MEIOSIS
Meiosis is a specialized cell cycle that generates recombinant, haploid

progeny cells from a diploid cell. The meiotic cell cycle differs from the
vegetative cell cycle in two outstanding respects. First, the S phase that
occurs prior to meiosis (premeiotic S) is followed by two successive rounds
of chromosome segregation, rather than alternating between S phase and
mitosis. Second, the first meiotic division is reductional, resulting in the
maintenance of cohesion between sister chromatids but the separation and
segregation of homologous chromosomes. The preparation for this modified
cell division cycle involves lengthy interaction between homologous
chromosomes during the prophase stage of meiosis I but is likely to initiate
as early as premeiotic S phase.
Premeiotic S phase is longer than S phase in vegetative cells in most
organisms [26]. The cause of this difference is unclear, since experiments in
budding yeast suggest that the same replication origins are active in vege-
tative and meiotic cells [30]. However, other experiments in S. cerevisiae
suggest that meiosis-specific chromosomal factors required during prophase
might assemble during premitotic STET [26].
Recent studies in S. pombe have addressed the question of whether the
replication machinery that functions during premeiotic S phase is the same
as that utilized in the vegetative cell cycle [48, 103, 128]. S. pombe proteins
required for the actual synthesis of the DNA in vegetative cells, such as
DNA polymerase α and ribonucleotide reductase, also are essential for
premeiotic S phase [48]. In contrast, mutants defective in initiation of DNA
replication, such as the mcm mutants and cdc18, display different
phenotypes in meiosis and mitosis. In mitosis, conditional alleles of these
mutants allow bulk DNA replication but cause cells to arrest in late S phase
[49, 81, 102]. In contrast, in similar conditions these mutants can proceed
through the meiotic divisions and sporulate [48]. With more extreme
conditions, these mutants delay replication and the subsequent meiotic
divisions [103, 128]. This may reflect a quantitative difference: meiotic cells
may tolerate a lower amount of certain replication proteins than that needed
during the vegetative cell cycle. It is also possible that other meiotic factors,
perhaps recombination proteins, can contribute to premeiotic replication.
The S. pombe MCM protein complex is associated with chromatin during
premeiotic S phase [103], consistent with the hypothesis that these proteins
function in premeiotic DNA replication. However, MCM proteins are not

localized to chromatin in between the meiotic divisions, when an additional
round of DNA replication is suppressed [103].
Similar to the vegetative cell cycle, the meiotic cell cycle is subject to
checkpoint controls. Fission yeast cells that have been induced to enter
meiosis block the cell cycle when treated with HU [48, 128]. As in the
vegetative cell cycle, HU-induced arrest during meiosis is likely to be
checkpoint-dependent. Future work should resolve the components of this
response, which are likely to be essential for the viability of gametes.
Importantly, premeiotic S phase is closely coupled to the downstream
events of meiosis such as recombination [47]. This has been best demon-
strated in budding yeast, where blocks to premeiotic DNA synthesis prevent
meiotic recombination and changes in the timing of premeiotic replication
result in corresponding changes in the timing of initiation of meiotic
recombination [18]. The molecular mechanism by which this occurs is still
unclear [47].
10 REPLICATION: A CONSERVED PROCESS
Most, if not all, of the proteins involved or implicated in DNA replication

in S. pombe have homologs in other eukaryotes including human cells.
Analysis of these proteins in S. pombe and other model organisms has
greatly facilitated the identification and characterization of the human
counterparts. However, many of the details of regulation of the replication
process differ between yeast and human cells, which may reflect internal
differences in cell cycle control between the different organisms as well as
the additional complexity required in multicellular organisms.
S. pombe cells maintain genome stability and order of the cell cycle
through multiple and overlapping pathways. In particular, the process of
DNA replication itself is tightly regulated so that the genome is duplicated
once, entirely and accurately, in each cell cycle. Mechanisms are also in
place to restrain passage into mitosis until replication is complete. It is
possible that additional mechanisms, as yet uncovered, also act to maintain
the integrity of the genetic material. S. pombe cells that have lost this control
capability may re-replicate DNA, lose chromosomes, and die. In compa-
rison, deregulation of S-phase control in human cells may result in genome
instability and contribute to cancer progression.
11 PATHWAYS TO CANCER FROM REPLICATION

DEFECTS
The recent completion of the S. pombe genome [205] revealed an

impressive number of fission yeast genes with human homologs implicated
in cancers. Interestingly, many of these genes have known or implied
functions in DNA replication in S. pombe (Table 1). Current evidence
suggests that multiple pathways of control of eukaryotic DNA replication
can be disrupted to result in genome instability and predisposition to cancer
[133]. Thus, deregulation of CDK activity, impaired origin firing, changes in
the timing of firing, loss of control in the order of S phase and M phase, and
inability to limit replication to once per cell cycle are all mechanisms that
may lead to changes in chromosome structure and gene function [17, 80]. In
addition, defects in the checkpoint response to replication blocks, and the
inability to respond appropriately to stalled replication forks, also contribute
to genome instability. Ultimately, the gain or loss of genetic information
may lead to inappropriate expression of proto-oncogenes or loss of
tumor-suppressor function [59].
When the normal timing of origin firing is disrupted, cells are susceptible
to deregulated cell cycle progression. This could result either through
refiring of origins in a single cell cycle, or through firing late origins of
replication under conditions where they are normally prevented from firing.
Treatment of S. cerevisiae cells with the antitumor drug adozelesin changes
the normal pattern of replication such that active replication forks are
blocked, but silent origins are activated [200]. Translocations or amplifi-
cations of mammalian chromosomes also may alter replication timing of a
particular sequence [165, 177]. Conversely, uncontrolled cell proliferation
may result in deregulation of replication timing. This is observed both in
checkpoint mutants in S. pombe [86] and in human cancers [38]. Thus,
disruption of the timing and coordination of replication is one pathway
toward genome instability.
Cells extend multiple, overlapping control mechanisms to restrict DNA
replication to once per cell cycle. In S. pombe, this is accomplished by
regulation of CDK kinase activity, phosphorylation, and destruction of
Cdc18, and phosphorylation of STET [80]. In human cells, the Cdc18
equivalent Cdc6 is also negatively regulated by CDK phosphorylation [115],
suggesting that regulation of human Cdc6 likewise contributes to prevention
of re-replication. The MCM proteins are another CDK target, at least in
some organisms [17]. There are several examples of deregulated CDK
activity associated with cancers [42, 174].
Table 1. Fission yeast replication-related proteins and their human homologs

S. pombe protein Human equivalent Role in cancer (References)
PreRC
Orp1–6 ORC1–6
Upregulated in cancer cells [4, 64, 154, 167, 183,
MCM2–7 MCM2–7 202]; Mcm2 (Bm28/CDCL1) is associated with
acute myeloid leukemia [122, 190]
Cdc18 Cdc6 Upregulated in proliferating cells [202]
Cdt1 Cdt1 Upregulated in cancer cells [5]
Initiation
Rad4/Cut5 TopBP1 BRCT domains found in BRCT and XRCC1 [19]
Sld3 ?
Sna41 Cdc45
Drc1 ?
Hsk1 Cdc7 Upregulated in cancer cells [63]
Dfp1 Dbf4, ASK
Synthesis
Pol1/Swi7 Pol α
Cdc6 Pol δ
Cdc20 Pol ε
Cdc17 DNA ligase Upregulated in cancer cells [176]
PCNA PCNA Upregulated in proliferating cells [183, 202]
RFC1–5 RFC1–5
RPA RPA Overproduced in cancer cells [191]
Rad2 FEN1
Cdc24 ?
Checkpoint/Cell Cycle
Rad3 ATR, ATM Ataxia telangiectasia [159]
Cds1 Rad53/CHK2 Li-Fraumeni [12]
Chk1 CHK1
Rad17 RAD17 Overexpressed in cancer cells [9]
Cdc2 CDC2 [42]
Mrc1 Claspin
Swi1 ?
Recombination/Repair
Rad50 RAD50
Ataxia-telangiectasia-like disorder (Mre11),
Mre11 MRE11
Nijmegen syndrome (Nbs1) [35]
Nbs1 NBS1
Rhp51 RAD51
Rhp54 RAD54
Bloom’s syndrome (BLM), Werner’s syndrome
Rqh1 BLM, WRN, RecQL4 (WRN), Rothmund-Thomson Syndrome (RecQL4)
[51, 196]
Cohesion/Chromatin
Swi6 HP1 Downregulated in cancer cells [89]
Rad21 Rad21 Downregulated in cancer cells [83]
Eso1 Rad30/Pol µ Xeroderma pigmentosum variant [71, 113]
Overexpression of certain replication proteins, such as Cdt1, can promote

tumor formation in mammals [5]. In addition, many replication proteins are
specifically upregulated in cancer cells. Human Cdc7 (the homolog of the
S. pombe Hsk1 kinase) is overexpressed in certain tumor cells [63].
Furthermore, human MCM proteins are specifically expressed (or
overexpressed) in cycling cells and are not detectable in quiescent cells. An
important consequence of these findings is that the presence of the MCM

proteins in cells provides a sensitive diagnostic marker for proliferating cells.
MCM proteins are detected in cells that have exited quiescence and
reentered the cell cycle; thus, MCM proteins are detected in precancerous
cells as well as in tumor cells [4, 64, 154, 183, 190, 202]. MCM transcription
is further upregulated by activated oncogenes [167]. Interestingly, human
BM28/CDCL1 (the homolog of Mcm2), maps to a chromosomal locus
associated with acute myeloid leukemia, suggesting BM28/CDCL1 as a
candidate oncogene [122].
Damage tolerance and repair mechanisms are also essential to prevent
genome instability. In S. pombe, Rqh1 is needed for recovery from
replication blocks [131, 175]. Human cells have at least five Rqh1
homologs, three of which are linked with cancer susceptibility syndromes.
Mutations in BLM are associated with Bloom’s syndrome, mutations in
WRN lead to Werner’s syndrome, and mutation of RecQL4 results in
Rothmund-Thomson syndrome [51, 196]. Hyperrecombination and cancer
susceptibility are characteristic of both Bloom’s and Werner’s syndromes.
Inappropriate recombination due to the loss of other S-phase functions may
generate deletions or expansions in the genetic information, as has been
demonstrated in S. cerevisiae [134, 135]. Polymerase slippage may
contribute to the formation of triplet repeat sequences, which are associated
with several disorders including Huntington’s disease [93].
Checkpoint genes are important gatekeepers of genome stability [59].
Mutations in the ATM checkpoint kinase are linked to ataxia telangiectasia
[159], and mutations in the checkpoint kinase Cds1 (also called CHK2) are
found in a subset of patients with Li-Fraumeni syndrome [10, 12]. In
addition, Rad17 (one of the checkpoint rad proteins) is overexpressed in
certain types of human cancers [9]. The corresponding S. pombe proteins
(Rad3, Cds1, and Rad17) are all involved in the cellular response to
replication blocks [25]. The S. pombe Rad4/Cut5 protein, which also has a
role in cellular checkpoints [156], contains a BRCT motif that is also present
in the human BRCA1 tumor suppressor and the XRCC1 DNA repair protein
[44, 157]. Thus, mutations that disrupt function of the replication checkpoint
are also implicated in predisposition to cancer.
Genomic instability leading to cancers may also result from chromosome
structure defects caused by errors in S-phase processes linked to DNA
replication. In S. pombe, the Eso1 protein is needed to activate cohesion so
that sisterchromatids are held together until mitosis [186]. Part of the Eso1
protein is homologous to DNA polymerase η (Rad30), which is defective
in the xeroderma pigmentosum variant syndrome characterized by
predisposition to skin cancers [71, 113]. In addition, human securin, which
normally prevents premature sister-chromatid separation, can induce cell

transformation and tumorigenesis when overexpressed [211].
Expression of the Rad21 cohesin is downregulated in certain tumors [83].
In addition, HP1 is downregulated in breast cancer cells that are metastatic
or invasive [89]. The S. pombe homolog of HP1, Swi6, recruits Rad21 to
centromeres and other regions of heterochromatin [14, 144]. Recently,
phosphorylation of another cohesin subunit, Smc1, has been shown to be
required for the S-phase checkpoint in human cells [87, 207]. Taken
together, these connections suggest a direct role for chromatin structure in
maintenance of genome stability.
Work from multiple experimental systems in recent years has revealed
striking similarities in basic cellular processes among eukaryotes. In
addition, synergy between studies of DNA replication, recombination, and
chromosome structure has provided important insights into how DNA
replication is integrated with other cellular processes. Studies of DNA
replication in S. pombe and in other model systems has provided the proteins
and pathways that serve as a framework for identification and character-
ization of the human counterparts. Collections of yeast mutants, as well as
information about which genes together are essential for a process (synthetic
lethality), are an important resource for understanding how mutations in
distinct genes can result in similar phenotypes such as predisposition to
cancers [60]. Several recent studies have supported the validity of yeast
mutants with chromosome instability defects as models for the cellular
response to cancer treatment drugs [76, 151, 168]. These simple eukaryotes
continue to lead the way in fundamental understanding of normal and
abnormal cell division.
ACKNOWLEDGMENTS
Work in our laboratory was supported by grants from the NIH, NSF, and
the American Cancer Society. S.L.F. is a Stohlman Scholar of the Leukemia
& Lymphoma Society. J.M.B. is a fellow of the Damon Runyon Cancer
Research Fund (DRG-1634). We thank Michael Catlett and Han-Kuei
Huang for comments on the manuscript.
REFERENCES
1. Adachi, Y., J. Usukura, and M. Yanagida. 1997. A globular complex formation by

Nda1 and the other five members of the MCM protein family in fission yeast. Genes to
Cells 2:467–479.
2. Ahmed, S., S. Saini, S. Arora, and J. Singh. 2001. Chromodomain protein Swi6-
mediated role of DNA polymerase alpha in establishment of silencing in fission yeast.
J. Biol. Chem. 276:47814–47821.
3. Alcasabas, A. A., A. J. Osborn, J. Bachant, F. Hu, P. J. Werler, K. Bousset,
K. Furuya, J. F. Diffley, A. M. Carr, and S. J. Elledge. 2001. Mrc1 transduces signals
of DNA replication stress to activate Rad53. Nat. Cell Biol. 3:958–965.
4. Alison, M. R., T. Hunt, and S. J. Forbes. 2002. Minichromosome maintenance
(MCM) proteins may be pre-cancer markers. Gut 50:290–291.
5. Arentson, E., P. Faloon, J. Seo, E. Moon, J. M. Studts, D. H. Fremont, and K. Choi.
2002. Oncogenic potential of the DNA replication licensing protein CDT1. Oncogene
21:1150–1158.
6. Arroyo, M. P., K. M. Downey, A. G. So, and T. S. F. Wang. 1996.
Schizosaccharomyces pombe proliferating cell nuclear antigen mutations affect DNA
polymerase delta processivity. J. Biol. Chem. 271:15971–15980.
7. Aves, S. J., N. Tongue, A. J. Foster, and E. A. Hart. 1998. The essential
Schizosaccharomyces pombe cdc23 DNA replication gene shares structural and
functional homology with the Saccharomyces cerevisiae DNA43 (MCM10) gene. Curr.
Genet. 34:164–171.
8. Bailis, J. M., P. Bernard, R. Antonelli, R. C. Allshire, and S. L. Forsburg. 2002. In
preparation.
9. Bao, S. D., M. S. Chang, D. Auclair, Y. P. Sun, Y. B. Wang, W. K. Wong, J. Y.
Zhang, Y. Liu, X. Q. Qian, R. Sutherland, C. Magi-Galluzi, E. Weisberg, E. Y. S.
Cheng, L. N. Hao, H. Sasaki, M. S. Campbell, S. K. Kraeft, M. Loda, K. M. Lo, and
L. B. Chen. 1999. HRad17, a human homologue of the Schizosaccharomyces pombe
checkpoint gene rad17, is overexpressed in colon carcinoma. Cancer Res. 59:2023–
2028.
10. Bartek, J., J. Falck, and J. Lukas. 2001. CHK2 kinase – a busy messenger. Nat. Rev.
Mol. Cell. Biol. 2:877–886.
11. Beach, D., B. Durkacz, and P. Nurse. 1982. Functionally homologous cell cycle
control genes in budding and fission yeast. Nature 300:706–709.
12. Bell, D. W., J. M. Varley, T. E. Szydlo, D. H. Kang, D. C. R. Wahrer, K. E.
Shannon, M. Lubratovich, S. J. Verselis, K. J. Isselbacher, J. F. Fraumeni, J. M.
Birch, F. P. Li, J. E. Garber, and D. A. Haber. 1999. Heterozygous germ line hCHK2
mutations in Li-Fraumeni syndrome. Science 286:2528–2531.
13. Bell, S. P. 2002. The origin recognition complex: from simple origins to complex
functions. Genes Dev. 16:659–672.
14. Bernard, P., J. F. Maure, J. F. Partridge, S. Genier, J. P. Javerzat, and R. C.
Allshire. 2001. Requirement of heterochromatin for cohesion at centromeres. Science
294:2539–2542. Published online 11 Oct 2001(10.1126/science.1064027).
15. Birkenbihl, R. P., and S. Subramani. 1992. Cloning and characterization of rad21 an
essential gene of Schizosaccharomyces pombe involved in DNA double-strand-break
repair. Nucleic Acids Res. 20:6605–6611.
16. Blow, J. J. 2001. Control of chromosomal DNA replication in the early Xenopus
embryo. EMBO. J. 20:3293–3297.
17. Blow, J. J., and B. Hodgson. 2002. Replication licensing – origin licensing: defining
the proliferative state? Trends Cell Biol. 12:72–78.
18. Borde, V., A. S. H. Goldman, and M. Lichten. 2000. Direct coupling between meiotic
DNA replication and recombination initiation. Science 290:806–809.
19. Bork, P., K. Hofmann, P. Bucher, A. F. Neuwald, S. F. Altschul, and E. V. Koonin.
1997. A superfamily of conserved domains in DNA damage-responsive cell cycle
checkpoint proteins. FASEB J. 11:68–76.
20. Bousset, K., and J. F. X. Diffley. 1998. The Cdc7 protein kinase is required for origin
firing during S phase. Genes Dev. 12:480–490.
21. Broek, D., R. Bartlett, K. Crawford, and P. Nurse. 1991. Involvement of p34cdc2 in
establishing the dependency of S phase on mitosis. Nature 349:388–393.
22. Brown, G. W., and T. J. Kelly. 1999. Cell cycle regulation of Dfp1, an activator of the
Hsk1 protein kinase. Proc. Natl. Acad. Sci. USA 96:8443–8448.
23. Brown, G. W., and T. J. Kelly. 1998. Purification of Hsk1, a minichromosome
maintenance protein kinase from fission yeast. J. Biol. Chem. 273:22083–22090.
24. Caddle, M. S., and M. P. Calos. 1994. Specific initiation at an origin of replication
from Schizosaccharomyces pombe. Mol. Cell. Biol. 14:1796–1805.
25. Caspari, T., and A. M. Carr. 1999. DNA structure checkpoint pathways in
Schizosaccharomyces pombe. Biochimie 81:173–181.
26. Cha, R. S., B. M. Weiner, S. Keeney, J. Dekker, and N. Kleckner. 2000. Progression
of meiotic DNA replication is modulated by interchromosomal interaction proteins,
negatively by Spo11p and positively by Rec8p. Genes Dev. 14:493–503.
27. Chuang, R.-Y., and T. J. Kelly. 1999. The fission yeast homologue of Orc4p binds to
replication origin DNA via multiple AT-hooks. Proc. Natl. Acad. Sci. USA 96:2656–
2661.
28. Chuang, R. Y., L. Chretien, J. Dai, and T. J. Kelly. 2002. Purification and
characterization of the Schizosaccharomyces pombe origin recognition complex:
Interaction with origin DNA and Cdc18 protein. J. Biol. Chem. 277:16920–16927, Feb
15 [epub ahead of print].
29. Clyne, R. K., and T. J. Kelly. 1995. Genetic analysis of an ARS element from the
fission yeast Schizosaccharomyces pombe. EMBO J. 14:6348–6357.
30. Collins, I., and C. S. Newlon. 1994. Chromosomal DNA replication initiates at the
same origins in meiosis and mitosis. Mol. Cell. Biol. 14:3524–3534.
31. Correa-Bordes, J., and P. Nurse. 1995. p25rum1 orders S phase and mitosis by acting
as an inhibitor of the p23cdc2 mitotic kinase. Cell 83:1001–1009.
32. Costanzo, V., K. Robertson, M. Bibikova, E. Kim, D. Grieco, M. Gottesman,
D. Carroll, and J. Gautier. 2001. Mre11 protein complex prevents double-strand break
accumulation during chromosomal DNA replication. Mol. Cell 8:137–147.
33. Cox, M. M., M. F. Goodman, K. N. Kreuzer, D. J. Sheratt, S. J. Sandler, and K. J.
Marians. 2000. The importance of repairing stalled replication forks. Nature 202:37–41.
34. Coxon, A., K. Maundrell, and S. E. Kearsey. 1992. Fission yeast cdc21+ belongs to a
family of proteins involved in an early step of chromosome replication. Nucl. Acids Res.
20:5571–5577.
35. D’Amours, D., and S. P. Jackson. 2002. The mre11 complex: at the crossroads of
DNA repair and checkpoint signalling. Nat. Rev. Mol. Cell. Biol. 3:317–327.
36. Datta, A., J. L. Schmeits, N. S. Amin, P. J. Lau, K. Myung, and R. D. Kolodner.
2000. Checkpoint-dependent activation of mutagenic repair in Saccharomyces
cerevisiae pol3-01 mutants. Mol. Cell 6:593–603.
37. Donaldson, A. D., W. L. Fangman, and B. J. Brewer. 1998. Cdc7 is required
throughout the yeast S phase to activate replication origins. Genes Dev. 12:491–501.
38. Dotan, Z. A., A. Dotan, T. Litmanovitch, Y. Ravia, N. Oniashvili, I. Leibovitch,

J. Ramon, and L. Avivi. 2000. Modification in the inherent mode of allelic replication
in lymphocytes of patients suffering from renal cell carcinoma: a novel genetic
alteration associated with malignancy. Genes Chromosomes Cancer 27:270–277.
39. Dubey, D. D., S.-M. Kim, I. T. Todorov, and J. A. Huberman. 1996. Large, complex
modular structure of a fission yeast DNA replication origin. Curr. Biol. 6:467–473.
40. Dubey, D. D., J. Zhu, D. L. Carlson, K. Sharma, and J. A. Huberman. 1994. Three
ARS elements contribute to the ura4 replication origin in the fission yeast
Schizosaccharomyces pombe. EMBO J. 13:3638–3647.
41. Dutta, A., and S. P. Bell. 1997. Initiation of DNA replication in eukaryotic cells. Annu.
Rev. Cell Biol. 13:293–332.
42. Dutta, A., R. Chandra, L. M. Leiter, and S. Lester. 1995. Cyclins as markers of
tumor proliferation: immunocytochemical studies in breast cancer. Proc. Natl. Acad. Sci.
USA 92:5386–5390.
43. Ehrenhofer-Murray, A. E., R. T. Kamakaka, and J. Rine. 1999. A role for the
replication proteins PCNA, RF-C polymerase e and Cdc45 in transcriptional silencing in
Saccharomyces cerevisiae. Genetics 153:1171–1182.
44. Fenech, M., A. M. Carr, J. Murray, F. Z. Watts, and A. R. Lehmann. 1991. Cloning
and characterization of the rad4 gene of Schizosaccharomyces pombe: a gene showing
short regions of sequence similarity to the human XRCC1 gene. Nucl. Acids Res.
19:6737–6741.
45. Fisher, D. L., and P. Nurse. 1996. A single fission yeast mitotic cyclin B p34(Cdc2)
kinase promotes both S-phase and mitosis in the absence of G1 cyclins. EMBO J.
15:850–860.
46. Forsburg, S. L. 1999. The best yeast. Trends Genet. 15:340–344.
47. Forsburg, S. L. 2002. Only connect: linking meiotic DNA replication to chromosome
dynamics. Mol. Cell 9:703–711.
48. Forsburg, S. L., and J. A. Hodson. 2000. Mitotic replication initiation proteins are not
required for pre-meiotic S phase. Nat. Genet. 25:263–268.
49. Forsburg, S. L., D. A. Sherman, S. Ottilie, J. R. Yasuda, and J. A. Hodson. 1997.
Mutational analysis of Cdc19p, a Schizosaccharomyces pombe MCM protein. Genetics
147:1025–1041.
50. Fox, C. A., A. E. Ehrenhofer-Murray, S. Loo, and J. Rine. 1997. The origin
recognition complex, SIR1, and the S phase requirement for silencing. Science
276:1547–1551.
51. Frei, C., and S. M. Gasser. 2000. Hypothesis: RecQ-like helicases: the DNA
replication checkpoint connection. J. Cell Sci. 113:2641–2646.
52. Gomez, E. B., M. G. Catlett, and S. L. Forsburg. 2002. Different phenotypes in vivo
are associated with ATPase motif mutations in Schizosaccharomyces pombe MCM
proteins. Genetics 160.
53. Gomez, M., and F. Antequera. 1999. Organization of DNA replication origins in the
fission yeast genome. EMBO J. 18:5683–5690.
54. Gould, K. L., C. G. Burns, A. Feoktistova, C. P. Hu, S. G. Pasion, and S. L.
Forsburg. 1998. Fission yeast cdc24+ encodes a novel replication factor required for
chromosome integrity. Genetics 149:1221–1233.
55. Grallert, B., and P. Nurse. 1996. The ORC1 homolog orp1 in fission yeast plays a key
role in regulating onset of S phase. Genes Dev. 10:2644–2654.
56. Griffiths, D. J. F., V. F. Liu, P. Nurse, and T. S.-F. Wang. 2001. Role of fission yeast
primase catalytic subunit in the replication checkpoint. Mol. Biol. Cell 12:115–128.
57. Guacci, V., D. Koshland, and A. Strunnikov. 1997. A direct link between sister
chromatid cohesion and chromosome condensation revealed through the analysis of
MCD1 in S. cerevisiae. Cell 91:47–57.
58. Hartsuiker, E., E. Vaessen, A. M. Carr, and J. Kohli. 2001. Fission yeast Rad50
stimulates sister chromatid recombination and links cohesion with repair. EMBO J.
20:6660–6671.
59. Hartwell, L. H., and M. B. Kastan. 1994. Cell cycle control and cancer. Science
266:1821–1828.
60. Hartwell, L. H., P. Szankasi, C. J. Roberts, A. W. Murray, and S. H. Friend. 1997.
Integrating genetic approaches into the discovery of anticancer drugs. Science
278:1064–1068.
61. Hayles, J., D. Fisher, A. Woollard, and P. Nurse. 1994. Temporal order of S phase
and mitosis in fission yeast is determined by the state of the p34cdc2 mitotic B cyclin
complex. Cell 78:813–822.
62. Hendrickson, M., M. Madine, S. Dalton, and J. Gautier. 1996. Phosphorylation of
MCM4 by cdc2 protein kinase inhibits the activity of the minichromosome maintenance
complex. Proc. Natl. Acad. Sci. USA 93:12223–12228.
63. Hess, G. F., R. F. Drong, K. L. Weiland, J. L. Slightom, R. A. Sclafani, and R. E.
Hollingsworth. 1998. A human homolog of the yeast CDC7 gene is overexpressed in
some tumors and transformed cell lines. Gene 211:133–140.
64. Hiraiwa, A., M. Fujita, T. Nagasaka, A. Adachi, M. Ohashi, and M. Ishibashi.
1997. Immunolocalization of hCDC47 protein in normal and neoplastic human tissues
and its relation to growth. Int. J. Cancer 74:180–184.
65. Hofmann, J. F. X., and D. Beach. 1994. cdt1+ is an essential target of the Cdc10/Sct1
transcription factor: requirement for DNA replication and inhibition of mitosis. EMBO
J. 13:425–434.
66. Homesley, L., M. Lei, Y. Kawasaki, S. Sawyer, T. Christensen, and B. K. Tye. 2000.
Mcm10 and the MCM2–7 complex interact to initiate DNA synthesis and to release
replication factors from origins. Genes Dev. 14:913–926.
67. Ishimi, Y. 1997. A DNA helicase activity is associated with an MCM4, –6, and –7
protein complex. J. Biol. Chem. 272:24508–24513.
68. Jallepalli, P. V., G. W. Brown, M. Muzi-Falconi, D. Tien, and T. J. Kelly. 1997.
Regulation of the replication initiator protein p65cdc18 by CDK phosphorylation. Gen.
Dev. 11:2767–2779.
69. Jallepalli, P. V., D. Tien, and T. J. Kelly. 1998. sud1+ targets cyclin-dependent
kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted
diploidization in fission yeast. Proc. Natl. Acad. Sci. USA 95:8159–8164.
70. Jares, P., A. Donaldson, and J. J. Blow. 2000. The Cdc7/Dbf4 protein kinase: target of
the S phase checkpoint? EMBO Rep. 1:319–322.
71. Johnson, R. E., C. M. Kondratick, S. Prakash, and L. Prakash. 1999. hRAD30
mutations in the variant form of xeroderma pigmentosum. Science 285:263–265.
72. Johnston, L., D. Barker, and P. Nurse. 1986. Molecular cloning of the
Schizosaccharomyces pombe DNA ligase gene cdc17 and an associated sequence
promoting high frequency plasmid transformation. Gene 41:321–325.
73. Johnston, L., H. Masai, and A. Sugino. 1999. First the CDKs, now the DDKs. Trends
Cell Biol. 9:249–252.
74. Kamimura, Y., Y.-S. Tak, A. Sugino, and H. Araki. 2001. Sld3, which interacts with
Cdc45 (Sld4) functions for chromosomal DNA replication in Saccharomyces cerevisiae.
EMBO J. 20:2097–2107.
75. Kang, H.-Y., E. Choi, S.-H. Bae, K.-H. Lee, B.-S. Gim, H.-D. Kim, C. Park, S. A.
MacNeill, and Y.-S. Seo. 2000. Genetic analyses of Schizosaccharomyces pombe
dna2+ reveal that Dna2 plays an essential role in Okazaki fragment metabolism.
Genetics 155:1055–1067.
76. Karran, P. 2001. Mechanisms of tolerance to DNA damaging therapeutic drugs.
Carcinogenesis 22:1931–1937.
77. Kawasaki, Y., S.-I. Hiraga, and A. Sugino. 2000. Interactions between Mcm10p
and other replication factors are required for proper initiation and elongation of
chromosomal DNA replication in Saccharomyces cerevisiae. Genes to Cells 5:975–989.
78. Kearsey, S. E., and K. Labib. 1998. MCM proteins: evolution, properties, and role in
DNA replication. Bba-Gene Struct. Express 1398:113–136.
79. Kearsey, S. E., S. Montgomery, K. Labib, and K. Linder. 2000. Chromatin binding
of the fission yeast replication factor Mcm4 occurs during anaphase and requires ORC
and Cdc18. EMBO J. 19:1681–1690.
80. Kelly, T. J., and G. W. Brown. 2000. Regulation of chromosome replication. Annu.
Rev. Biochem. 69:829–880.
81. Kelly, T. J., G. S. Martin, S. L. Forsburg, R. J. Stephen, A. Russo, and P. Nurse.
1993. The fission yeast cdc18+ gene product couples S phase to START and mitosis.
Cell 74:371–382.
82. Kihara, M., W. Nakai, S. Asano, A. Suzuki, K. Kitada, Y. Kawaski, L. H. Johnston,
and A. Sugino. 2000. Characterization of the yeast Cdc7p/Dbf4p complex purified from
insect cells: its protein kinase activity is regulated by Rad53p. J. Biol. Chem.
275:35051–35062.
83. Kim, M. S., J. H. Baek, M. K. Bae, and K. W. Kim. 2001. Human rad21 gene,
hHR21(SP), is downregulated by hypoxia in human tumor cells. Biochem. Biophys.
Res. Commun. 281:1106–1112.
84. Kim, S. M., and J. A. Huberman. 1999. Influence of a replication enhancer on the
hierarchy of origin efficiencies within a cluster of DNA replication origins. J. Mol. Biol.
288:867–882.
85. Kim, S. M., and J. A. Huberman. 1998. Multiple orientation-dependent,
synergistically interacting, similar domains in the ribosomal DNA replication origin of
the fission yeast, Schizosaccharomyces pombe. Mol. Cell Biol. 18:7294–7303.
86. Kim, S. M., and J. A. Huberman. 2001. Regulation of replication timing in fission
yeast. EMBO J. 20:6115–6126.
87. Kim, S. T., B. Xu, and M. B. Kastan. 2002. Involvement of the cohesin protein, Smc1,
in Atm-dependent and independent responses to DNA damage. Genes Dev. 16:560–570.
88. Kirchmaier, A. L., and J. Rine. 2001. DNA replication-independent silencing in
S. cerevisiae. Science 291:646–649.
89. Kirschmann, D. A., R. A. Lininger, L. M. Gardner, E. A. Seftor, V. A. Odero,
A. M. Ainsztein, W. C. Earnshaw, L. L. Wallrath, and M. J. Hendrix. 2000. Down-
regulation of HP1Hs alpha expression is associated with the metastatic phenotype in
breast cance. Cancer Res. 60:3359–3363.
90. Kong, D., and M. L. DePamphilis. 2001. Site-specific DNA binding of the
Schizosaccharomyces pombe origin recognition complex is determined by the Orc4
subunit. Mol. Cell. Biol. 21:8095–8103.
91. Koonin, E. V. 1993. A common set of conserved motifs in a vast variety of putative
nucleic acid-dependent ATPases including MCM proteins involved in the initiation of
eukaryotic DNA replication. Nucl. Acids Res. 21:2541–2547.
92. Kraus, E., W.-Y. Leung, and J. E. Haber. 2001. Break-induced replication: a review
and an example in budding yeast. Proc. Natl. Acad. Sci. USA 98:8255–8262.
93. Kunkel, T. A., and K. Bebenek. 2000. DNA replication fidelity. Annu. Rev. Biochem.
69:497–529.
94. Labib, K., J. A. Tercero, and J. F. X. Diffley. 2000. Uninterrupted MCM2–7 function
required for DNA replication fork progression. Science 288:1643–164
95. Leatherwood, J., A. Lopezgirona, and P. Russell. 1996. Interaction of cdc2 and cdc18
with a fission yeast Orc2-like protein. Nature 379:360–363.
96. Lee, J.-K., and J. Hurwitz. 2000. Isolation and characterization of various complexes
of the minichromosome maintenance proteins of Schizosaccharomyces pombe. J. Biol.
Chem. 275:18871–18878.
97. Lee, M. G., and P. Nurse. 1987. Complementation used to clone a human homologue
of the fission yeast cell cycle control gene cdc2. Nature 327:31–35.
98. Lei, M., and B. Tye. 2001. Initiating DNA synthesis: from recruiting to activating the
MCM complex. J. Cell Sci. 114:1447–1454.
99. Li, Y.-C., T.-H. Cheng, and M. R. Gartenberg. 2001. Establishment of transcriptional
silencing in the absence of DNA replication. Science 291:650–653.
100. Liang, D. T., and S. L. Forsburg. 2001. Characterization of Schizosaccharomyces
pombe mcm7(+) and cdc23(+) (MCM10) and interactions with replication checkpoints.
Genetics 159:471–86.
101. Liang, D. T., and S. L. Forsburg. 2001. A novel allele of S. pombe mcm7+ requires
replication checkpoints for viability. Genetics 159:471–486.
102. Liang, D. T., J. A. Hodson, and S. L. Forsburg. 1999. Reduced dosage of a single
fission yeast MCM protein causes genetic instability and S phase delay. J. Cell Sci.
112:559–567.
103. Lindner, K., J. Gregan, S. Montgomery, and S. E. Kearsey. 2002. Essential role of
MCM proteins in premeiotic DNA replication. Mol. Biol. Cell 13:435–444.
104. Lindsay, H. D., D. J. F. Griffiths, R. J. Edwards, P. U. Christensen, J. M. Murray,
F. Osman, N. Walworth, and A. M. Carr. 1998. S-phase-specific activation of Cds1
kinase defines a subpathway of the checkpoint response in Schizosaccharomyes pombe.
Genes Dev. 12:382–395.
105. Liu, V. F., D. Bhaumik, and T. S. F. Wang. 1999. Mutator phenotype induced by
aberrant replication. Mol. Cell Biol. 19:1126–1135.
106. Lopes, M., C. Cotta-Ramusino, A. Pellicioli, G. Liberi, P. Plevani, M. Muzi-Falconi,
C. S. Newlon, and M. Foiani. 2001. The DNA replication checkpoint response
stabilizes stalled replication forks. Nature 412:557–561.
107. Lygerou, Z., and P. Nurse. 1999. The fission yeast origin recognition complex is
constitutively associated with chromatin and is differently modified through the cell
cycle. J. Cell Sci. 112:3703–3712.
108. Maiorano, D., G. Blom van Assendelft, and S. E. Kearsey. 1996. Fission yeast cdc21,
a member of the MCM protein family, is required for onset of S phase and located in the
nucleus throughout the cell cycle. EMBO J. 15:861–872.
109. Maiorano, D., and M. Mechali. 2002. Many roads lead to the origin. Nat. Cell Biol.
4:E58–E59.
110. Masai, H., and K. Arai. 2002. Cdc7 kinase complex: a key regulator in the initiation of
DNA replication. J. Cell Physiol. 190:287–296.
111. Masai, H., T. Miyake, and K.-I. Arai. 1995. hsk1+, a Schizosaccharomyces pombe
gene related to Saccharomyces cerevisiae CDC7, is required for chromosomal
replication. EMBO J. 14:3094–3104.
112. Masumoto, H., A. Sugino, and H. Araki. 2000. Dpb11 controls the association
between DNA polymerases a and e and the autonomously replicating sequence region of
budding yeast. Mol. Cell. Biol. 20:2809–2817.
113. Masutani, C., R. Kusumoto, A. Yamada, N. Dohmae, M. Yokoi, M. Yuasa,

M. Araki, S. Iwai, K. Takio, and F. Hanaoka. 1999. The XPV (xeroderma
pigmentosum variant) gene encodes human DNA polymerase eta. Nature 399:700–704.
114. Maundrell, K., A. Hutchison, and S. Shall. 1988. Sequence analysis of ARS elements
in fission yeast. EMBO J. 7:2203–2209.
115. Mendez, J., and B. Stillman. 2000. Chromatin association of human origin recognition
complex, cdc6, and minichromosome maintenance proteins during the cell cycle:
assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20:8602–8612.
116. Merchant, A. M., Y. Kawasaki, Y. R. Chen, M. Lei, and B.-K. Tye. 1997. A lesion in
the DNA replication initiation factor Mcm10 induces pausing of elongation forks
through chromosomal replication origins in Saccharomyces cerevisiae. Mol. Cell. Biol.
17:3261–3271.
117. Merrill, B. J., and C. Holm. 1999. A requirement for recombinational repair in
Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants.
Genetics 153:595–605.
118. Michaelis, C., R. Ciosk, and K. Nasmyth. 1997. Cohesins: chromosomal proteins that
prevent premature separation of sister chromatids. Cell 91:35–45.
119. Michel, B. 2000. Replication fork arrest and DNA recombination. Trends Biochem. Sci.
25:173–178.
120. Michel, B., M.-J. Flores, E. Viguera, G. Grompone, M. Seigneur, and V. Bidnenko.
2001. Rescue of arrested replication forks by homologous recombination. Proc. Natl.
Acad. Sci. USA 98:8181–8188.
121. Miller, A. M., and K. A. Nasmyth. 1984. Role of DNA replication in the repression of
silent mating type loci in yeast. Nature 312:247–251.
122. Mincheva, A., I. Todorov, D. Werner, T. M. Finke, and P. Lichter. 1994. The human
gene for nuclear protein BM28 (CDCL1), a new member of the early S phase family of
proteins, maps to chromosome band 3q21. Cytogenet. Cell Genet. 65:276–277.
123. Miyake, S., N. Okishio, I. Samejima, Y. Hiraoka, T. Toda, I. Saitoh, and
M. Yanagida. 1993. Fission yeast genes nda1+ and nda4+, mutations of which lead to
S-phase block, chromatin alteration and Ca2+ suppression, are members of the
CDC46/MCM2 family. Mol. Biol. Cell 4:1003–1015.
124. Miyake, S., and S. Yamashita. 1998. Identification of sna41+ gene, which is the
suppressor of nda4 mutation and is involved in DNA replication in Schizosaccharomyces
pombe [In Process Citation]. Genes Cells 3:157–166.
125. Mondesert, O., C. H. McGowan, and P. Russell. 1996. Cig2, a B-type cyclin,
promotes the onset of S in Schizosaccharomyces pombe. Mol. Cell. Biol. 16:1527–1533.
126. Moreno, S., and P. Nurse. 1994. Regulation of progression through the G1 phase of the
cell cycle by the rum1+ gene. Nature 367:236–242.
127. Moser, B. A., and P. Russell. 2000. Cell cycle regulation in Schizosaccharomyces
pombe. Curr. Opin. Microbiol. 3:631–636.
128. Murakami, H., and P. Nurse. 2001. Regulation of premeiotic S phase and
recombination-related double-strand DNA breaks during meiosis in fission yeast. Nat.
Genet. 28:290–300.
129. Murakami, H., and H. Okayama. 1995. A kinase from fission yeast responsible for
blocking mitosis in S phase. Nature 374:817–819.
130. Muris, D. F. R., K. Vreeken, A. M. Carr, J. M. Murray, C. Smit, P. H. M. Lohman,
and A. Pastink. 1996. Isolation of the Schizosaccharomyces pombe rad54 homologue,
rhp54+, a gene involved in the repair of radiation damage and replication fidelity. J. Cell
Sci. 109:73–81.
131. Murray, J. M., H. D. Lindsay, C. A. Munday, and A. M. Carr. 1997. Role of

Schizosaccharomyces pombe RecQ homolog, recombination, and checkpoint genes in
UV damage tolerance. Mol. Cell. Biol. 17:6868–6875.
132. Murray, J. M., M. Tavassoli, R. Al-Harithy, K. S. Sheldrick, A. R. Lehman, A. M.
Carr, and F. Z. Watts. 1994. Structural and functional conservation of the human
homologue of the Schizosaccharomyces pombe rad2 gene, which is required for
chromosome segregation and recovery from DNA damage. Mol. Cell. Biol. 14:4878–
4888.
133. Myung, K., C. Chen, and R. D. Kolodner. 2001. Multiple pathways cooperate in the
suppression of genome instability in Saccharomyces cerevisiae. Nature 411:1073–1076.
134. Myung, K., A. Datta, and R. D. Kolodner. 2001. Suppression of spontaneous
chromosomal rearrangements by S phase checkpoint functions in Saccharomyces
cerevisiae. Cell 104.
135. Myung, K., and R. D. Kolodner. 2002. Inaugural article: suppression of genome
instability by redundant S-phase checkpoint pathways in Saccharomyces cerevisiae.
Proc. Natl. Acad. Sci. USA 99:4500–4507.
136. Nakajima, R., and H. Masukata. 2002. SpSld3 is required for loading and
maintenance of SpCdc45 on chromatin in DNA replication in fission yeast. Mol. Biol.
Cell 13:1462–1472.
137. Nakamura, T. M., J. P. Cooper, and T. R. Cech. 1998. Two modes of survival of
fission yeast without telomerase. Science 282:493–496.
138. Nakayama, J., R. C. Allshire, A. J. Klar, and S. I. Grewal. 2001. A role for DNA
polymerase alpha in epigenetic control of transcriptional silencing in fission yeast.
EMBO J. 20:2857–2866.
139. Nasmyth, K. 2001. Disseminating the genome: joining, resolving, and separating sister
chromatids during mitosis and meiosis. Annu. Rev. Genet. 35:673–745.
140. Nguyen, V. Q., C. Co, and J. J. Li. 2001. Cyclin-dependent kinases prevent DNA re-
replication through multiple mechanisms. Nature 411:1068–1072.
141. Nishitani, H., Z. Lygerou, T. Nishimoto, and P. Nurse. 2000. The Cdt1 protein is
required to license DNA for replication in fission yeast. Nature 404:625–628.
142. Nishitani, H., and P. Nurse. 1995. p65cdc18 plays a major role controlling the
initiation of DNA replication in fission yeast. Cell 83:397–405.
143. Noguchi, E., P. Shanahan, C. Noguchi, and P. Russell. 2002. CDK phosphorylation
of Drc1 regulates DNA replication in fission yeast. Curr. Biol. 12:599–605.
144. Nonaka, N., T. Kitajima, S. Yokobayashi, G. Xiao, M. Yamamoto, S. I. S. Grewal,
and Y. Watanabe. 2002. Recruitment of cohesin to heterochromatic regions by
Swi6/HP1 in fission yeast. Nat. Cell Biol. 4:89–93. Published online 26 November 2001
(DOI:10.1038/ncb739).
145. Nougarede, R., D. Seta, P. Zarzov, and E. Schwob. 2000. Hierarchy of S-phase
promoting factors: yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin dependent
kinase activation. Mol. Cell. Biol. 20:3795–3806.
146. Ogawa, Y., T. Takahashi, and H. Masukata. 1999. Association of fission yeast Orp1
and Mcm6 proteins with chromosomal replication origins. Mol. Cell. Biol. 19:7228–
7236.
147. Okuno, Y., T. Okazaki, and H. Masukata. 1997. Identification of a predominant
replication origin in fission yeast. Nucl. Acid Res. 25:530–536.
148. Pasion, S. G., and S. L. Forsburg. 2001. Deconstructing a conserved protein family:
the role of MCM proteins in eukaryotic DNA replication. In J. K. Setlow (ed.), Genetic
Engineering, Principles and Methods, vol. 23. Kluwer Academic/Plenum Press,
Dordrecht.
149. Pasion, S. G., and S. L. Forsburg. 1999. Nuclear localization of fission yeast
Mcm2/Cdc19p requires MCM complex formation. Mol. Biol. Cell 10:4043–4057.
150. Pellicioli, A., C. Lucca, G. Liberi, F. Marini, M. Lopes, P. Plevani, A. Romano,
P. Di Fiore, and M. Foiani. 1999. Activation of Rad53 kinase in response to DNA
damage and its effect in modulating phosphorylation of the lagging strand DNA
polymerase. EMBO J. 18:6561–6572.
151. Perego, P., G. S. Jimenez, L. Gatti, S. B. Howell, and F. Zunino. 2000. Yeast mutants
as a model system for identification of determinants of chemosensitivity. Pharmacol.
Rev. 52:477–492.
152. Pereverzeva, I., E. Whitmire, B. Khan, and M. Coue. 2000. Distinct phosphoisoforms
of the Xenopus Mcm4 protein regulate the function of the Mcm complex. Mol. Cell.
Biol. 20:3667–3676.
153. Raghuraman, M. K., E. A. Winzeler, D. Collingwood, S. Hunt, L. Wodicka,
A. Conway, D. J. Lockhart, R. W. Davis, B. J. Brewer, and W. L. Fangman. 2001.
Replication dynamics of the yeast genome. Science 294:115–121.
154. Ramnath, N., F. J. Hernandez, D. F. Tan, J. A. Huberman, N. Natarajan, A. F.
Beck, A. Hyland, I. T. Todorov, J. S. Brooks, and G. Bepler. 2001. MCM2 is an
independent predictor of survival in patients with non-small-cell lung cancer. J. Clin.
Oncol. 19:4259–4266.
155. Reynolds, N., E. Warbrick, P. A. Fantes, and S. A. MacNeill. 2000. Essential
interaction between the fission yeast DNA polymerase d subunit Cdc27 and Pcn1
(PCNA) mediated through a C-terminal p21 Cip1-like PCNA binding motif. EMBO J.
19:1108–1118.
156. Saka, Y., P. Fantes, T. Sutani, C. McInerny, J. Creanor, and M. Yanagida. 1994.
Fission yeast cut5 links nuclear chromatin and M phase regulator in the replication
checkpoint control. EMBO J. 13:5319–5329.
157. Saka, Y., and M. Yanagida. 1993. Fission yeast cut5+, required for S-phase onset and M-
phase restraint, is identical to the radiation-damage repair gene rad4+. Cell 74:383–393.
158. Santocanale, C., and J. F. X. Diffley. 1998. A Mec1- and Rad53-dependent checkpoint
controls late-firing origins of DNA replication. Nature 395:615–618.
159. Savitsky, K., A. Bar-Shira, S. Gilad, G. Rotman, Y. Ziv, L. Vanagaite, D. A. Tagle,
S. Smith, T. Uziel, and S. e. a. Sfez. 1995. A single ataxia telanglectasia gene with a
product similar to PI-3 kinase. Science 268:1749–1753.
160. Schwacha, A., and S. P. Bell. 2001. Interactions between two catalytically distinct
MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication.
Mol. Cell 8:1093–1104.
161. Sclafani, R. A. 2000. Cdc-7p-Dbf4p becomes famous in the cell cycle. J. Cell Sci.
113:2111–2117.
162. Sherman, D. A., and S. L. Forsburg. 1998. S. pombe Mcm3p, an essential nuclear
protein, associates tightly with Nda4p (Mcm5p). Nucl. Acids Res. 26:3955–3961.
163. Sherman, D. A., S. G. Pasion, and S. L. Forsburg. 1998. Multiple domains of fission
yeast Cdc19p (MCM2) are required for its association with the core MCM complex.
Mol. Biol. Cell 9:1833–1845.
164. Shimada, M., D. Okuzaki, S. Tanaka, T. Tougan, K. K. Tamai, C. Shimoda, and
H. Nojima. 1999. Replication factor C3 of Schizosaccharomyces pombe, a small subunit
of replication factor C complex, plays a role in both replication and damage checkpoints.
Mol. Biol. Cell 10:3991–4003.
165. Shimizu, N., T. Ochi, and K. Itonaga. 2001. Replication timing of amplified genetic
regions relates to intranuclear localization but not to genetic activity or G/R band. Exp.
Cell. Res. 268:201–210.
166. Shirahige, K., Y. Hori, K. Shiraishi, M. Yamashita, K. Takahashi, C. Obuse,

T. Tsurimoto, and H. Yoshikawa. 1998. Regulation of DNA-replication origins during
cell-cycle progression. Nature 395:618–621.
167. Shohet, J. M., M. J. Hicks, S. E. Plon, S. M. Burlingame, S. Stuart, S. Y. Chen,
M. K. Brenner, and J. G. Nuchtern. 2002. Minichromosome maintenance protein
MCM7 is a direct target of the MYCN transcription factor in neuroblastoma. Cancer
Res. 62:1123–1128.
168. Simon, J. A., P. Szankasi, D. K. Nguyen, C. Ludlow, H. M. Dunstan, C. J. Roberts,
E. L. Jensen, L. H. Hartwell, and S. H. Friend. 2000. Differential toxicities of
anticancer agents among DNA repair and checkpoint mutants of Saccharomyces
cerevisiae. Cancer Res. 60:328–333.
169. Sjögren, C., and K. Nasmyth. 2001. Sister chromatid cohesion is required for
postreplicative double-strand break repair in Saccharomyces cerevisiae. Curr. Biol.
11:991–995.
170. Smith, J. G., M. S. Caddle, G. H. Bulboaca, J. G. Wohlgemuth, M. Baum,
L. Clarke, and M. P. Calos. 1995. Replication of Centromere II of
Schizosaccharomyces pombe. Mol. Cell. Biol. 15:5165–5172.
171. Snaith, H. A., G. Brown, and S. L. Forsburg. 2000. S. pombe Hsk1p is a potential
Cds1p target required for genome integrity. Mol. Cell. Biol. 20:7922–7932.
172. Snaith, H. A., and S. L. Forsburg. 1999. Rereplication in fission yeast requires MCM
proteins and other S phase genes. Genetics 152:839–851.
173. Sonoda, E., M. S. Sasaki, J. M. Buerstedde, O. Bezzubova, A. Shinohara, H. Ogawa,
M. Takata, Y. Yamaguchi-Iwai, and S. Takeda. 1998. Rad51-deficient vertebrate cells
accumulate chromosomal breaks prior to cell death. EMBO J. 17:598–608.
174. Spruck, C. H., K. A. Won, and S. I. Reed. 1999. Deregulated cyclin E induces
chromosome instability. Nature 401:297–300.
175. Stewart, E., C. R. Chapman, F. Al-Khodairy, A. M. Carr, and T. Enoch. 1997.
rqh1+, a fission yeast gene related to the Bloom’s and Werner’s syndrome genes, is
required for reversible S phase arrest. EMBO J. 16:2682–2692.
176. Sun, D., R. Urrabaz, M. Nguyen, J. Marty, S. Stringer, E. Cruz, L. Medina-
Gundrum, and S. Weitman. 2001. Elevated expression of DNA ligase I in human
cancers. Clin. Cancer Res. 7:4143–4148.
177. Sun, Y., R. T. Wyatt, A. Bigley, and T. G. Krontiris. 2001. Expression and replication
timing patterns of wildtype and translocated BCL2 genes. Genomics 73:161–170.
178. Takahashi, K., H. Yamada, and M. Yanagida. 1994. Fission yeast minichromosome
loss mutants mis cause lethal aneuploidy and replication abnormality. Mol. Biol. Cell
5:1145–1158.
179. Takahashi, T., and H. Masukata. 2001. Interaction of fission yeast ORC with essential
adenine/thymine stretches in replication origins. Genes Cells 6:837–849.
180. Takeda, T., K. Ogino, E. Matsui, M. K. Cho, H. Kumagai, T. Miyake, K. Arai, and
H. Masai. 1999. A fission yeast gene, him1(+)/dfp1(+), encoding a regulatory subunit
for Hsk1 kinase, plays essential roles in S-phase initiation as well as in S-phase
checkpoint control and recovery from DNA damage. Mol. Cell. Biol. 19:5535–5547.
181. Takeda, T., K. Ogino, K. Tatebayashi, H. Ikeda, K. Arai, and H. Masai. 2001.
Regulation of initiation of S phase, replication checkpoint signaling, and maintenance of
mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast. Mol.
Biol. Cell 12:1257–1274.
182. Takisawa, H., S. Mimura, and Y. Kubota. 2000. Eukaryotic DNA replication: from
pre-replication complex to initiation complex. Curr. Opin. Cell. Biol. 12:690–696.
183. Tan, D. F., J. A. Huberman, A. Hyland, G. M. Loewen, J. S. Brooks, A. F. Beck,

I. T. Todorov, and G. Bepler. 2001. MCM2 – a promising marker for premalignant
lesions of the lung: a cohort study. BMC Cancer 1:6.
184. Tanaka, H., K. Tanaka, H. Murakami, and H. Okayama. 1999. Fission yeast Cdc24
is a replication factor C- and prolifering cell nuclear anitgen-interacting factor essential
for S-phase completion. Mol. Cell. Biol. 19:1038–1048.
185. Tanaka, K., and P. Russell. 2001. Mrc1 channels the DNA replication arrest signal to
checkpoint kinase Cds1. Nat. Cell Biol. 3:966–972.
186. Tanaka, K., T. Yonekawa, Y. Kawasaki, M. Kai, K. Furuya, M. Iwasaki,
H. Murakami, M. Yanagida, and H. Okayama. 2000. Fission yeast Eso1p is required
for establishing sister chromatid cohesion during S Phase. Mol. Cell. Biol. 20:3459–
3469.
187. Tatebayashi, K., J. Kato, and H. Ikeda. 1998. Isolation of a Schizosaccharomyces
pombe rad21ts mutant that is aberrant in chromosome segregation, microtubule function,
DNA repair and sensitive to hydroxyurea: possible involvement of Rad21 in ubiquitin-
mediated proteolysis. Genetics 148:49–57.
188. Tercero, J. A., and J. F. X. Diffley. 2001. Regulation of DNA replication fork
progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412:553–557.
189. Tercero, J. A., K. Labib, and J. F. Diffley. 2000. DNA synthesis at individual
replication forks requires the essential initiation factor Cdc45p. EMBO J. 19:2082–2093.
190. Todorov, I. T., B. A. Werness, H. Q. Wang, L. N. Buddharaju, P. D. Todorova,
H. K. Slocum, J. S. Brooks, and J. A. Huberman. 1998. HsMCM2/BM28: a novel
proliferation marker for human tumors and normal tissues. Lab. Invest. 78:73–78.
191. Tomkiel, J. E., H. Alansari, N. Tang, J. B. Virgin, X. Yang, P. VandeVord, R. L.
Karvonen, L. Granda, M. J. Kraut, J. F. Ensley, and F. Fernandez-Madrid. 2002.
Autoimmunity to the M(r) 32,000 subunit of replication protein A in breast cancer. Clin.
Cancer Res. 8:752–758.
192. Tomonaga, T., K. Nagao, Y. Kawasaki, K. Furuya, A. Murakami, J. Morishita,
T. Yuasa, T. Sutani, S. E. Kearsey, F. Uhlmann, K. Nasmyth, and M. Yanagida.
2000. Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21
phophorylated in the S phase. Genes Dev. 14:2757–2770.
193. Tye, B. K. 1999. MCM Proteins in DNA Replication. Annu. Rev. Biochem. 68:649–686.
194. Uchiyama, M., K. Arai, and H. Masai. 2001. Sna41goa1, a novel mutation causing
G1/S arrest in fission yeast, is defective in a CDC45 homolog and interacts genetically
with pol alpha. Mol. Genet. Genomics 265:1039–1049.
195. Uchiyama, M., D. Griffiths, K. Arai, and H. Masai. 2001. Essential role of
Sna41/Cdc45 in loading of DNA polymerase alpha onto minichromosome maintenance
proteins in fission yeast. J. Biol. Chem. 276:26189–26196.
196. Van Brabant, A. J., R. Stan, and N. A. Ellis. 2000. DNA helicases, genomic
instability, and human genetic disease. Annu. Rev. Genomics Hum. Genet. 1:409–459.
197. Vas, A., W. Mok, and J. Leatherwood. 2001. Control of DNA rereplication via Cdc2
phosphorylation sites in the origin recognition complex. Mol. Cell. Biol. 21:5767–5777.
198. Waga, S., and B. Stillman. 1998. The DNA replication fork in eukaryotic cells. Annu.
Rev. Biochem. 67:721–751.
199. Walter, J., and J. Newport. 2000. Initiation of eukaryotic DNA replication: origin
unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase
alpha. Mol. Cell 5:617–627.
200. Wang, Y., T. A. Beerman, and D. Kowalski. 2001. Antitumor drug adozelesin
differentially affects active and silent origins of DNA replication in yeast checkpoint
kinase mutants. Cancer Res. 61:3787–3794.
201. Weinreich, M., and B. Stillman. 1999. Cdc7p-Dbf4p kinase binds to chromatin during
S phase and is regulated by both the APC and the RAD53 checkpoint pathway. EMBO
J. 18:5334–5346.
202. Williams, G. H., P. Romanowski, L. Morris, M. Madine, A. D. Mills, K. Stoeber,
J. Marr, R. A. Laskey, and N. Coleman. 1998. Improved cervical smear assessment
using anitbodies against proteins that regulate DNA replication. Proc. Natl. Acad. Sci.
USA 95:14932–14937.
203. Wohlgemuth, J. G., G. H. Bulboaca, M. Moghadam, M. S. Caddle, and M. P.
Calos. 1994. Physical mapping of origins of replication in the fission yeast
Schizosaccharomyces pombe. Mol. Biol. Cell 5:839–849.
204. Wohlschlegel, J. A., S. K. Dhar, T. A. Prokhorova, A. Dutta, and J. C. Walter.
2002. Xenopus Mcm10 binds to origins of DNA replication after Mcm2–7 and
stimulates origin binding of Cdc45. Mol. Cell 9:233–240.
205. Wood, V., R. Gwilliam, M. Rajandream, M. Lyne, R. Lyne, A. Stewart, J. Sgouros,
N. Peat, J. Hayles, S. Baker, D. Basham, S. Bowman, K. Brooks, D. Brown,
S. Brown, T. Fraser, S. Gentles, A. Goble, N. Hamlin, D. Harris, J. Hidalgo,
G. Hodgson, S. Holroyd, S. Holroyd, T. Hornsby, S. Howarth, E. J. Huckle,
S. Hunt, K. Jagels, K. James, L. Jones, M. Jones, S. Leather, S. Mcdonald,
J. McLean, S. Moule, K. Mungail, L. Murphy, D. Niblett, C. Odell, K. Oliver,
S. O’Neil, D. Pearson, M. A. Quail, E. Rabbinowitsch, K. Rutherford, S. Rutter,
D. Saunders, K. Seeger, S. Sharp, J. skelton, M. Simmonds, R. Squares, S. Squares,
K. Stevens, K. Taylor, R. G. Taylor, S. Walsh, T. Warren, S. Whitehead,
J. Woodward, G. Volckaerv, R. Aert, J. Robben, B. Grymonprez, I. Weltjens,
E. Vanstreels, M. Rieger, M. Schofer, S. Muller-Auer, C. Gabel, M. Fuchs,
C. Fritze, E. Holzer, D. Moestl, H. Hilbert, K. Borzym, I. Langer, A. Beck,
H. Lehrach, R. Reinhardt, T. M. Pohl, P. Eger, W. Zimmermann, H. Wedler,
R. Wambutt, B. Purnelle, A. Goffeau, E. Cadieu, S. Dréano, S. Gloux, V. Lelaure,
S. Mottier, F. Galibert, S. J. Aves, Z. Xiang, C. Hunt, K. Moore, S. M. Hurst, and
M. Lucas, et al. 2002. The genome sequence of Schizosaccharomyces pombe. Nature
415:871–880.
206. Wyrick, J. J., J. G. Aparicio, T. Chen, J. D. Barnett, E. G. Jennings, R. A. Young,
S. P. Bell, and O. M. Aparicio. 2001. Genome-wide distribution of ORC and MCM
proteins in S. cerevisiae: high-resolution mapping of replication origins. Science
294:2357–2360.
207. Yazdi, P. T., Y. Wang, S. Zhao, N. Patel, E. Y. Lee, and J. Qin. 2002. SMC1 is a
downstream effector in the ATM/NBS1 branch of the human S-phase checkpoint. Genes
Dev. 16:571–582.
208. Zannis-Hadjopoulos, M., and G. B. Price. 1999. Eukaryotic DNA Replication. J. Cell.
Biochem. Suppl. 32/33:1–14.
209. Zhang, Z., K. Shibahara, and B. Stillman. 2000. PCNA connects DNA replication to
epigenetic inheritance in yeast. Nature 408:221–225.
210. Zhu, J., D. L. Carlson, D. D. Dubey, K. Sharma, and J. A. Huberman. 1994.
Comparison of two major ARS elements of the ura4 replication origin region with other
ARS elements in the fission yeast Schizosaccharomyces pombe. Chromosoma 103:414–
422.
211. Zou, H., T. J. McGarry, T. Bernal, and M. W. Kirschner. 1999. Identification of a
vertebrate sister-chromatid separation inhibitor involved in transformation and
tumorigenesis. Science 285:418–422.
ADDENDUM
Since this chapter was written, several important discoveries have advanced
our understanding of how DNA replication contributes to genome stability.
First, progress has been made toward uncovering the functions of proteins
associated with replication origins. Unexpectedly, an additional, conserved
protein complex essential for DNA replication has been identified. The
GINS complex consists of four subunits (Sld5/Cdc105, Psf1/Cdc101,
Psf2/Cdc102, and Psf3) that associate with replication origins and are
required for replication initiation and elongation [10, 12, 21]. In S. pombe,
the Psf2 subunit was identified in a screen for mutants that prevent
rereplication; similar to its homologs in other organisms, S. pombe Psf2 is
required for normal S-phase progression [4]. Other recent reports have
uncovered details about the requirements for replication protein association
with origins and activity on their targets. The S. pombe Cdc23 protein has
been shown to be required for Hsk1-dependent phosphorylation of the MCM
complex [13], as well as for the recruitment of Cdc45 to chromatin [6].
Cdc45 appears to be one of the last proteins to associate with the prerepli-
cative complex, as its localization requires formation of the prereplicative
complex, as well as Hsk1 and Rad4 [3, 6]. Cdc45 has been suggested to
travel with the replication fork in S. cerevisiae [1, 22] and is required for the
in vitro helicase activity of the heterohexameric MCM complex [14].
A second area of research progress has been in understanding how cells
respond to S-phase damage and replication blocks. S. pombe Cds1, like its
homolog Rad53 in S. cerevisiae, is required to prevent replication fork
collapse in the presence of HU [15]. Importantly, two proteins that mediate
Cds1 function, Mrc1 and Swi1/Tof1, have been shown to associate with the
replication fork even in the absence of DNA damage, suggesting a
mechanism to couple detection of damage by the moving fork [11, 15, 17].
In mammalian cells, HU-induced checkpoint activation leads to the
association of MCM7 with the Rad17 and ATRIP/ATR checkpoint proteins
[23] and phosphorylation of MCM2 and MCM3 [2, 9, 24]. Interestingly, the
mammalian checkpoint kinases appear to affect not only fork stabilization
during damage, but also the timing of origin firing during normal S-phase
progression [19]. Together, these findings strongly suggest a central,
conserved link between S-phase checkpoints, MCM proteins, and replication
fork stability. Interestingly, fission yeast checkpoint mutants predicted to
cause replication fork collapse do not cause cell inviability in meiosis [18].
Meiotic cells may tolerate a higher level of DNA damage because pro-
grammed DNA damage is generated during meiotic recombination.
Lastly, our understanding of the links between replication and human
disease has been strengthened. Seckel syndrome, a human disease that
predisposes to chromosome instability and shares phenotypes with DNA
repair disorders [16] is associated with mutation of the checkpoint protein

kinase ATR (Rad3 in fission yeast). Genes involved in the Fanconi anemia
pathway (such as BRCA and Rad51) are required for the response to
replication blocks [8]. Interestingly, Rad51, which is implicated in leukemia
[20] has been shown to influence the rate of replication fork progression
during S-phase DNA damage [7]. Additionally, there is an ever-broadening
literature on the use of MCMs and other conserved replication proteins as
markers of hyperproliferating cells, and as measures of cancer prognosis
(rev. in [5]). Many of these recent discoveries have been carried out in
human cells, but clearly build on basic research in yeast that continues to
identify new genes and regulatory interactions. Thus, studying DNA
replication in simple model systems continues to provide a crucial foun-
dation for understanding pathways of cancer initiation and progression.
REFERENCES
1. Aparicio, O. M., D. M. Weinstein, and S. P. Bell. 1997. Components and dynamics of
DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and
Cdc45p during S phase. Cell 91:59–69.
2. Cortez, D., G. Glick, and S. J. Elledge. 2004. Minichromosome maintenance proteins
are direct targets of the ATM and ATR checkpoint kinases. Proc. Nat. Acad. Sci. USA
101:10078–10083. Epub 2004 Jun 21.
3. Dolan, W. P., D. A. Sherman, and S. L. Forsburg. 2004. Schizosaccharomyces pombe
replication protein Cdc45/Sna41 requires Hsk1/Cdc7 and Rad4/Cut5 for chromatin
binding. Chromosoma 113:145–156. Epub 2004 Aug 03.
4. Gómez, E. B., V. T. Angeles, and S. L. Forsburg. 2005. A Screen for
Schizosaccharomyces pombe mutants defective in rereplication identifies new alleles of
rad4+, cut9+ and psf2+. Genetics 169:77–89. Epub 2004 Sep 30.
5. Gonzalez, M. A., K. E. Tachibana, R. A. Laskey, and N. Coleman. 2005. Innovation:
control of DNA replication and its potential clinical exploitation. Nat. Rev. Cancer
5:135–141.
6. Gregan, J., K. Lindner, L. Brimage, R. Franklin, M. Namdar, E. A. Hart, S. J.
Aves, and S. E. Kearsey. 2003. Fission yeast Cdc23/Mcm10 functions after pre-
replicative complex formation to promote Cdc45 chromatin binding. Mol. Biol. Cell
14:3876–3887. Epub 2003 Jun 13.
7. Henry-Mowatt, J., D. Jackson, J. Y. Masson, P. A. Johnson, P. M. Clements, F. E.
Benson, L. H. Thompson, S. Takeda, S. C. West, and K. W. Caldecott. 2003.
XRCC3 and Rad51 modulate replication fork progression on damaged vertebrate
chromosomes. Mol. Cell 11:1109–1117.
8. Howlett, N. G., T. Taniguchi, S. G. Durkin, A. D. D’Andrea, and T. W. Glover.
2005. The Fanconi anemia pathway is required for the DNA replication stress response
and for the regulation of common fragile site stability. Hum. Mol. Genet. Epub ahead of
print.
9. Ishimi, Y., Y. Komamura-Kohno, H. J. Kwon, K. Yamada, and M. Nakanishi.
2003. Identification of MCM4 as a target of the DNA replication block checkpoint
system. J. Biol. Chem. 278:24644–24650. Epub 2003 Apr 24.
10. Kanemaki, M., A. Sanchez-Diaz, A. Gambus, and K. Labib. 2003. Functional

proteomic identification of DNA replication proteins by induced proteolysis in vivo.
Nature 423:720–724.
11. Katou, Y., Y. Kanoh, M. Bando, H. Noguchi, H. Tanaka, T. Ashikari, K. Sugimoto,
and K. Shirahige. 2003. S-phase checkpoint proteins Tof1 and Mrc1 form a stable
replication-pausing complex. Nature 424:1078–1083.
12. Kubota, Y., Y. Takase, Y. Komori, Y. Hashimoto, T. Arata, Y. Kamimura,
H. Araki, and H. Takisawa. 2003. A novel ring-like complex of Xenopus proteins
essential for the initiation of DNA replication. Genes Dev. 17:1141–1152.
13. Lee, J. K., Y. S. Seo, and J. Hurwitz. 2003. The Cdc23 (Mcm10) protein is required
for the phosphorylation of minichromosome maintenance complex by the Dfp1-Hsk1
kinase. Proc. Nat. Acad. Sci. USA 100:2334–2339. Epub 2003 Feb 25.
14. Masuda, T., S. Mimura, and H. Takisawa. 2003. CDK- and Cdc45-dependent
priming of the MCM complex on chromatin during S-phase in Xenopus egg extracts:
possible activation of MCM helicase by association with Cdc45. Genes Cells 8:145–
161.
15. Noguchi, E., C. Noguchi, W. H. McDonald, J. R. R. Yates, and P. Russell. 2004.
Swi1 and Swi3 are components of a replication fork protection complex in fission yeast.
Mol. Cell Biol. 24:8342–8355.
16. O’Driscoll, M., V. L. Ruiz–Perez, C. G. Woods, P. A. Jeggo, and J. A. Goodship.
2003. A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related
protein (ATR) results in Seckel syndrome. Nat. Genet. 33:497–501. Epub 2003 Mar 17.
17. Osborn, A. J., and S. J. Elledge. 2003. Mrc1 is a replication fork component whose
phosphorylation in response to DNA replication stress activates Rad53. Genes Dev.
17:1755–1767.
18. Pankratz, D. G., and S. L. Forsburg. 2005. Meiotic S-phase damage activates
recombination without checkpoint arrest. Mol. Biol. Cell 2005 Feb 2; [Epub ahead of
print].
19. Shechter, D., V. Costanzo, and J. Gautier. 2004. ATR and ATM regulate the timing
of DNA replication origin firing. Nat. Cell Biol. 6:648–655. Epub 2004 Jun27.
20. Slupianek, A., C. Schmutte, G. Tombline, M. Nieborowska-Skorska, G. Hoser,
M. O. Nowicki, A. J. Pierce, R. Fishel, and T. Skorski. 2001. BCR/ABL regulates
mammalian RecA homologs, resulting in drug resistance. Mol. Cell 8:795–806.
21. Takayama, Y., Y. Kamimura, M. Okawa, S. Muramatsu, A. Sugino, and H. Araki.
2003. GINS, a novel multiprotein complex required for chromosomal DNA replication
in budding yeast. Genes Dev. 17:1153–1165.
22. Tercero, J. A., K. Labib, and J. F. Diffley. 2000. DNA synthesis at individual
replication forks requires the essential initiation factor Cdc45p. EMBO J. 19:2082–2093.
23. Tsao, C. C., C. Geisen, and R. T. Abraham. 2004. Interaction between human MCM7
and Rad17 proteins is required for replication checkpoint signaling. EMBO J. 23:4660–
4669. Epub 2004 Nov 11.
24. Yoo, H. Y., A. Shevchenko, A. Shevchenko, and W. G. Dunphy. 2004. Mcm2 is a
direct substrate of ATM and ATR during DNA damage and DNA replication checkpoint
responses. J. Biol. Chem. 279: 53353–53364.
Chapter 2
DISSECTING LAYERS OF MITOTIC
REGULATION ESSENTIAL FOR
MAINTAINING GENOMIC STABILITY
Jennifer S. Searle 1 and Yolanda Sanchez 2

1
Department of Molecular Genetics, Biochemistry and Microbiology, The University of
Cincinnati, 231 Ablert Sabin Way, Cincinnati, OH 45267-0524; 2Department of Pharmacology
and Toxicology, Dartmouth Medical School, 7650 Remsen, Hanover, NH 03755, USA
Cell division is the process by which a cell copies its DNA and cellular
constituents and gives rise to two cells. One complete cell division is termed
a cell cycle. (Although you can have a cell cycle without cell division.) This
process is divided into four distinct phases for most eukaryotic cells: G1, S,
G2, and M (Figure 1A). S phase (for synthesis) is the stage in which the
DNA is replicated and M phase, (for mitosis), is the stage in which the
replicated DNA condenses and the chromosomes are divided equally into
two cells. G1 and G2 are “gap phases” that separate DNA replication and
mitosis. This chapter is focused on how yeast have served as a useful model
system that was instrumental in the identification of proteins that promote
cell cycle progression in all eukaryotes. We will discuss the pioneering
work of individuals that used budding yeast to reveal the layers of regulation
that coordinate progression through the cell cycle. In addition, we will
discuss how this model organism that is amenable for genetics has been used
to identify proteins that regulate cell cycle, and in particular mitosis, in order to
safeguard the integrity of the genome. We will end by linking this work to the
specific problem of cancer, and mention the utility of this system in research
designed to establish and validate targets for anticancer drug discovery.
37
© 2007 Springer.
38 J. S. Searle and Y. Sanchez
1 THE MANY LAYERS OF CELL CYCLE

REGULATION
1.1 The cell cycle engine is composed of a cyclin

and a cyclin-dependent kinase (Cdk)
For many years, scientists sought to identify the molecular components

that regulate the duplication and segregation of chromosomes. Several
groups of biochemists working on clam, sea urchin, and frog eggs had been
studying the activity of maturation-promoting factor (MPF), which causes
immature eggs to undergo meiosis and prepares them for fertilization [78,
110]. MPF from many species could drive mitosis when injected into cells
that were at stages other than mitosis. MPF could also induce replication
and condensation cycles of sperm DNA, mimicking the chromosome
division cycle in a cell-free system [76]. MPF activity rose in cells that were
entering meiosis or mitosis and would then drop precipitously after cell
division. A new burst of activity was followed by another chromosomal
division [34]. At the time, MPF was coined as the “cell cycle clock” and
many groups attempted to purify its components but failed. Tim Hunt
reasoned that a protein functioning to promote mitosis should have a pattern
of expression that coincided with the induction of mitosis in a cell-free
extract system. Hunt used sea urchin and Xenopus extracts to identify a
protein whose accumulation levels cycled (cyclin) in a similar pattern as did
the activity of the MPF, and the translation of which was required for
Xenopus extracts to enter mitosis [28, 83].
Meanwhile, the geneticists interested in this problem took a different
approach. They postulated that if proteins had a role in the progression
through the cell cycle by either promoting a transition (G1 to S or G2 to M),
or by acting as a substrate for the next transition to occur, then the genes
encoding these proteins could be identified in a genetic screen. The screen
consisted of randomly mutagenizing yeast and selecting for conditional
mutants (functional at one temperature, and inactive at a higher or restrictive
temperature) that would cause the cells to arrest at particular stages of the
cell cycle when the cells were shifted to the restrictive temperature. Budding
yeast was the optimal organism for geneticists doing cell cycle research
because the cells divide by budding and the size of the bud indicates their
position in the cell cycle. Yeast is also easy to grow and is amenable to
genetic manipulation. Leland Hartwell carried out a genetic screen to
identify conditional mutants in the budding yeast Saccharomyces cerevisiae.
Since many of the components that drive cell division are essential (the cell
Chapter 2: Checkpoints and Genomic Stability 39
cannot live without them), some of the mutants that Hartwell identified had
defects in cell cycle progression (cdc = cell division cycle mutants). Hartwell
identified dozens of mutants that arrested at different stages of the cell cycle
when the cells were raised to the restrictive temperature. The arrest point
was easy to identify as the cells arrested with no bud (G1) or with a uniform
bud size (Figure 1B). One such mutant, cdc28, was defective in the
initiation of DNA replication [43]. The cdc28 mutation caused cells to arrest
without a bud and with unreplicated DNA at the restrictive temperature.
Paul Nurse and Pierre Thuriaux carried out a screen similar to Hartwell’s
using the fission yeast Schizosaccharomyces pombe. They identified a
mutant, cdc2, which arrested prior to mitosis under restrictive conditions
[92]. When the CDC2 gene was cloned, it was found to encode a protein
with homology to kinases. Protein kinases are enzymes that modify other
proteins by adding phosphate groups, a common mechanism used by the cell
to transmit information or regulate protein activity. David Beach, while
working with Nurse, also found that the cdc2 mutation was in a gene
homologous to the budding yeast CDC28 gene identified by Hartwell [5] and
cloned earlier by Nasmyth and Reed [87].
In the late 1980s, investigators in the fields of biochemistry and genetics
came together and initiated an explosion of cell cycle research. At that
point, Manfred Lohka and James Maller succeeded in the purification of
MPF by monitoring an activity that could promote chromosome conden-
sation. Jean Gautier and Maller’s group joined with Nurse, and William
Dunphy and John Newport joined with David Beach and both groups
discovered that MPF is composed of two subunits; a kinase that is the
product of the CDC28 and cdc2+ genes in the yeasts and the mitotic cyclin
[27, 33]. Thus, it was found that the cell cycle engine is universal or
conserved, and that in its bare bones is a complex, composed of a cyclin-
dependent kinase (Cdk) and an activating partner, the cyclin [27, 33, 65, 72].
Interaction of the Cdk and cyclin activates the complex to phosphorylate
substrates, allowing the cell to move into the next cell cycle stage. The
cyclin partner not only activates the Cdk but also provides specificity for
substrates that will drive a particular cell cycle transition. For example, a
complex containing a Cdk and a member of the G1 cyclin family promotes
entry into a new cell division cycle; whereas a Cdk/Mitotic cyclin complex
promotes entry into mitosis. Thus, the availability of the cyclins serves to
regulate the cell cycle stage-specific activity of the Cdks.
The 2001 Nobel Prize for medicine was awarded to three individuals who
used biochemistry and genetics to discover the Cdk complex. Tim Hunt
discovered the mitotic cyclin and, importantly, he found that degradation of
the cyclin coincided with the end of mitosis and, identified a layer of
regulation of the cell cycle engine. Leland Hartwell carried out the famous
genetic screen that led to the identification of the Cdc28/Cdk. Paul Nurse
identified cdc2+ in a similar genetic screen and found that mammals had a
gene that could function in the place of cdc2+. In doing so, he showed that
the proteins that comprise the cell cycle engine are conserved between yeast
and mammals [68]. These individuals, combined forces with other
biochemists studying the activity of the cell cycle engine, which sparked the
intensity of the field that studies how cell division, chromosome duplication,
and segregation are coordinated. [86, 98].
Many years after the discovery of the Cdk complex, Pavletich and co-
workers analyzed the crystal structure of the cyclin/Cdk complex, which
revealed the mechanism by which the cyclin activates the Cdk. It turns out
that the helix containing a conserved amino acid involved in ATP binding is
positioned away from the active site of the Cdk. The cyclin, by virtue of its
binding, realigns the residues involved in ATP binding and keeps domains
from blocking the catalytic cleft of the enzyme. Thus, binding of the cyclin
removes a stearic block that allows the cdk to phosphorylate its substrates
[55]. This is the reason why MPF activity coincides with a peak of cyclin
levels. Phosphorylation of the activation loop is required to stabilize the
active form of the Cdk complexed with the cyclin, and thus adds another
level of regulation to this complex [95].
Figure 1A. The eukaryotic cell cycle and the universal cell cycle engine. The eukaryotic cell
cycle (with the exception of germ cells and early embryonic cells) is divided into four distinct
phases G1, S, G2, and M. S phase (for synthesis) is the stage in which the DNA is replicated
and M phase, mitosis, is the stage in which the replicated DNA condenses and the
chromosomes are divided equally into two daughter cells. G1 and G2 are “gap phases” that
separate DNA replication and mitosis. The cyclin-dependent kinase/cyclin (Cdk/cyclin)
complex which drives cell cycle transitions is regulated by phosphorylation of the Cdk.
Figure 1B. Progression of Saccharomyces cerevisiae cells through the cell cycle can be
monitored by bud and nuclear morphology. Bud emergence coincides with the initiation of
DNA synthesis (DNA replication), G2 cells have large buds and an undivided nucleus at the
neck. Cells in anaphase have an elongated bipolar nucleus (dumbbell shape) and elongated
spindles. Telophase cells have two distinctly divided nuclei in the mother and daugher cells.
Cytokinesis gives rise to two unbudded cells that are in G1. Indicated is the morphology with
which the cdc mutants arrest at each point in the cell cycle.
2 LAYERS OF REGULATION: PART I. HOW

YEAST GENETICS AND BIOCHEMISTRY
REVEALED THE FIRST LAYER
OF REGULATION
2.1 Phosphorylation and proteolysis regulate the activity

of the cell cycle engine
Using genetic and biochemical analyses, researchers in the last 15 years

have deciphered a complex regulatory web, involving phosphorylation,
ubiquitination-mediated proteolysis and control of subcellular localization
that functions to achieve the exquisite order and timing of progression
through the stages of the cell cycle. For the remainder of this chapter, we
will focus on the layers of regulation that ensure that mitosis results in the
transmission of genetic information with high fidelity. Therefore, it is
important to review here the stages of mitosis that result in the segregation of
chromosomes and the generation of two daughter cells.
Following activation of MPF and chromosome condensation, and prior to

separation of the replicated chromosomes, the cells are in the metaphase
stage of mitosis (see Figure 2).
Metaphase. The condensed chromosomes are attached to the spindle and
the sister chromatids remain associated to each other via their centromeres
(kinetochores), and by the presence of cohesin and condensing complexes
(see Figure 2) [39, 46, 62]. In metazoans the chromosomes align them-
selves in the mid-zone in a structure called the metaphase plate.
Anaphase. The cohesion between the sister chromatids is lost and the
sisters separate due to the force exerted by the spindle, which is the physical
structure that separates the chromosomes so that the two daughter cells
receive a complete copy of the genome [62]. This is achieved by the
cleavage of the cohesin complexes by the enzyme separase (Esp1 in yeast)
[16].
Telophase. The separated chromatids move to opposite poles of the cell.
Mitotic exit. The mitotic Cdk/B-type cyclin complexes are inactivated via
many mechanisms (discussed below). This allows the pre-replication
complexes to assemble on the chromatin so that the cell is ready to begin
DNA replication at the next S phase.
Cytokinesis. The physical separation of the cytoplasm that results in the
two daughter cells.
Figure 2. Progression through mitosis. Following activation of MPF and chromosome

condensation and prior to separation of the replicated chromosomes, the cells are in the
metaphase stage of mitosis. In metaphase, the condensed chromosomes are attached to the
spindle, and the sister chromatids remain associated to each other via their centromeres
(kinetochores), and by the presence of cohesin complexes (Scc1, Smc1, and Smc3). Ubiquitin-
mediated destruction of securin (Pds1) leads to release of separase (Esp1), which cleaves
cohesin complexes. The cohesion between the sister chromatids is lost which triggers
anaphase where the sisters separate due to the force exerted by the spindle. The separated
chromatids move to opposite poles of the cell. The mitotic Cdk/B-type cyclin complexes are
inactivated via many mechanisms, which allows the pre-replication complexes to assemble on
the chromatin so that the cell is ready to begin DNA replication at the next S phase.
2.1.1 Regulation of the Cdk/cyclin complex by phosphorylation
Biochemical and genetic experiments identified kinases and phosphatases

(enzymes that remove the phosphates added by the kinases) that regulate
Cdk complexes (see Figure 1). The Cdk/cyclin complex can be regulated by
(1) positive (see above) and negative phosphorylation on the Cdk subunit;
(2) the expression and destruction of the cyclin component; (3) binding of
inhibitors (Cdk inhibitors=CKI’s); and (4) regulating the subcellular
localization of the complex [95]. Experiments carried out in the fission yeast
S. pombe, and the Xenopus extract system provided the first clues as to how
this complex is regulated by phosphorylation. The studies of fission yeast
mutants carried out by Nurse identified the Wee1 family of kinases, that
function to maintain the Cdk/cyclin complex in an inactive state until the cell
is ready to enter the next phase of cell division [91]. Genetic and
biochemical studies also led to the identification of the Cdc25 family of
phosphatases, which catalyze the removal of the inhibitory phosphates added
by the Wee1 kinases to the Cdk subunit [32, 103]. For example, during the
late stages of DNA replication, the mitotic cyclins are expressed and
assemble with their cdk partner (Cdk1); however, this complex is maintained
in an inactive state by inhibitory phosphorylation carried out by the Wee1
kinases. At the end of DNA replication, and before the onset of mitotic
events, the inhibitory phosphates are removed by the Cdc25 family of
phosphatases, thus setting off an amplification of mitotic Cdk/Cyclin
activity. This regulatory network is utilized in the fission yeast and
metazoans to prevent premature onset of mitosis [9, 96, 105, 111, 130] and
in the budding yeast to regulate mitosis until the cell has reached a critical
bud size [45, 79]. In response to perturbations in bud construction, Swe1
(budding yeast Wee1) and Mih1 (budding yeast Cdc25 phosphatase) are
regulated to delay mitotic activation of Cdc28 [118]. Failure to block mitosis
when bud construction or critical size have not been completed results in
binucleate cells [42, 45, 112].
2.1.1.1 Regulation of the cell cycle by ubiquitin-mediated proteolysis

The biochemists Hunt, Kirchner, and Ruderman, among others [4, 28, 60,
85] had identified that the cyclins were regulated by proteolysis, and more
importantly, that degradation of the B-type cyclin was required for cycling
Xenopus extracts to exit mitosis. Expression of a nondegradable cyclin

mutant would lock the extracts in mitosis [85]. Using purification and
fractionation approaches, these investigators identified an activity that would
ligate ubiquitin to the cyclin component and trigger its destruction. A
structure called the proteosome would then recognize the proteins with poly-
ubiquitin chains and process them down to their amino acid components
(degradation). This activity of ligating ubiquitin to the B-type cyclin was
given the name of the anaphase-promoting complex or cyclosome (APC/C)
[116, 4].
Many of the mutants identified by Hartwell exhibited an arrest either
prior to DNA replication (but were able to bud) or exhibited a large-budded
arrest, which indicated that the cells were able to replicate their DNA, but
were unable to complete mitosis. These mutants turned out to be deficient in
components of the multi-protein complexes involved in the ordered
destruction of cell cycle regulators.
There are three critical cell cycle transitions that are regulated by
proteolysis: the G1 to S, metaphase to anaphase, and anaphase to G1
transitions. Several of the cdc mutants that arrested at the G1 to S transition
had high protein levels of the G1 cyclins (Clns) and of the Cdk kinase
inhibitor (CKI) Sic1. Similarly, mutants that arrested in mitosis before
anaphase did so with high levels of the B-type (mitotic) cyclin Clb2 and,
presumably, with high levels of an anaphase inhibitor [54]. Mutants that had
completed anaphase but arrested at telophase did so with high levels of the
B-type cyclins, which mimicked the block to mitotic exit observed with the
nondegradable B-type cyclin in metazoan extracts (Figure 3). Since cyclin
degradation was a requirement for mitotic exit in the cell-free extract system,
scientists predicted that the strains that arrested with high levels of the
mitotic cyclin carried deficiencies in components of multi-protein complexes
involved in ligating ubiquitin to cell cycle regulators in order to trigger their
ordered destruction.
There are two complexes that regulate the cell cycle via ubiquitin-mediated
proteolysis: the Skip1, Cdc53, F box complex (SCF) and the APC/C. We
will focus our attention on the APC, or cyclosome, which is a multi-protein
complex that acts as a ubiquitin ligase and is active in mitosis and G1. Once
again, the geneticists and biochemists joined efforts in order to identify the
enzymatic activity of a large multi-protein complex that is required for
progression through mitosis in all eukaryotes. Stefan Irniger, when in
Nasmyth’s laboratory, showed that two of the cdc mutants that showed a
pre-anaphase arrest phenotype, cdc16 and cdc23, carried mutations in genes
encoding proteins that were required for the proteolysis of the B-type cyclins
[54]. Around the same time, Hieter’s group found that the Cdc16 and Cdc23
proteins formed a complex with Cdc27, another protein also required for
proteolysis of the B-type cyclin [66]. Then Kirschner’s and Hieter’s groups
joined forces to determine that Cdc27 and Cdc16 are indeed components of a
large complex that catalyzes the conjugation of ubiquitin to the B-type
cyclins and are thus, components of the evolutionarily conserved APC/C
[60].
The biochemical experiments with the nondegradable B-type cyclin
mentioned above always led to a block in mitotic exit but not to chromosome
segregation at anaphase indicating that proteolysis of the B-type cyclins was
not required for anaphase to occur. The observation that APC mutants that
arrested in pre-anaphase did so with high levels of the B-type cyclin Clb2,
and that B-type cyclin degradation is not required for anaphase to take place
(biochemical experiments with nondegradable B-type cyclin and [118]),
suggested that the APC/C had different substrates in order to promote
anaphase and mitotic exit. The anaphase substrate of the APC/C, Pds1, was
identified in a genetic screen carried out by Doug Koshland’s group that
identified genes encoding proteins involved in chromosome segregation
[135, 136]. In this screen, the investigators were looking for mutations that
caused the cell to separate sister chromatids prematurely (Pds = precocious
dissociation of the sisters). Orna Cohen-Fix, while in Koshland’s laboratory,
showed that Pds1 was destroyed prior to anaphase and that it was a substrate
of the APC/C [20]. The APC/C components and its substrates are conserved,
thus Pds1 orthologues are now called securin, due to their function in
keeping sister chromatids together until anaphase. The mechanism by which
Pds1 keeps sister chromatids together is by acting as an inhibitor of a
caspase-like protease Esp1 (also known as separase). Esp1 cleaves Scc1, a
protein that is part of the cohesin complex (Scc1, Smc1, Smc3, and Scc3)
that forms during DNA replication to keep the sister chromatids together.
Cleavage of Scc1 by Esp1 leads to loss of cohesion along the arms of the
chromosomes, and the tension provided by the spindle apparatus is then able
to pull the sister chromatids apart at anaphase (see Figure 2). In summary,
the APC promotes progression through mitosis by the ordered ubiquitin-
mediated proteolysis of the anaphase inhibitor, Pds1 (securin), and the
B-type (mitotic) cyclins, Clb2 and Clb5 [66, 97, 123, 126, 127].
The APC relies on proteins that act as specificity factors in order to
ubiquitinate the correct substrates in an ordered and timely manner. These
proteins are the WD repeat (tryptophan and aspartic acid repeats) containing
proteins Cdc20 and Hct1/Cdh1. Thus, cdc20, a mutant identified in the
Hartwell screen that arrests prior to anaphase, is defective in the ubiquitination
and destruction of both Pds1 (securin) and Clb2 (B-type cyclin). Since
destruction of the B-type cyclins is not required for anaphase [118], this
finding suggested that there existed a regulatory step that ensured that the
destruction of securin preceded the destruction of the cyclins in order to

ensure that the sister chromatids separate before the cell exits mitosis. This
finding also explains why cells with defects in the destruction of securins
would arrest with high levels of the B-type cyclins [19, 121].
In summary, entrance into mitosis is regulated at the level of activation of
the Cdk/mitotic cyclin complex (Figures 1 and 3). High activity of the
mitotic Cdk complex triggers mitosis but is inhibitory to the pathway that
promotes exit from mitosis (Figure 3). Exit from mitosis requires the
inactivation of this complex and reversal of Cdk phosphorylation, achieved
by the APC-mediated proteolysis of the mitotic cyclins and release of the
phosphatase Cdc14 from the nucleolus [107, 127, 127].
The mechanism that regulates the interdependence of anaphase and
mitotic exit has recently come to light from work carried out in the
laboratories of Kim Nasmyth, Angelika Amon, Doug Koshland, David
Morgan, Uttam Surana, and Ray Deshaies among others. We will simplify
the intricate regulatory network that regulates mitotic exit (MEN), however,
the details can be found in the following papers and references therein [56,
97, 107, 114, 137]. Following a successful anaphase, the initial degradation
of the B-type cyclins, Clb5 and Clb2 [109], leads to the activation of a
signaling network that results in the release of the phosphatase Cdc14 from
the nucleolus. This involves the activation of Tem1, a GTP-binding protein
of the Ras superfamily, which is a positive regulator of Clb destruction
(Figure 3). Tem1 is negatively regulated by a heteromeric GTPase-activating
protein (GAP) composed of Bfa1 and Bub2. Inactivation of Bfa1 and Bub2
allows activation of Tem1 and triggers the release of Cdc14 from the
nucleolus. Activation of the polo-like kinase Cdc5, also promotes Cdc14
release from the nucleolus. Cdc14 then reaches its targets to remove
phosphates that have been added by the Cdk complex [127]. The activity of
Cdc14 allows full activation of Hct1/Cdh1, the APC specificity factor for
Clb2 ubiquitination, and dephosphorylation of Sic1, which causes this Cdk
inhibitor to accumulate. These two events serve to lower the activity of the
mitotic Cyclin/Cdk complexes even further, allowing mitotic exit and the
establishment of pre-replication complexes [123, 126, 127]. cdc5 and cdc14
were identified in the Hartwell screen as mutations that caused cells to arrest
after anaphase but before cytokinesis (telophase arrest, see Figure 1) and the
proteins encoded by the genes mutated in these strains are positive regulators
of Clb2 inactivation and mitotic exit.
2.2 Why does the cell have so many proteins to regulate

cell division?
Cells must replicate chromosomes with high fidelity and segregate

identical copies to two cells [17]. They must also duplicate their cellular
constituents and maintain a critical size. In order to carry out this feat every
time, cells must overcome two problems:
The alternation problem: DNA must be replicated once per cell cycle.
One example of a control that addresses the alternation problem is the
exquisite regulation of Cdk kinase activity by the APC during mitotic
progression, which ensures that cells complete mitosis prior to the initiation
of another round of DNA replication. When these mechanisms fail, cells go
through more than one replication cycle without an intervening mitosis.
This results in hyperploidy, or too many sets of chromosomes. In some
specialized cell types, such as trophoblasts, DNA replication without mitosis
leads to the formation of tetraploid cells.
The completion problem: Initiating a cell cycle transition is dependent
upon the completion of the previous stage. For example, cells must finish
DNA replication before beginning mitosis. When this dependence fails, cells
will attempt to segregate incompletely replicated chromosomes, which can
lead, in a worst case scenario, to a catastrophic or lethal mitosis.
The first evidence that the cell had evolved mechanisms to address these
two problems came from elegant experiments carried out in the 1970s by
Potu Rao and Robert Johnson. These scientists used cell fusion experiments
to show that there was an activity in S-phase cells that could promote
initiation of DNA replication in a G1 cell in a fashion similar to MPF
promotion of mitosis. That is, when a cell in the replication stage of the cell
cycle (S phase) was fused to a G1 cell the G1 nucleus in the heterokaryon
(the cell resulting from the fusion that had two nuclei) would begin DNA
replication. Today we know that this S-phase-promoting factor is none other
than Cdk complexes with S-phase cyclins. When Rao and Johnson fused a
cell in S phase with a cell that had completed DNA replication but had not
initiated its mitotic program (G2 cell) they made a very important discovery.
The first observation was that the G2 nucleus in the heterokaryon would
delay entry into mitosis until DNA replication was completed in the S-phase
nucleus. The second observation was that the G2 nucleus did not reinitiate
DNA replication. These observations indicated that there was a signal from
the S-phase nucleus that would delay mitosis until replication was completed
(completion problem); and that there was a mechanism in place in the G2
nucleus that prevented the reinitiation of DNA replication until the cell had
gone through mitosis (the alternation problem). Therefore, with these now
historical studies, Rao and Johnson demonstrated that cells had mechanisms
in place that regulated the order and timing of cell cycle transitions. These
mechanisms are referred to as checkpoint controls [98].
The controls identified by Rao and Johnson become essential when the
relative timing of cell cycle transitions is challenged and cannot be merely
regulated by an oscillator, or the “cell cycle clock”; such is the case when
DNA replication is prolonged due to DNA damage or other stress conditions
(Figures 4 and 5). Elegant experiments carried out by Smythe and Newport
with cell free extracts from Xenopus eggs provided biochemical evidence of
this response [111] by showing that incompletely replicated DNA blocks the
activation of the mitotic Cdk/cyclin complex by maintaining the Cdk subunit
in a phosphorylation form. The mechanisms that are activated when the
relative timing of cell cycle transitions is altered are also important when the
chromosomes are not correctly attached or oriented on the spindle or when a
cell has not reached a critical size prior to division. These checkpoint
controls are mediated by signal transduction pathways that ensure the
interdependence of cell cycle events. For example, during DNA replication
a signal is produced (probably at the replication fork, or when forks pause or
stall] that will cause the cell to delay mitosis until all forks have completed
replication. Because of their function, these biochemical pathways are called
checkpoints, a term coined by Hartwell, and they represent the next layer of
regulation of the cell cycle that is covered in this chapter.
Figure 3. Phosphorylation and ubiquitin-mediated proteolysis regulate ordered progression

through mitosis. Entrance into mitosis is regulated by the abundance of the cyclin and by
positive and negative phosphorylation of the Cdk subunit. The activation of the mitotic Cdk
complex triggers mitosis but is inhibitory to the pathway that promotes exit from mitosis. The
APC promotes progression through mitosis by the ordered ubiquitin-mediated proteolysis of
the anaphase inhibitor, Pds1 (securin), and the B-type (mitotic) cyclins, Clb2, and Clb5. The
APC/Cdc20 targets Pds1 for ubiquitination, this leads to activation of the separase and loss of
cohesion (see Figure 2). The APC/Cdc20 then targets the Clbs for ubiquitination leading to
the activation of the mitotic exit network (MEN) and release of the phosphatase Cdc14 from
the nucleolus. Dephosphorylation of Hct1 and Sic1 serve to further inactivate the mitotic
Cdk/Cyclin complexes and allows the assembly of pre-replication complexes and exit from
mitosis.
3 LAYERS OF REGULATION: PART II.

THE YEAST MODEL SYSTEM WAS
INSTRUMENTAL IN THE DISCOVERY
OF CHECKPOINT PATHWAYS
The genetic material of all organisms is constantly challenged by DNA-

damaging agents from endogenous sources, such as the byproducts of
respiration and from exogenous sources, such as UV radiation and environ-
mental toxicants. In addition, during every S phase a number of the repli-
cation forks stall or collapse invoking mechanisms under study today to
reactivate the fork or fix the damage resulting from the collapsed fork [23,
139]. Accurate transmission of chromosomes to each daughter cell requires
that cells do not begin anaphase until all chromosomes have been completely
replicated and correctly aligned on the spindle. Similarly, cells that have
incurred DNA damage in G1 delay DNA replication until the damage has
been repaired. Cells that have incurred damage in S phase slow down DNA
replication and repair damage before they progress through mitosis [17, 44,
74] (Figure 4). Genetic analyses in yeast have shown that checkpoint
pathways regulate cell cycle transitions in response to perturbations and
solve the completion and alternation problem [17, 74].
3.1 The S phase and DNA damage checkpoints.

The genetic screens that uncovered the signal
transduction pathways
The Rad screen. By the late 1960s, many groups had carried out screens
to identify yeast mutants that were sensitive to ionizing radiation, UV
radiation, or both; these mutants were named rad mutants. Brian Cox, John
Game, and Robert Mortimer among others, identified a collection of
radiation sensitive mutants [22]. In 1970 scientists came to an agreement that
mutations that conferred sensitivity to ionizing radiation would continue to
be named rad mutants, numbered from RAD50 upward and they would be
loci separate from those identified in screens for UV-sensitive mutants.
Game and Mortimer took a collection of mutants sensitive to ionizing

radiation and decided to assign x-ray sensitive mutants to genetic loci and to
name them according to the new nomenclature. In doing so, they assigned
the RAD50 to RAD57 loci.
There are at least three explanations as to why mutations would lead to a
radiation-sensitive phenotype. The mutated gene could encode a protein that
is required in order for the cell to: (1) repair DNA damage; (2) stop the cell
cycle before a critical transition (i.e., mitosis) in order to allow DNA repair;
or (3) regulate the increased expression of proteins involved in DNA repair
or cell cycle arrest. Mutation in the second class of genes usually confers
sensitivity to both types of radiation, although this cannot be used as the only
criterion for assigning them to the cell cycle arrest category.
The Mec screen. Although many groups embarked on studies of the rad
mutants that affected repair of the UV and IR-induced lesions, Lee Hartwell
and Ted Weinert analyzed the rad mutants for their ability to stop the cell
cycle in G2 in response to DNA damage and called the surveillance
mechanism responsible for monitoring the successful completion of cell
cycle events checkpoints. Investigators had long noted that yeast cells
delayed cell cycle progression in response to DNA damage (refs in [133],
but the genes required for the G2/M arrest had not been uncovered. Ted
Weinert, while working with Lee Hartwell, characterized the rad mutations
that were required for cell cycle arrest following a DNA damage signal. He
also carried out a screen that would identify mutations in additional genes
involved in this checkpoint response and named them mec mutants (mitotic
entry checkpoint). Using cdc mutants that accumulate damage lesions in G2
at the restrictive temperature, Weinert showed that a rad9 mutation
(originally identified as a UV-sensitive mutant [22] and subsequently
mutations in RAD17 and RAD24 allowed cells to go through mitosis in the
presence of DNA damage. Weinert then used a genetic screen with the
damage-inducing cdc mutants and identified the mec1, mec2, and mec3
mutants as defective for the G2/M checkpoint [132].
The Sad and Dun Screens. Meanwhile Stephen Elledge’s group carried
out a screen to identify mutants that were defective for the S phase
checkpoint-induced arrest (Sad = S phase arrest defective) that would lead to
catastrophic mitosis of unreplicated chromosomes. That screen identified
SAD1 and SAD3 as essential components for this response [2]. The Elledge
lab also carried out a screen for mutants that failed to turn on the trans-
criptional response as measured by the upregulation of ribonucleotide
reductase 3 mRNA (RNR3). These mutants were called dun (DNA damage
uninducible) and led to the identification of DUN1 and DUN2 as genes
required for the transcriptional response following replication blocks and
DNA damage [89, 142].
At around the same time, Kato and Ogawa characterized a mutant named
esr1, which showed sensitivity to the alkylating agent methyl–methane–
sulfonate (MMS) and to UV radiation [57]. In addition, the esr1 mutants
displayed meiotic defects. David Stern’s group took a biochemical approach
and isolated Spk1 in a screen that used a bacterial system to identify dual
specificity kinases (kinases that can phosphorylate proteins on serines/
threonine and tyrosine residues) from yeast [141].
When all of the genes mutated in these strains were cloned it turned out
that many alleles of the same genes had been identified in the different
screens and this pointed not only to their important role in various responses
but also provided hints as to their biochemical functions. From the genes
that were cataloged by Game and Mortimer as x-ray sensitive [30], RAD51,
RAD52, and RAD54–RAD57 encoded proteins involved in recombination-
mediated DNA repair. However, RAD53 encoded an essential kinase that is
required for the cell to arrest not only in response to DNA damage but also
when DNA replication is slowed down or blocked [2, 133, 141]. RAD53
alleles were identified not only in the rad screens but also in the sad (sad1),
and mec (mec2) screens and Rad53 was the kinase identified in David
Stern’s screen for dual specificity kinases (Spk1) (Table II). Another
essential kinase that regulates this response is Mec1, and it was identified in
the mec screen, sad screen (sad3) and in Ogawa’s screen as the gene mutated
in esr1.
Dun1 turned out to be another kinase [143] that shared phosphor–peptide
recognition domains (FHA forked-head associated domains [47]) with
Rad53 outside of the kinase domain. Dun2 is the catalytic subunit of DNA
polymerase epsilon [89]. The fact that a component of DNA polymerase
showed defects in a checkpoint response sparked enthusiasm and speculation
that the DNA polymerase complexes, by the nature of their function, made
attractive candidates for sensors and scanning machines [88]. Three kinases
had been identified that are required in order to signal DNA damage and
replication blocks. Many groups began to organize the rad, mec, and sad
mutants (and other mutants that displayed sensitivity to agents that damage
DNA or cause replication blocks) into the DNA repair category or as
components of signal transduction pathways that signal the presence of these
lesions.
There were several criteria and several readouts that were used by many
laboratories in order to piece together the checkpoint signal transduction
pathways. The readouts included the effect of mutations on: (1) the cell’s
ability to delay cell cycle progression when encountering damage at different
stages of the cell cycle; (2) the activation of the kinases by phosphorylation;
and (3) the upregulation of RNR3 mRNA and other damage-inducible gene
transcripts. Using these readouts, proteins required for the checkpoint
response with similarity to proteins involved in DNA replication (DNA

polymerase or polymerase-associated proteins) or lesion processing (proteins
with homology to nucleases or other DNA associated complexes) were
considered to act as sensors that would trigger the response. Proteins that
acted by regulating phosphorylation status of targets, such as protein kinases,
were assigned the role of transducers due to their biochemical function. The
proteins phosphorylated by the kinases (and presumably also regulated by
phosphatases) were considered as effectors. These turned out to be crude
assignments since most signal transduction pathways involve feedback loops
that cause the kinases to phosphorylate upstream components. Nevertheless,
at the time, it was a good plan of attack.
3.2 The S phase and DNA damage checkpoints.

The biochemical properties of the components
of the signal transduction pathways
Although the response to different forms of DNA damage employ

common transducers, like Mec1 and Rad53, the sensors and downstream
effectors differ depending on the signal type and the cell cycle stage during
which the lesion is encountered (see below and [88]). An important aspect
of checkpoint function concerns the mechanisms by which the transducers
integrate different signals and regulate different cell cycle transitions. Here
we will discuss the pathways that regulate progression through S phase and
mitosis in order to prevent the segregation of incompletely replicated
or damaged chromosomes. In 1995, Yosef Shilo’s group cloned the gene
mutated in patients with a cancer-prone syndrome, ataxia telangectasia.
These patients also display sensitivity to radiation and their cells have
checkpoint defects [94, 108]. The product of the ATM gene is a large protein
with homology to phosphatidylinositol 3-kinases (PI(3)-kinase-related
protein kinases) and belongs to a family of proteins that include the
checkpoint kinases Mec1 and Tel1 [84] from S. cerevisiae and Rad3 from S.
pombe [7]. This discovery sparked renewed interest in the yeast checkpoint
field since once again it was shown, as was the case for CDC28 and cdc2+,
that the checkpoint pathways are also conserved between yeast and humans
(Table II).
The sensors: The first sensors identified for the S-phase checkpoint are
proteins associated with the replication machinery [58, 88]. Proteins other
than those associated with the replication machinery have also been ascribed
a role as sensors for the S-phase checkpoint. Three of these proteins are part
of a trimeric complex: Mre11, Rad50, and Xrs2 (Nibrin in mammals), and
have been implicated in lesion processing for DNA repair [10, 15].
Mutations in the genes encoding these proteins leads to checkpoint signaling

defects in yeast [24, 36] and in humans mutations in NIBRIN and MRE11
lead to a cancer-prone syndrome similar to that of ataxia telangiectasia [13,
115]. The proteins in this complex are also targets of the Mec1 kinase and
its orthologues. It remains to be determined whether other proteins that are
involved in lesion processing and repair are also involved in checkpoint
signaling, and if this role is different from their role in generating the single-
stranded DNA that could act as the signal [124] to recruit other sensor
complexes (see below).
The picture of how the DNA damage sensor complexes activate the
checkpoint kinases following DNA damage has begun to emerge. So goes
the model: due to their association with DNA and predicted protein
structure, there are at least three complexes that have been assigned the role
of DNA damage sensors in yeast and mammals (Figure 4). Two complexes
include the Rad24-RFC “clamp loading” complex (Table 2) [37], which is
associated with replication Factor C-like proteins, and the Ddc1-Mec3-
Rad17 (“clamp like” complex [14, 63, 64, 113, 128], which has been
proposed to act like a PCNA clamp. The clamp complex is referred to as the
9-1-1 complex due to the nomenclature of the S. pombe (and mammalian)
orthologous proteins (S. pombe and human Rad9, Hus1 and Rad1, see Table
II), where the similarity to the PCNA clamp was originally observed [126].
A third complex that contains Mec1 and Ddc2/Lcd1/Pie1 (Atr/Atrip in
mammals, see Table II) requires both the Rad24 and Mec3 complexes for
activation of the DNA damage response [61, 81, 101, 129].
Following recognition or processing of the lesion, the Rad24 complex
recruits the Ddc1-Mec3-Rad17 clamp complex to DNA in an analogous
mechanism to replication factor C-mediated loading of PCNA onto DNA at
the primer-template junction during DNA replication, where it serves to act
as a platform for the processive DNA polymerase complexes [122]. The
Ddc1 protein is phosphorylated by Mec1 [93], and the human orthologue of
Rad24 becomes phosphorylated by the Mec1 orthologue in a Mec3-dependent
manner [144]. The Jackson laboratory used yeast genetics to define the role
of Ddc2/Lcd1/Pie1 (Atrip homologue) in localizing Mec1 to chromatin
[101]. Although Mec1 and the human homologue Atr localize to chromatin
independently of the Rad24 complex, the phosphorylation of the yeast Mec1
and mammalian Atr substrates, and thus checkpoint signaling, is regulated
by the sensor complexes containing Rad24 and Mec3 [21, 81, 144]. The fact
that the kinases phosphorylated the sensor complexes generated confusion as
to which component was upstream; however, work published in 2001 and
2002 (reviewed in [80]) gave rise to the model that there are at least three
chromatin associated complexes: Mec1/Ddc2/Lcd1, Rad24-RFC and the
Mec3 complex. The Rad24 and Mec3 complexes are thought to provide
substrate specificity to the central checkpoint kinases of the Atm family,

which include Mec1 and mammalian Atm and Atr [144] (Figure 4), although
phosphorylation of a subset of Mec1 substrates (like Ddc2) does not require
Rad24 and Mec3. All of these stepwise interactions argue that the activation
of the DNA damage response could be lesion, cell cycle, and protein
complex specific. Future work will determine all the factors and interactions
that provide such specificity.
The checkpoint responses triggered during DNA replication have been
classified as the intra-S phase and S/M checkpoints. The former, monitors
replication fork integrity, serves to stabilize replication forks, and prevents
the firing of late replication origins. The S/M checkpoint blocks mitosis
until DNA replication has been completed. What is the nature of the signal
for activating the intra-S phase and the S/M checkpoints?
Recent analyses of the structures arising from stalled replication forks
point to single-stranded DNA that accumulates at a stalled fork as the
possible signal. Single-stranded DNA has previously been proposed as the
signal that activates the DNA damage response [31, 75]. The trimeric Rpa
complex and Rad51 protein, which can bind single-stranded DNA, could be
the molecules that trigger a response when they interact with the sensor
complexes in the context of a stalled or collapsed replication fork, which
have been estimated to occur every cell cycle [101]. rpa mutations in yeast
confer S phase and G2/M checkpoint defects [58], and Ddc1 (the S.
cerevisiae homologue of hRad9) has been found to co-localize with Rad51,
during meiosis in yeast [48].
Work carried out with both yeast and the Xenopus systems put forth the
idea that the primase enzyme, which is part of DNA Polα complex and
synthesizes the RNA primer during DNA replication, was required for the S
phase checkpoint signal to be generated [77, 82]. This suggested that the
synthesis of the RNA primer itself could act as the signal that elicits the
checkpoint response. This is in agreement with the fact that the Rfc and
PCNA-like molecules, which require the RNA primer to load on to DNA are
also required for checkpoint signaling [138]. Newport’s and Dunphy’s
groups have used a Xenopus in vitro system to study the association of the
Atm-related protein Atr and other checkpoint proteins with chromatin. An
interesting find from these and other laboratories was that DNA replication
is required for Atr and Hus1, a component of the clamp, to associate with
chromatin [138]. Depletion experiments showed that Rpa was necessary for
Atr and Hus1 to associate with chromatin and drug experiments suggested
that DNA Polα was required for Hus1 association with chromatin [138].
However, these studies do not rule out the possibility that the effect of Rpa
depletion is due to the inhibition of DNA replication, which is required for
these proteins to associate with chromatin. These studies also do not
differentiate between the scenarios where the RNA primer is the signal or
where the synthesis of the primer is a step required for the sensor complexes
to assemble on DNA [138] (Figure 5). Nevertheless, with some of the known
players in hand, the answer to these questions may be around the corner.
The transducers. In S. cerevisiae, replication blocks and DNA damage
which occur in S-phase activate a checkpoint response regulated by the kinase
Mec1 (Atr orthologue), which acts upstream of the kinase Rad53 to stabilize
replication forks, prevent firing of late replicating origins and prevent
anaphase entry [2, 106, 120, 133]. A role for Rad53 in the regulation of repli-
cation origin stability and firing would suggest that Rad53 complexes may
also be localized in close proximity to proteins involved in DNA replication.
Mec1 has also been shown to mediate the DNA-damage checkpoint that
regulates mitotic progression by signaling to both Chk1 and Rad53. While in
the Elledge laboratory we showed that Chk1 and Rad53 via Dun1 form
parallel branches in the DNA-damage checkpoint that function downstream
of Mec1 to regulate anaphase entry and mitotic exit, respectively (Figure 4,
[103]). This is accomplished by blocking the destruction of two substrates
of the anaphase-promoting complex or cyclosome.
The effectors. The effectors of the S-phase checkpoint that mediate a
block to anaphase in the budding yeast remain elusive, although in the
fission yeast and vertebrates Chk1 and Cds1/Chk2 (Rad53 orthologue) target
the Cdc25 phosphatase to elicit a G2/M arrest. However the effectors of the
DNA damage checkpoint in the budding yeast have been identified.
The DNA damage-induced checkpoint arrest that prevents progression
through mitosis in S. cerevisiae occurs after the formation of a spindle,
a stage that requires high activity of the mitotic cyclin/Cdk kinase
(Clb2/Cdc28). At the checkpoint-induced arrest, which occurs at the
metaphase to anaphase transition (M–A), the sister chromosomes are paired,
held together by cohesins (such as Scc1, Mcd1) and can presumably interact
with the bipolar pre-anaphase (short) spindle that has assembled at this stage.
Progression through mitosis in all eukaryotic cells is regulated by the activity
of the APC or cyclosome, which triggers chromosome segregation and
mitotic exit through the ubiquitin-mediated degradation of anaphase
inhibitors, such as Pds1 and mitotic cyclins, respectively. Orna Cohen-Fix,
when in Koshland’s laboratory, showed that Pds1 was phosphorylated in
response to DNA damage in a Mec1-dependent manner [18]. Phosphorylation
of Pds1 correlates with accumulation of Pds1 following DNA damage [104].
While in the Elledge laboratory, we also showed that Chk1 inhibits the M–A
transition by phosphorylation and stabilization of Pds1 and Rad53 blocks
Clb2 degradation and possibly exit from mitosis by blocking the activation
of the MEN. The Elledge laboratory continued to look for the targets of the
Rad53 pathway and found that Bfa1, a component of the MEN pathway is
regulated by Rad53 and Dun1. Phosphorylation of Bfa1 in a Rad53-
dependent manner inhibits the activity of the MEN pathway to help maintain
the levels of Clb2 and thus allow cells to maintain their arrest in mitosis by
maintaining active Cdk/B-type complexes [50]. However, the first wave of
Clb2 destruction occurs at or right after anaphase, and the Cross laboratory
showed that the first wave of Clb2 destruction could be sufficient for mitotic
exit [131] indicating that Rad53 must have other targets in order to maintain
high levels of Clb2/Cdk activity.
Other roles of the checkpoint pathways involve the transcriptional
induction of genes encoding products involved in DNA metabolism and
DNA repair. The ribonucleotide reductase (RNR) genes are transcriptional
targets of the S phase and DNA damage checkpoints that have been studied
extensively in yeast and are conserved in mammals. The Rnr proteins
catalyze the rate-limiting step of DNA synthesis, which is the generation of
the deoxyribonucleotide pools. The Sphase and DNA damage checkpoint
mediated by Mec1, Rad53, and Dun1 controls the transcriptional upregu-
lation of the RNR genes in yeast [51, 139, 142]. Two of the effectors for the
checkpoint-induced upregulation of Rnr activity are the transcriptional
repressor Crt1 and the Rnr regulatory subunit Sml1 [139, 143]. In response
to DNA damage or stalled replication forks, Crt1 is hyperphosphorylated in
a Dun1-dependent manner and released from the DNA allowing
transcription of the RNR genes [51]. In addition, phosphorylation of Sml1 in
a Dun1-dependent fashion allows the activation of ribonucleotide reductase,
presumably by mediating the degradation of Sml1 [141].
The Chk1 and Rad53 (Cds1/Chk2) proteins from both S. pombe and
S. cerevisiae function downstream of the Atm homologues Rad3 and Mec1
and regulate mitotic progression following DNA damage, albeit through
different mechanisms. Although S. cerevisiae displays a unique cell cycle
organization, orthologues of both Pds1 and Esp1 have been identified in
S. pombe (cut2 and cut1, respectively) and mammals (separin and securin,
see Table II).
Figure 4. The DNA damage checkpoint regulates mitotic progression by blocking the
ubiquitin-mediated destruction of Pds1 and the inactivation of the mitotic Cdk/Cyclin
complexes. Checkpoint activation is achieved by the interaction of two complexes that are
independently recruited to sites of DNA damage Mec1-Ddc2/Lcd1/Pie1 and Mec3-Ddc1-
Rad17 (9-1-1 complex in mammals). The Rad24-RFC “clamp loading” complex recruits the
Ddc1-Mec3-Rad17 “clamp like” complex following recognition or processing of the lesion.
Mec1 and Ddc2/Lcd1/Pie1 requires both the Rad24 and Mec3 complexes for activation of the
DNA damage response. The Ddc1 and Ddc2 proteins are phosphorylated by Mec1. The
Rad24 and Mec3 complexes provide substrate specificity to the central checkpoint kinase
Mec1 to direct phosphorylation of substrates such as Rad9, and the effector kinases Rad53
and Chk1, which then associate with Rad9. Chk1 and Rad53 form parallel branches in the
DNA damage checkpoint to regulate anaphase entry and mitotic exit. Chk1 phosphorylates
and blocks the ubiqutin-mediated destruction of the securin Pds1, and Rad53 activates Dun1
to block the inactivation of Bfa1 and activation of MEN. Dun1 also phosphorylates the Crt1
transcriptional repressor and the RNR inhibitor Sml1 (not shown) in order to elicit a
transcriptional response.
3.3 The spindle checkpoint, another checkpoint

mechanism that monitors the integrity of the spindle
3.3.1 The genetic screens
Andrew Murray, who has studied the regulation of mitosis using both
biochemistry (MPF) and yeast genetics, identified mitotic arrest deficient
(mad) mutants with increased sensitivity to anti-microtubule drugs. These
mutants were defective in the mitotic checkpoint arrest in response to a drug
that interferes with mitotic spindle assembly by depolymerizing micro-
tubules [70]. Murray identified mad1, mad2, and mad3 in this screen.
Andrew Hoyt also identified mutants that were sensitive to microtubule
destabilizing agents and failed to restrain subsequent cell cycles as
evidenced by budding and DNA replication when in the presence of an anti-
microtubule drug; these mutants were named bub (budding uninhibited by
benzimidazole) [49]. Bub1 was later shown by Hoyt to be a kinase, again
pointing to a signal transduction pathway in checkpoint control that is
activated in response to microtubule perturbation. Mark Winey identified
mutants defective in the duplication of the spindle poles. Duplication of
spindle pole bodies is a requisite step in order to form a bipolar spindle that
will separate the chromosomes at mitosis. He called his mutants mps for
(monopolar spindle) [134]. One of the proteins identified by Whiney, Mps1,
is a kinase [67], and the function of Mps1 is required for both spindle pole
body duplication and the spindle checkpoint.
The spindle checkpoint pathways, like the DNA damage checkpoint,
bifurcate to control anaphase and mitotic exit [1, 8, 69, 119]. Epistasis
analyses determined that the Mad2 and Bub2 acted in two separate
pathways: Mad/Bub pathway functions to block M–A and the Bub2 pathway
functions to block mitotic exit. The kinase Mps1 is thought to act upstream
of the identified spindle checkpoint proteins as its overexpression activates
both the Mad/Bub pathway and the Bub2 pathway, and causes cells to arrest
both in metaphase and before mitotic exit [41].
Figure 5. What is the signal that activates the S/M checkpoint during DNA replication and in
response to replication problems? Some of the same proteins involved in the DNA damage
response, such as the clamp loader proteins and the clamp-like complex, are involved in
activation of the S/M phase checkpoint during DNA replication. During DNA replication the
primase and DNA polymerase α complex synthesizes an RNA primer followed by short
stretch of DNA synthesis. This acts to recruit the PCNA clamp via the RFC complex to the
primer/template junction. PCNA and other proteins have a role in recruiting the processive
DNA polymerase enzymes δ and ε. Genetic and biochemical experiments implicate proteins
that are located at the replication fork (Rpa, DNA polymerase α/Primase complex) in the
recruitment of both the clamp loader, clamp and Atr (Mec1) kinases in order to signal ongoing
replication and stalled forks and to activate a checkpoint response that will block mitotic
progression until DNA replication has been completed.
3.3.2 The biochemical pathways that are emerging from the studies
of the genes identified in the genetic screens
As was the case for the DNA damage and S-phase-checkpoint proteins,
the biochemical properties of the proteins that regulate the spindle
checkpoint comprise a signal transduction pathway. Because the spindle and
DNA damage checkpoints both regulate mitotic progression, they share
some of the same targets, however, the transducers and sensors are different
due to the nature and location of the signal. In the spindle checkpoint the
signal seems to be generated at least in part by an unattached or mono-

oriented kinetochore (the structure on the chromosome that allows
attachment to the spindle, see Figure 6) (reviewed in [11]). Both Mad2,
which recognizes unattached kinetochores, and the Aurora Kinase Ipl1,
which is activated by mono-oriented kinetochores, activate the downstream
signaling pathway to arrest cells in mitosis.
The precise composition of the signaling complexes that elicit a
metaphase arrest has yet to be elucidated, however, many of the proteins
involved have been identified via genetic screens. Mad1, 2, and 3, as well as
Bub1 and Bub3, comprise the Mad/Bub pathway, and these proteins are
involved in the metaphase arrest in response to a spindle-activated
checkpoint. Bub1, a kinase in the Mad/Bub pathway, acts interdependently
with Mps1 to inhibit anaphase. Mad1 is hyperphosphorylated by Mps1
and this phosphorylation is dependent on Bub1 as well as the proteins
Mad2 and Bub3 (reviewed in [3]). Mad2 binds to the APC specificity
factor Cdc20 and this interaction is thought to inhibit the APC/C from
targeting Pds1 for degradation, thereby inhibiting anaphase [53, 59]. cdc20
mutants that cannot bind Mad2 are deficient in the spindle checkpoint [40,
53]. Other components of the Mad/Bub pathway, including Mad3 and Bub3,
are also found in a complex with Cdc20. Whether the interaction of Mad3
and Bub3 with Cdc20 results in inhibition of the APC/C has yet to be
determined. Therefore, Cdc20 acts as the effector of the Mad/Bub pathway
in response to spindle checkpoint activation. Mitotic exit is regulated by a
second pathway, the Bub2 pathway, which also acts downstream of Mps1.
Bfa1 is the effector of the DNA damage checkpoint and it interacts with
Bub2 to form the GTPase activating protein involved in the MEN pathway.
Recently, the Bfa1-interacting protein Ibd2 was identified and was shown to
act upstream of Bub2/Bfa1 in the spindle checkpoint [52]. Activation of
Bub2 by unattached or mono-oriented kinetochores inhibits the MEN
pathway and the cells arrest before exiting mitosis (Figure 6). Thus, the
Bub2/Bfa1 complex integrates different signals in order to block activation
of the MEN. It remains to be determined whether other pathways that
coordinate cell cycle transitions also regulate this complex.
Figure 6. The spindle checkpoint blocks mitotic progression when the chromosomes are not
attached or bi-oriented to the spindle. Unattached kinetochores as well as mono-oriented
kinetochores (not bi-oriented) activate the spindle checkpoint. The spindle checkpoint is a
signal transduction pathway that bifurcates to control anaphase and mitotic exit. Mps1 is
thought to act upstream and it signals to both the Mad/Bub pathway and the Bub2 pathway, to
bock anaphase and mitotic exit. Mps1 phosphorylates Mad1, and this phosphorylation is
dependent on Bub1, Mad2, and Mad3. Phosphorylated Mad1 interacts with Mad2 and Bub3,
Mad3 and Mad2, can bind to Cdc20. Interaction of these proteins with Cdc20 prevents the
activation of the APC/Cdc20 blocking the ubiquitination of Pds1 and the Clbs. Mps1
activation of Bub2 blocks activation of the MEN. Ipl1, an Aurora kinase that localizes to
kinetochres, activates the spindle checkpoint when the spindles bound to the kinetochores are
not connected to opposite spindle pole bodies. The signal for this event is the lack of tension
on the spindles bound to the sister chromatids. There are two possible mechanisms for Ipl1
mediated activation of the spindle checkpoint, (a) Ipl1 causes the dissociation of the spindle
from a kinetochore which is detected by Mad2 and activates the Mps1-dependent checkpoint,
and (b) Ipl1 could activate the Mad2 checkpoint directly. The detailed mechanisms of this
surveillance pathway remain to be determined.
3.4 Roles of the S phase, DNA damage checkpoints

and spindle (mitotic) checkpoints
From the studies carried out in S. cerevisiae, the following roles have
been elucidated for the checkpoint that monitors DNA replication status and
DNA integrity during S phase:
The sensors are part of or associated with the replication machinery, or
their structure mimics the complexes involved in DNA replication. The
kinases Mec1 and Rad53 regulate effectors that (1) stabilize replication forks
and allow reinitiation of DNA replication once the problem has been
overcome [26, 73, 120]; (2) prevent firing of late replication origins [106];
(3) block anaphase until DNA replication is complete [2, 133]; and (4)
through regulation of Dun1, activate transcription of genes encoding proteins
involved in DNA metabolism and DNA repair [51, 139, 142, 143].
The DNA damage checkpoint pathway that operates at G1/S functions to
block entry into S phase until the damage is repaired. The targets of this
response have been better characterized in metazoans than in yeast. Due to
differences in the organization of the cell cycle, the effectors for the DNA
damage-induced arrest at G2/M differ between the budding yeast and
mammals. In the budding yeast, the arrest occurs at the metaphase to
anaphase transition (Figure 4); whereas in metazoans and fission yeast, the
Chk1 and Cds1/Chk2 (sp and hRad53)-mediated arrest occurs in G2 by
inactivation of the Cdc25 phosphatase and activation of the Wee1 kinases
[29, 38, 96, 100, 105]. Budding yeast cells do activate a G2/M checkpoint in
response to perturbations in bud construction and/or bud size [45, 79];
therefore, it remains to be determined whether mammalian cells also regulate
the metaphase to anaphase transition in response to DNA damage.
The mitotic or spindle checkpoint monitors the proper attachment and
orientation of chromosomes on the spindle. This ensures the accurate
transmission of genetic material to daughter cells.
The signals that activate the S phase and spindle checkpoints originate at
the replication fork or the kinetochore, and serve to indicate lack of
completion of that particular cell cycle stage, much like the checkpoint
mechanisms identified by Rao Johnson. Similarly the checkpoints that
monitor replication forks and DNA integrity use similar mechanisms, and
sometimes some of the same proteins, to delay cell cycle transitions, and in
addition play an important role in the stability of replication forks and in
DNA repair.
One aspect of checkpoint signaling that eludes investigators is the

mechanism by which these proteins provide specificity to activate the kinases in
a cell cycle and lesion-specific context. For example, in S. cerevisiae Rad9 is
involved in the amplification of the signal to both Rad53 and Chk1, however
Chk1 is not activated in G1/S, whereas Rad53 is. In addition, Mec1 and the
paralogue Tel1 can both signal to Rad53 and possibly to Chk1 but the
pattern of Chk1 phosphorylation differs depending on the type of lesion that
activates the response. Cells use a relatively limited number of molecules to
integrate a variety of signals to coordinate cell cycle progression. As in all
signal transduction studies, the burning question in checkpoint signaling is
how specificity is achieved in these responses.
4 THE CANCER CONNECTION
Mutations in checkpoint genes, compromise the response to DNA

damage at the cellular level and at the level of the organism lead to a
predisposition to cancer. The low complexity of architecture of the yeast
genome along with its amenability to genetic manipulation has allowed rapid
alternation between the yeast and vertebrate model systems to elucidate the
circuitry of the DNA damage checkpoints in eukaryotic cells. An important
question in the study of checkpoints that has become the challenge for the
next few years concerns the organization of the signal transduction pathways
and the possible crosstalk or relationship between the checkpoint response
and the proteins that carry out DNA repair.
One of the hallmarks of tumor cells is that they have accumulated
mutations that not only relieve the cells from growth regulatory signals but
also allow them to invade the circulatory system, avoid the immune
response, and grow at distant sites usually in a different tissue environment.
The current hypothesis for the genetic changes in tumor cells is that they
have gone through a period of genomic instability and loss of checkpoint
control [71]. It is not surprising then that human orthologues of the
checkpoint genes identified in yeast Atm (Mec1/Tel1), Chk2 (Rad53), Brca1
(Rad9) are tumor suppressor genes and that mutations in these genes
predispose individuals to cancer [12].
Another characteristic of tumor cells is that they are aneuploid (too many
chromosomes or missing chromosomal regions). This is not surprising since
some cancer cells have mutations in spindle checkpoint genes that cause
defects in the surveillance of the correct orientation and attachment of
chromosomes to the mitotic spindle [12].
4.1 Therapeutic targets for cancer therapy
Most cancer cells are partially compromised in their ability to respond to

DNA damage or replication blocks, which limits the effectiveness of current
cancer treatments that involve the use of DNA-damaging agents such as
chemotherapy and radiation. Therefore, manipulation of these pathways
may allow us to design more effective therapeutic regimes for the treatment
of cancer. For example, cells could be rendered more sensitive to genotoxic
agents by inactivation of an additional branch or signaling point of the
checkpoint pathway. Our studies with the yeast pathways regulated by
Rad53/Dun1 and Chk1 have shown that indeed this is the case. Such
treatments would render cancer cells more sensitive to chemotherapy or
radiation treatment. Several investigators are using both S. cerevisiae and S.
pombe to test this hypothesis by using the yeasts as genetic tools to inactivate
interactions among different players in the response to DNA damage.
By the year 2001, not only had the yeast genome been sequenced but
there was a collection of deletion mutants in every nonessential yeast gene
which has been used in screens to identify genes involved in several
physiological processes, including DNA repair. Michael Resnick’s group,
who had identified some of the original RAD genes in standard genetic
screens, used this collection of deletion mutants to identify additional genes
involved in the response to ionizing radiation [6]. This screen uncovered old
favorites and new mutants that had varying degrees of sensitivity to ionizing
radiation and to other agents that damage DNA or block DNA replication.
The sensitive strains had mutations in genes that encode proteins involved in
DNA repair, cell cycle arrest, chromatin remodeling, nuclear architecture,
and endocytosis. In these studies the budding yeast system, proved once
again, to be a powerful genetic tool for the identification of proteins that
function in pathways that regulate cell cycle progression and genomic
stability. And the lessons that we have learned from yeast include the finding
that the efficacy of treatment depends on the integrity of checkpoint response
in the cell.
Due to the conservation of the pathways that guard the integrity of the
genome, we have the tools today that allow investigators to use the budding
yeast in genome-wide approaches to screen peptide and chemical agents as
potential therapeutic agents. The utility of this system for the development
of peptide inhibitors that target signaling and checkpoint pathways has
already been demonstrated in a screen that identified peptides that speci-
fically inhibited components of MAP kinase signaling and mitotic
checkpoint pathways [90].
Checkpoint pathways, by virtue of their role in protecting the integrity of
the genome, are essential to mammals for the avoidance of diseases such as
cancer. When the checkpoint pathways go awry, they become targets for
cancer drug discovery. Thus, studies that shed light on the mechanisms of
the DNA damage response in mammalian cells will allow us to further
understand both the therapeutic effects and the individual risk of exposure to
genotoxic agents, chemotherapeutic agents, and to radiation.
ACKNOWLEDGMENTS
In this chapter we have covered the work of many laboratories spanning

nearly 40 years. Broad strokes were necessary to convey the big picture of
how the players of the many layers of regulation of mitosis were identified.
We apologize to our colleagues whose work we did not cite due to limited
space. We are grateful to Ted Weinert, Craig Tomlinson, Todd Stukenberg,
and members of the Sanchez laboratory for helpful comments and discus-
sions and to Doug Kellogg for communicating data prior to publication. We
thank Eunice Ablordeppey for doing the footwork necessary to gather many
of the published papers included in the references.
REFERENCES
1. Alexandru, G., W. Zachariae, A. Schleiffer, and K. Nasmyth. 1999. Sister chromatid

separation and chromosome re-duplication are regulated by different mechanisms in
response to spindle damage. EMBO J. 18:2707–2721.
2. Allen, J. B., Z. Zhou, W. Siede, E. C. Friedberg, and S. J. Elledge. 1994. The
SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced
transcription in yeast. Genes Dev. 8:2401–2415.
3. Amon, A. 1999. The spindle checkpoint. Curr. Opin. Genet. Dev. 9:69–75.
4. Aristarkhov, A., E. Eytan, A. Moghe, A. Admon, A. Hershko, and J. V. Ruderman.
1996. E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of
mitotic cyclins. Proc. Natl. Acad. Sci. USA 93:4294–4299.
6. Bennett, C. B., L. K. Lewis, G. Karthikeyan, K. S. Lobachev, Y. H. Jin, J. F.
Sterling, J. R. Snipe, and M. A. Resnick. 2001. Genes required for ionizing radiation
resistance in yeast. Nat. Genet. 29:426–434.
7. Bentley, N. J., D. A. Holtzman, G. Flaggs, K. S. Keegan, A. DeMaggio, J. C. Ford,
M. Hoekstra, and A. M. Carr. 1996. The Schizosaccharomyces pombe rad3 checkpoint
gene. EMBO J. 15:6641–6651.
8. Biggins, S., and A. W. Murray. 2001. The budding yeast protein kinase Ipl1/Aurora
allows the absence of tension to activate the spindle checkpoint. Genes Dev. 15:3118–
3129.
9. Boddy, M. N., B. Furnari, O. Mondesert, and P. Russell. 1998. Replication
checkpoint enforced by kinases Cds1 and Chk1. Science 280:909–912.
10. Bressan, D. A., B. K. Baxter, and J. H. Petrini. 1999. The Mre11-Rad50-Xrs2 protein
complex facilitates homologous recombination-based double-strand break repair in
Saccharomyces cerevisiae. Mol. Cell. Biol. 19:7681–7687.
11. Burke, D. J. 2000. Complexity in the spindle checkpoint. Curr. Opin. Genet. Dev.
10:26–31.
12. Cahill, D. P., C. Lengauer, J. Yu, G. J. Riggins, J. K. Willson, S. D. Markowitz,
K. W. Kinzler, and B. Vogelstein. 1998. Mutations of mitotic checkpoint genes in
human cancers. Nature 392:300–303.
13. Carney, J. P., R. S. Maser, H. Olivares, E. M. Davis, M. Le Beau, J. R. Yates, 3rd,
L. Hays, W. F. Morgan, and J. H. Petrini. 1998. The hMre11/hRad50 protein
complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the
cellular DNA damage response. Cell 93:477–486.
14. Caspari, T., M. Dahlen, G. Kanter-Smoler, H. D. Lindsay, K. Hofmann,
K. Papadimitriou, P. Sunnerhagen, and A. M. Carr. 2000. Characterization of
Schizosaccharomyces pombe Hus1: a PCNA-related protein that associates with Rad1
and Rad9. Mol. Cell. Biol. 20:1254–62.
15. Chen, L., K. Trujillo, W. Ramos, P. Sung, and A. E. Tomkinson. 2001. Promotion of
Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes.
Mol. Cell 8:1105–1115.
16. Ciosk, R., W. Zachariae, C. Michaelis, A. Shevchenko, M. Mann, and K. Nasmyth.
1998. An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the
metaphase to anaphase transition in yeast. Cell 93:1067–1076.
17. Clarke, D. J., and J. F. Gimenez-Abian. 2000. Checkpoints controlling mitosis.
Bioessays 22:351–363.
18. Cohen-Fix, O., and D. Koshland. 1997. The anaphase inhibitor of Saccharomyces
cerevisiae Pds1p is a target of the DNA damage checkpoint pathway. Proc. Natl. Acad.
Sci. USA 94:14361–14366.
19. Cohen-Fix, O., and D. Koshland. 1999. Pds1p of budding yeast has dual roles:
inhibition of anaphase initiation and regulation of mitotic exit. Genes Dev. 13:1950–
1959.
20. Cohen-Fix, O., J. M. Peters, M. W. Kirschner, and D. Koshland. 1996. Anaphase
initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation
of the anaphase inhibitor Pds1p. Genes Dev. 10:3081–3093.
21. Cortez, D., S. Guntuku, J. Qin, and S. J. Elledge. 2001. ATR and ATRIP: partners in
checkpoint signaling. Science 294:1713–1716.
22. Cox, B. S., and J. M. Parry. 1968. The isolation, genetics and survival characteristics
of ultraviolet light-sensitive mutants in yeast. Mutat. Res. 6:37–55.
23. Cox, M. M., M. F. Goodman, K. N. Kreuzer, D. J. Sherratt, S. J. Sandler, and K. J.
24. D’Amours, D., and S. P. Jackson. 2001. The yeast Xrs2 complex functions in S phase
checkpoint regulation. Genes Dev. 15:2238–2249.
25. de la Torre-Ruiz, M. A., C. M. Green, and N. F. Lowndes. 1998. RAD9 and RAD24
define two additive, interacting branches of the DNA damage checkpoint pathway in
budding yeast normally required for Rad53 modification and activation. Embo. J.
17:2687–2698.
26. Desany, B. A., A. A. Alcasabas, J. B. Bachant, and S. J. Elledge. 1998. Recovery
from DNA replicational stress is the essential function of the S-phase checkpoint
pathway. Genes Dev. 12:2956–2970.
27. Dunphy, W. G., L. Brizuela, D. Beach, and J. Newport. 1988. The Xenopus cdc2
protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell 54:423–431.
28. Evans, T., E. T. Rosenthal, J. Youngblom, D. Distel, and T. Hunt. 1983. Cyclin: a
protein specified by maternal mRNA in sea urchin eggs that is destroyed at each
cleavage division. Cell 33:389–396.
29. Furnari, B., N. Rhind, and P. Russell. 1997. Cdc25 mitotic inducer targeted by chk1
DNA damage checkpoint kinase [see comments]. Science 277:1495–1497.
30. Game, J. C., and R. K. Mortimer. 1974. A genetic study of x-ray sensitive mutants in
yeast. Mutat. Res. 24:281–292.
31. Garvik, B., M. Carson, and L. Hartwell. 1995. Single-stranded DNA arising at
telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint.
Mol. Cell. Biol. 15:6128–6138.
32. Gautier, J., T. Matsukawa, P. Nurse, and J. Maller. 1989. Dephosphorylation
and activation of Xenopus p34cdc2 protein kinase during the cell cycle. Nature
339:626–629.
33. Gautier, J., C. Norbury, M. Lohka, P. Nurse, and J. Maller. 1988. Purified
maturation-promoting factor contains the product of a Xenopus homolog of the fission
yeast cell cycle control gene cdc2+. Cell 54:433–439.
34. Gerhart, J., M. Wu, and M. Kirschner. 1984. Cell cycle dynamics of an M-phase-
specific cytoplasmic factor in Xenopus laevis oocytes and eggs. J. Cell Biol. 98:1247–
1255.
35. Gilbert, C. S., C. M. Green, and N. F. Lowndes. 2001. Budding yeast Rad9 is an
ATP-dependent Rad53 activating machine. Mol. Cell 8:129–136.
36. Grenon, M., C. Gilbert, and N. F. Lowndes. 2001. Checkpoint activation in response
to double-strand breaks requires the Mre11/Rad50/Xrs2 complex. Nat. Cell Biol.
3:844–847.
37. Griffiths, D. J., N. C. Barbet, S. McCready, A. R. Lehmann, and A. M. Carr. 1995.
Fission yeast rad17: a homologue of budding yeast RAD24 that shares regions of
sequence similarity with DNA polymerase accessory proteins. EMBO J. 14:5812–5823.
38. Guo, Z., A. Kumagai, S. X. Wang, and W. G. Dunphy. 2000. Requirement for atr in
phosphorylation of chk1 and cell cycle regulation in response to DNA replication blocks
and UV-damaged DNA in xenopus egg extracts. Genes Dev. 14:2745–2756.
39. Haering, C. H., J. Lowe, A. Hochwagen, and K. Nasmyth. 2002. Molecular
architecture of SMC proteins and the yeast cohesin complex. Mol. Cell 9:773–788.
40. Hardwick, K. G., R. C. Johnston, D. L. Smith, and A. W. Murray. 2000. MAD3
encodes a novel component of the spindle checkpoint which interacts with Bub3p,
Cdc20p, and Mad2p. J. Cell Biol. 148:871–882.
41. Hardwick, K. G., E. Weiss, F. C. Luca, M. Winey, and A. W. Murray. 1996.
Activation of the budding yeast spindle assembly checkpoint without mitotic spindle
disruption. Science 273:953–956.
42. Harrison, J. C., E. S. Bardes, Y. Ohya, and D. J. Lew. 2001. A role for the
Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint. Nat. Cell Biol.
3:417–420.
43. Hartwell, L. H. 1971. Genetic control of the cell division cycle in yeast. II. Genes
controlling DNA replication and its initiation. J. Mol. Biol. 59:183–194.
44. Hartwell, L. H., and T. A. Weinert. 1989. Checkpoints: controls that ensure the order
of cell cycle events. Science 246:629–634.
45. Harvey, S. L., and D. Kellogg. 2002. Conservation of mechanisms controlling entry
into mitosis: budding yeast Wee1 delays entry into mitosis and is required for cell size
control. submitted.
46. Hirano, T. 2002. The ABCs of SMC proteins: two-armed ATPases for chromosome
condensation, cohesion, and repair. Genes Dev. 16:399–414.
47. Hofmann, K, and P. Bucher. 1995. The FHA domain: a putative nuclear signalling
domain found in protein kinases and transcription factors. Trends Biochem. Sci.
20:347–349.
48. Hong, E. J., and G. S. Roeder. 2002. A role for Ddc1 in signaling meiotic double-
strand breaks at the pachytene checkpoint. Genes Dev. 16:363–376.
49. Hoyt, M. A., L. Totis, and B. T. Roberts. 1991. S. cerevisiae genes required for cell
cycle arrest in response to loss of microtubule function. Cell 66:507–517.
50. Hu, F., Y. Wang, D. Liu, Y. Li, J. Qin, and S. J. Elledge. 2001. Regulation of the
Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints. Cell 107:655–665.
51. Huang, M., Z. Zhou, and S. J. Elledge. 1998. The DNA replication and damage
checkpoint pathways induce transcription by inhibition of the Crt1 repressor. Cell
94:595–605.
52. Hwang, H. S., and K. Song. 2002. IBD2 Encodes a novel component of the Bub2p-
dependent spindle checkpoint in the budding yeast Saccharomyces cerevisiae. Genetics
161:595–609.
53. Hwang, L. H., L. F. Lau, D. L. Smith, C. A. Mistrot, K. G. Hardwick, E. S. Hwang,
A. Amon, and A. W. Murray. 1998. Budding yeast Cdc20: a target of the spindle
checkpoint. Science 279:1041–1044.
54. Irniger, S., S. Piatti, C. Michaelis, and K. Nasmyth. 1995. Genes involved in sister
chromatid separation are needed for B-type cyclin proteolysis in budding yeast. Cell
81:269–278.
55. Jeffrey, P. D., A. A. Russo, K. Polyak, E. Gibbs, J. Hurwitz, J. Massague, and N. P.
Pavletich. 1995. Mechanism of CDK activation revealed by the structure of a cyclinA-
CDK2 complex. Nature 376:313–320.
56. Jensen, S., M. Geymonat, and L. H. Johnston. 2002. Mitotic exit: delaying the end
without FEAR. Curr. Biol. 12:R221–223.
57. Kato, R, and H. Ogawa. 1994. An essential gene, ESR1, is required for mitotic cell
growth, DNA repair and meiotic recombination in Saccharomyces cerevisiae. Nucleic
Acids Res. 22:3104–3112.
58. Kim, H. S., and S. J. Brill. 2001. Rfc4 interacts with Rpa1 and is required for both
DNA replication and DNA damage checkpoints in Saccharomyces cerevisiae. Mol. Cell.
Biol. 21:3725–3737.
59. Kim, S. H., D. P. Lin, S. Matsumoto, A. Kitazono, and T. Matsumoto. 1998. Fission yeast
Slp1: an effector of the Mad2-dependent spindle checkpoint. Science 279:1045–1047.
60. King, R. W., J. M. Peters, S. Tugendreich, M. Rolfe, P. Hieter, and M. W.
Kirschner. 1995. A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-
specific conjugation of ubiquitin to cyclin B. Cell 81:279–288.
61. Kondo, T., T. Wakayama, T. Naiki, K. Matsumoto, and K. Sugimoto. 2001.
Recruitment of Mec1 and Ddc1 checkpoint proteins to double-strand breaks through
distinct mechanisms. Science 294:867–870.
62. Koshland, D. E., and V. Guacci. 2000. Sister chromatid cohesion: the beginning of a
long and beautiful relationship. Curr. Opin. Cell Biol. 12:297–301.
63. Kostrub, C. F., F. al-Khodairy, H. Ghazizadeh, A. M. Carr, and T. Enoch. 1997.
Molecular analysis of hus1+, a fission yeast gene required for S-M and DNA damage
checkpoints. Mol. Gen. Genet. 254:389–399.
64. Kostrub, C. F., K. Knudsen, S. Subramani, and T. Enoch. 1998. Hus1p, a conserved
fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response
to DNA damage. EMBO J. 17:2055–2066.
65. Labbe, J. C., J. P. Capony, D. Caput, J. C. Cavadore, J. Derancourt, M. Kaghad,
J. M. Lelias, A. Picard, and M. Doree. 1989. MPF from starfish oocytes at first
meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule
of cyclin B. EMBO J. 8:3053–3058.
66. Lamb, J. R., W. A. Michaud, R. S. Sikorski, and P. A. Hieter. 1994. Cdc16p, Cdc23p
and Cdc27p form a complex essential for mitosis. EMBO J. 13:4321–4328.
67. Lauze, E., B. Stoelcker, F. C. Luca, E. Weiss, A. R. Schutz, and M. Winey. 1995.
Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity
protein kinase. EMBO J. 14:1655–1663.
68. Lee, M. G., and P. Nurse. 1987. Complementation used to clone a human homologue
of the fission yeast cell cycle control gene cdc2. Nature 327:31–35.
69. Li, R. 1999. Bifurcation of the mitotic checkpoint pathway in budding yeast. Proc. Natl.
Acad. Sci. USA 96:4989–4994.
70. Li, R., and A. W. Murray. 1991. Feedback control of mitosis in budding yeast. Cell
66:519–531.
71. Loeb, L. A. 1991. Mutator phenotype may be required for multistage carcinogenesis.
Cancer Res 51:3075–3079.
72. Lohka, M. J., M. K. Hayes, and J. L. Maller. 1988. Purification of maturation-
promoting factor, an intracellular regulator of early mitotic events. Proc. Natl. Acad. Sci.
USA 85:3009–3013.
73. Lopes, M., C. Cotta-Ramusino, A. Pellicioli, G. Liberi, P. Plevani, M. Muzi-Falconi,
C. S. Newlon, and M. Foiani. 2001. The DNA replication checkpoint response
stabilizes stalled replication forks. Nature 412:557–561.
74. Lowndes, N. F., and J. R. Murguia. 2000. Sensing and responding to DNA damage.
Curr. Opin. Genet. Dev. 10:17–25.
75. Lydall, D., Y. Nikolsky, D. K. Bishop, and T. Weinert. 1996. A meiotic
recombination checkpoint controlled by mitotic checkpoint genes. Nature 383:840–843.
76. Maller, J. L. 1998. Recurring themes in oocyte maturation. Biol. Cell 90:453–460.
77. Marini, F., A. Pellicioli, V. Paciotti, G. Lucchini, P. Plevani, D. F. Stern, and
M. Foiani. 1997. A role for DNA primase in coupling DNA replication to DNA damage
response. EMBO J. 16:639–650.
78. Masui, Y., and C. L. Markert. 1971. Cytoplasmic control of nuclear behavior during
meiotic maturation of frog oocytes. J. Exp. Zool. 177:129–145.
79. McMillan, J. N., R. A. Sia, and D. J. Lew. 1998. A morphogenesis checkpoint
monitors the actin cytoskeleton in yeast. J. Cell Biol. 142:1487–1499.
80. Melo, J., and D. Toczyski. 2002. A unified view of the DNA-damage checkpoint. Curr.
Opin. Cell Biol. 14:237–245.
81. Melo, J. A., J. Cohen, and D. P. Toczyski. 2001. Two checkpoint complexes are
independently recruited to sites of DNA damage in vivo. Genes Dev. 15:2809–2821.
82. Michael, W. M., R. Ott, E. Fanning, and J. Newport. 2000. Activation of the DNA
replication checkpoint through RNA synthesis by primase. Science 289:2133–2137.
83. Minshull, J., J. J. Blow, and T. Hunt. 1989. Translation of cyclin mRNA is necessary
for extracts of activated xenopus eggs to enter mitosis. Cell 56:947–956.
84. Morrow, D. M., D. A. Tagle, Y. Shiloh, F. S. Collins, and P. Hieter. 1995. TEL1, an
S. cerevisiae homolog of the human gene mutated in ataxia telangiectasia, is
functionally related to the yeast checkpoint gene MEC1. Cell 82:831–840.
85. Murray, A. W., M. J. Solomon, and M. W. Kirschner. 1989. The role of cyclin
synthesis and degradation in the control of maturation promoting factor activity. Nature
339:280–286.
86. Nasmyth, K. 2001. A prize for proliferation. Cell 107:689–701.
87. Nasmyth, K. A., and S. I. Reed. 1980. Isolation of genes by complementation in yeast:
molecular cloning of a cell-cycle gene. Proc. Natl. Acad. Sci. USA 77:2119–2123.
88. Navas, T. A., Y. Sanchez, and S. J. Elledge. 1996. RAD9 and DNA polymerase
epsilon form parallel sensory branches for transducing the DNA damage checkpoint
signal in Saccharomyces cerevisiae. Genes Dev. 10:2632–2643.
89. Navas, T. A., Z. Zhou, and S. J. Elledge. 1995. DNA polymerase epsilon links the
DNA replication machinery to the S phase checkpoint. Cell 80:29–39.
90. Norman, T. C., D. L. Smith, P. K. Sorger, B. L. Drees, S. M. O’Rourke, T. R.
Hughes, C. J. Roberts, S. H. Friend, S. Fields, and A. W. Murray. 1999. Genetic
selection of peptide inhibitors of biological pathways. Science 285:591–595.
91. Nurse, P., and P. Thuriaux. 1980. Regulatory genes controlling mitosis in the fission
yeast Schizosaccharomyces pombe. Genetics 96:627–637.
92. Nurse, P., P. Thuriaux, and K. Nasmyth. 1976. Genetic control of the cell division
cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 146:167–178.
93. Paciotti, V., G. Lucchini, P. Plevani, and M. P. Longhese. 1998. Mec1p is essential
for phosphorylation of the yeast DNA damage checkpoint protein Ddc1p, which
physically interacts with Mec3p. EMBO J. 17:4199–4209.
94. Painter, R. B. 1981. Radioresistant DNA synthesis: an intrinsic feature of ataxia
telangiectasia. Mutat. Res. 84:183–190.
95. Pavletich, N. P. 1999. Mechanisms of cyclin-dependent kinase regulation: structures of
Cdks, their cyclin activators, and Cip and INK4 inhibitors. J. Mol. Biol. 287:821–828.
96. Peng, C. Y., P. R. Graves, R. S. Thoma, Z. Wu, A. S. Shaw, and H. Piwnica-
Worms. 1997. Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding
by phosphorylation of Cdc25C on serine-216. Science 277:1501–1505.
97. Peters, J. M. 2002. The anaphase-promoting complex: proteolysis in mitosis and
beyond. Mol. Cell 9:931–943.
98. Pulverer, B. 2001. Trio united by division as cell cycle clinches centenary Nobel.
Nature 413:553.
99. Rao, P. N., and R. T. Johnson. 1970. Mammalian cell fusion: studies on the regulation
of DNA synthesis and mitosis. Nature 225:159–164.
100. Rhind, N., B. Furnari, and P. Russell. 1997. Cdc2 tyrosine phosphorylation is required
for the DNA damage checkpoint in fission yeast. Genes Dev. 11:504–511.
101. Rothstein, R., B. Michel, and S. Gangloff. 2000. Replication fork pausing and
recombination or “gimme a break”. Genes Dev. 14:1–10.
102. Rouse, J., and S. P. Jackson. 2002. Lcd1p recruits mec1p to DNA lesions in vitro and
in vivo. Mol. Cell. 9:857–869.
103. Russell, P., and P. Nurse. 1986. cdc25+ functions as an inducer in the mitotic control
of fission yeast. Cell 45:145–153.
104. Sanchez, Y., J. Bachant, H. Wang, F. Hu, D. Liu, M. Tetzlaff, and S. J. Elledge.
1999. Control of the DNA damage checkpoint by chk1 and rad53 protein kinases
through distinct mechanisms. Science 286:1166–1171.
105. Sanchez, Y., C. Wong, R. S. Thoma, R. Richman, Z. Wu, H. Piwnica-Worms, and
S. J. Elledge. 1997. Conservation of the Chk1 checkpoint pathway in mammals: linkage
of DNA damage to Cdk regulation through Cdc25. Science 277:1497–1501.
106. Santocanale, C., and J. F. Diffley. 1998. A Mec1- and Rad53-dependent checkpoint
controls late-firing origins of DNA replication. Nature 395:615–618.
107. Saunders, W. S. 2002. The FEAR factor. Mol. Cell 9:207–209.
108. Savitsky, K., A. Bar-Shira, S. Gilad, G. Rotman, Y. Ziv, L. Vanagaite, D. A. Tagle,
S. Smith, T. Uziel, S. Sfez, and et al. 1995. A single ataxia telangiectasia gene with a
product similar to PI-3 kinase. Science 268:1749–1753.
109. Shirayama, M., A. Toth, M. Galova, and K. Nasmyth. 1999. APC(Cdc20) promotes
exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5. Nature
402:203–207.
110. Smith, L. D., and R. E. Ecker. 1971. The interaction of steroids with Rana pipiens
oocytes in the induction of maturation. Dev. Biol. 25:232–247.
111. Smythe, C., and J. W. Newport. 1992. Coupling of mitosis to the completion of S
phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates
p34cdc2. Cell 68:787–797.
112. Sreenivasan, A., and D. Kellogg. 1999. The elm1 kinase functions in a mitotic
signaling network in budding yeast. Mol. Cell Biol. 19:7983–7994.
113. St Onge, R. P., C. M. Udell, R. Casselman, and S. Davey. 1999. The human G2
checkpoint control protein hRAD9 is a nuclear phosphoprotein that forms complexes
with hRAD1 and hHUS1. Mol. Biol. Cell 10:1985–1995.
114. Stegmeier, F., R. Visintin, and A. Amon. 2002. Separase, polo kinase, the kinetochore
protein Slk19, and Spo12 function in a network that controls Cdc14 localization during
early anaphase. Cell 108:207–220.
115. Stewart, G. S., R. S. Maser, T. Stankovic, D. A. Bressan, M. I. Kaplan, N. G.
Jaspers, A. Raams, P. J. Byrd, J. H. Petrini, and A. M. Taylor. 1999. The DNA
double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-
telangiectasia-like disorder. Cell 99:577–587.
116. Sudakin, V., D. Ganoth, A. Dahan, H. Heller, J. Hershko, F. C. Luca, J. V.
Ruderman, and A. Hershko. 1995. The cyclosome, a large complex containing cyclin-
selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.
Mol. Biol. Cell 6:185–197.
117. Sun, Z., J. Hsiao, D. S. Fay, and D. F. Stern. 1998. Rad53 FHA domain associated
with phosphorylated Rad9 in the DNA damage checkpoint [see comments]. Science
281:272–274.
118. Surana, U., A. Amon, C. Dowzer, J. McGrew, B. Byers, and K. Nasmyth. 1993.
Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to
anaphase transition in budding yeast. EMBO J. 12:1969–1978.
119. Tanaka, T. U., N. Rachidi, C. Janke, G. Pereira, M. Galova, E. Schiebel, M. J.
Stark, and K. Nasmyth. 2002. Evidence that the Ipl1-Sli15 (Aurora kinase-INCENP)
complex promotes chromosome bi-orientation by altering kinetochore-spindle pole
connections. Cell 108:317–329.
120. Tercero, J. A., and J. F. Diffley. 2001. Regulation of DNA replication fork progression
through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412:553–557.
121. Tinker-Kulberg, R. L., and D. O. Morgan. 1999. Pds1 and Esp1 control both
anaphase and mitotic exit in normal cells and after DNA damage. Genes Dev.
13:1936–349.
122. Tsurimoto, T., and B. Stillman. 1991. Replication factors required for SV40 DNA
replication in vitro. I. DNA structure-specific recognition of a primer-template junction
by eukaryotic DNA polymerases and their accessory proteins. J. Biol. Chem.
266:1950–1960.
123. Tyers, M., and P. Jorgensen. 2000. Proteolysis and the cell cycle: with this RING I do
thee destroy. Curr. Opin. Genet. Dev. 10:54–64.
124. Usui, T., H. Ogawa, and J. H. Petrini. 2001. A DNA damage response pathway
controlled by tel1 and the mre11 complex. Mol. Cell 7:1255–1266.
125. Venclovas, C., and M. P. Thelen. 2000. Structure-based predictions of Rad1, Rad9,
Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes. Nucleic
Acids Res. 28:2481–2493.
126. Visintin, R., K. Craig, E. S. Hwang, S. Prinz, M. Tyers, and A. Amon. 1998. The
phosphatase Cdc14 triggers mitotic exit by reversal of Cdk- dependent phosphorylation.
Mol. Cell 2:709–718.
127. Visintin, R., E. S. Hwang, and A. Amon. 1999. Cfi1 prevents premature exit from
mitosis by anchoring Cdc14 phosphatase in the nucleolus [see comments]. Nature
398:818–823.
128. Volkmer, E., and L. M. Karnitz. 1999. Human homologs of Schizosaccharomyces
pombe rad1, hus1, and rad9 form a DNA damage-responsive protein complex. J. Biol.
Chem. 274:567–570.
129. Wakayama, T., T. Kondo, S. Ando, K. Matsumoto, and K. Sugimoto. 2001. Pie1, a
protein interacting with Mec1, controls cell growth and checkpoint responses in
130. Walworth, N., S. Davey, and D. Beach. 1993. Fission yeast chk1 protein kinase links
the rad checkpoint pathway to cdc2. Nature 363:368–371.
131. Wasch, R., and F. R. Cross. 2002. APC-dependent proteolysis of the mitotic cyclin
Clb2 is essential for mitotic exit. Nature 418:556–562.
132. Weinert, T. A. 1992. Dual cell cycle checkpoints sensitive to chromosome replication
and DNA damage in the budding yeast Saccharomyces cerevisiae. Radiat. Res.
132:141–143.
133. Weinert, T. A., G. L. Kiser, and L. H. Hartwell. 1994. Mitotic checkpoint genes in
budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev.
8:652–665.
134. Winey, M., L. Goetsch, P. Baum, and B. Byers. 1991. MPS1 and MPS2: novel yeast
genes defining distinct steps of spindle pole body duplication. J. Cell Biol. 114:745–754.
135. Yamamoto, A., V. Guacci, and D. Koshland. 1996. Pds1p is required for faithful
execution of anaphase in the yeast, Saccharomyces cerevisiae. J. Cell Biol. 133:85–97.
136. Yamamoto, A., V. Guacci, and D. Koshland. 1996. Pds1p, an inhibitor of anaphase in
budding yeast, plays a critical role in the APC and checkpoint pathway(s). J. Cell Biol.
133:99–110.
137. Yeong, F. M., H. H. Lim, C. G. Padmashree, and U. Surana. 2000. Exit from mitosis
in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of
Cdc20. Mol. Cell 5:501–511.
138. You, Z., L. Kong, and J. Newport. 2002. The role of single-stranded DNA and
polymerase alpha in establishing the ATR, Hus1 DNA replication checkpoint. J. Biol.
Chem. 277:27088–27093.
139. Zhao, X., E. G. Muller, and R. Rothstein. 1998. A suppressor of two essential
checkpoint genes identifies a novel protein that negatively affects dNTP pools [In
Process Citation]. Mol.Cell 2:329–340.
140. Zhao, X., and R. Rothstein. 2002. The Dun1 checkpoint kinase phosphorylates and
regulates the ribonucleotide reductase inhibitor Sml1. Proc. Natl. Acad. Sci. USA
99:3746–3751.
141. Zheng, P., D. S. Fay, J. Burton, H. Xiao, J. L. Pinkham, and D. F. Stern. 1993.
SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a
nuclear serine/threonine/tyrosine kinase. Mol. Cell. Biol. 13:5829–5842.
142. Zhou, Z., and S. J. Elledge. 1993. DUN1 encodes a protein kinase that controls the
DNA damage response in yeast. Cell 75:1119–1127.
143. Zhou, Z., and S. J. Elledge. 1992. Isolation of crt mutants constitutive for transcription
of the DNA damage inducible gene RNR3 in Saccharomyces cerevisiae. Genetics
131:851–866.
144. Zou, L., D. Cortez, and S. J. Elledge. 2002. Regulation of ATR substrate selection by
Rad17-dependent loading of Rad9 complexes onto chromatin. Genes Dev. 16:198–208.
Chapter 3
YEAST AS A TOOL IN CANCER RESEARCH:
NUCLEAR TRAFFICKING
Anita H. Corbett and Adam C. Berger

Department of Biochemistry and Graduate Program in Biochemistry, Cell and Developmental
Biology, Emory University School of Medicine, 1510 CLifton Rd., NE, Atlanta, GA 30322
Recently, scientists have begun to appreciate the important role that

dynamic intracellular trafficking plays in regulating the function of growth
regulatory factors that have been implicated in cancer. Importantly,
numerous examples of proteins whose function is regulated by entry into or
exit from the nucleus have been identified [107]. The classic example of this
regulation is the translocation of a transcription factor into the nucleus in
response to a signal [63]. One such example is the transcription factor p53,
which is transported into the nucleus, where it can act in various nucleic acid
transactions in response to DNA damage [27, 89]. Since p53 function is lost
in a large number of human tumors [110], understanding what mechanisms
regulate p53 function is obviously of critical importance to understanding
cell transformation. It has become clear that one of the mechanisms that
contributes to regulation of p53, as well as other proteins that have been
implicated in cancer, is the orchestrated movement of the protein between
the cytoplasm and the nucleus [27, 89]. Our understanding of the various
mechanisms that regulate such nuclear protein import and export has been
spurred by recent advances in the field of nucleocytoplasmic trafficking.
An eukaryotic cell is defined by the advantageous presence of a
membrane-bound nucleus, which separates the nuclear genetic material from
the cytoplasmic protein translation machinery to allow exquisite regulation
of gene expression. This separation dictates the need for a transport system
that can connect the cytoplasm to the nucleus. The basic machinery that
mediates this transport has been conserved from yeast to humans [82]. In
fact, studies of nuclear transport in yeast have identified critical regulators of
nuclear transport and provided important insight into the mechanism by
which the process is orchestrated.
75
© 2007 Springer.
76 A. H. Corbett and A. C. Berger
The goal of this chapter is to provide a brief overview of the recent

advances in the study of nuclear transport and describe how studies in yeast
model systems have contributed to these advances. The chapter primarily
focuses on the budding yeast, Saccharomyces cerevisiae, but also
incorporates findings from the fission yeast, Schizosaccharomyces pombe.
The chapter first presents a general overview of the mechanism and
mediators of the nuclear transport process. It then describes three specific
examples of critical growth regulatory yeast proteins that are modulated by
nuclear transport and are models for cancer-implicated processes. The first
example is the cell cycle-regulated localization of cyclin B, primarily
focusing on studies with S. cerevisiae cyclin B, Clb2p. The second example
is the S. pombe phosphatase, cdc25. Finally, the third example is the
S. cerevisiae Pho4p transcription factor, which serves as a paradigm for
signal-mediated transcription factor import and export. These examples
highlight the regulation that can be imparted by dynamic localization
between the cytoplasm and the nucleus.
1 THE NUCLEAR PORE COMPLEX
All macromolecular traffic between the cytoplasm and the nucleus flows
through a large channel called the nuclear pore complex (NPC). The NPC is
a large macromolecular machine that spans the double membrane of the
nuclear envelope (see [28, 98, 100] for recent reviews). Studies over the
years have revealed that nuclear pores have eightfold rotational symmetry
within the membrane but have distinct nuclear and cytoplasmic faces. The
cytoplasmic face has filaments that emanate from the pore and the nuclear
face has a basket-like structure. These distinct faces are thought to contribute
to the mechanism of targeting and transport through the pores.
Nuclear pores are composed of proteins termed nucleoporins or Nups. A
large number of these nucleoporins contain characteristic phenylalanine–glycine
(FG) repeat motifs that are likely to play a critical role in the translocation of
substrates through the pore. Several recent studies have identified all the
proteins that make up both the yeast and the vertebrate NPC. This work was
led by a pioneering study in S. cerevisiae where Rout and colleagues used a
proteomic approach to identify the proteins that comprise the yeast NPC
[86]. This work paved the way for a similar study in higher eukaryotes [19],
which revealed that although the vertebrate pore complexes are somewhat
larger than those in yeast, the number of distinct proteins is approximately
the same.
Spatial analysis of the yeast nuclear pore revealed that many of the
nuclear pore proteins exist in multiple copies that are symmetrically
distributed between the nuclear and cytoplasmic faces of the pore [86].
Chapter 3: Nuclear Trafficking in Yeast 77
Many of the FG-repeat-containing nucleoporins are predicted to line the

central channel of the NPC and make direct contact with transport receptors
(section 2.2) moving through the pore. In contrast, there are a few nucleoporins
that are asymmetrically localized to either the nuclear or cytoplasmic face of
the pore. These asymmetric proteins probably comprise the distinct structures
seen on either face of the pore, the nuclear basket and the cytoplasmic
filaments. It seems likely that these structures may serve as docking or
assembly/disassembly sites for the cargoes that are transported through the
pore. A more detailed understanding of the overall structure of the pore will
be required before the precise mechanism of movement through can be fully
defined.
There are several examples of gene translocation events involving genes
encoding nucleoporins that have been implicated in human cancers. The
most common translocations of this type, which occur in acute myelogenous
leukemia, involve the Nup98 nuclear pore protein [68]. There have been
very few studies to understand the functional importance of these translo-
cations and the resulting oncogenic fusion proteins. Another example of a
translocation that has been linked to leukemia is the translocation that places
the Met kinase under the control of the Tpr (Translocated promoter region)
nuclear pore protein [23].
2 PROTEIN TRAFFICKING
2.1 Nuclear transport targeting signals
As with most intracellular targeting mechanisms, trafficking between the

nucleus and the cytoplasm depends on amino acid sequences within the
protein cargoes to be transported. However, unlike other targeting mechanisms,
such as mitochondrial and ER targeting, nucleocytoplasmic trafficking
signals are not cleaved and remain an integral part of the protein. This
mechanism allows the cargo proteins to undergo multiple rounds of import
and/or export and allows the cell to exploit this cycling as a regulatory
mechanism.
Although there are still numerous nucleocytoplasmic-targeting signals that
are ill-defined or completely unknown, canonical signals for both nuclear
protein import and nuclear protein export have been identified and studied in
some detail. Nuclear import signals are generally termed nuclear localization
signals (NLS), while signals that target proteins for export from the nucleus
are termed nuclear export signals (NES).
The classical NLS consists of either a single cluster of basic amino acid
residues (monopartite) or two clusters of basic amino acid residues separated
by a 10–13 amino acid linker sequence (bipartite) [56, 85]. The monopartite
NLS sequence is typified by the NLS found in the SV40 large T antigen
(PKKKRKV), whereas the bipartite NLS is typified by the nucleoplasmin
NLS (KRPAATKKAGQAKKKK). These sequences mediate binding to a
classical nuclear import receptor, importin/karyopherin α (discussed in more
detail in section 2.2 below). Although numerous NLS sequences have been
identified, the best consensus sequence that has emerged thus far is:
K(K/R)x(K/R) where x is any amino acid [44].
The classical NES is comprised of a series of hydrophobic amino acids,
generally leucine, isoleucine, or valine [32]. The founding member of the
NES family is the protein kinase A inhibitor protein, which contains the NES
sequence LALKLAGLDI, where the underlined residues are critical for NES
function [108]. Numerous variations on this theme have been identified and
thus far the best consensus sequence for an NES is LxxxLxxLxL, where the
spacing between the leucines can vary and, in fact, the leucines can be
substituted with virtually any hydrophobic amino acid.
Obviously, it is possible to scan the sequence of any given protein and
identify putative classical NLS or NES sequences, however, substantial
experimental evidence is required to prove that an amino acid sequence is
both necessary and sufficient to serve as a functional targeting signal [21].
This is particularly critical given the rather weak consensus sequences for
the known nuclear targeting signals. Current efforts are being made to better
define the important characteristics of both NLS and NES sequences. Recent
studies have examined the interaction between NLS sequences and the
classical NLS receptor, importin α, by determining the binding energy
contributed by each residue within NLS sequences [44]. This study has been
extended by using budding yeast to determine how the binding affinity of the
NLS for the NLS receptor relates to the actual accumulation of the cargo
within the nucleus [43]. A complementary study used an in vivo rando-
mization-selection assay to determine which residues within an NES are
absolutely required for export function [11].
Mutations or modifications that alter the nuclear-targeting signals of
growth regulatory proteins are associated with some instances of cellular
transformation. There is evidence that phosphorylation that occurs proximal
to an NLS sequence can alter the nuclear targeting of the cargo protein [40,
51]. One notable example where an alteration in nuclear targeting is asso-
ciated with cellular transformation is the v-jun oncogene. Normal cellular jun
(c-jun) contains a classical NLS with a flanking cysteine [13]. In the v-jun
oncogene the sequence is altered such that the cysteine is replaced by a
serine [13, 102]. It seems that phosphorylation of this serine regulates the
nuclear import of v-jun and thus imparts an inappropriate function on the
oncoprotein [13, 102]. There are a number of other mechanisms that can
be used to regulate the function of a nuclear targeting signal. For example,
there are cases where an NLS is masked by a binding partner as in the case
of IκB binding to the transcription factor NFκB. IκB binds to NFκB and
masks the NLS and only when IκB is degraded in a signal-dependent
manner does NFκB enter the nucleus, trigger gene expression, and cause
changes in cell growth [71, 80]. As we learn more about the signals that
target cargo proteins to and from the nucleus, it is likely that we will uncover
other regulatory mechanisms that are critical for proper control of cell
growth. In fact, recent work with the Bcr–Abl oncoprotein indicates that
trapping this protein within the nucleus in its active state can induce
transformed cells to undergo apoptosis [105]. Thus, there is potential
therapeutic value in uncovering ways to alter the nuclear transport properties
of certain proteins. Experiments carried out in the yeast model system are
making important contributions to our understanding of nuclear targeting
signals.
2.2 Nuclear transport receptors
Targeting signals within cargo proteins are recognized by soluble

receptors that direct those cargoes to the nuclear pore for transport. These
receptors are a family of related molecules that are generally referred to as
either importins (for import receptors), exportins (for export receptors), or
generally as transport receptors or karyopherins [14, 35, 111]. The budding
yeast proteins are generally referred to as karyopherins, or Kaps, and are
coupled with their molecular weight to generate the standard yeast name
(e.g., Kap95 for the 95 kDa karyopherin). There are 14 of these functionally
and structurally related Kap proteins in S. cerevisiae (see Table 1) and 23
that have been identified thus far in humans [99]. In many cases, the cargoes
bind directly to these Kap proteins for import into the nucleus. However, an
additional adaptor is required to mediate recognition and binding of the
classical basic NLS [34]. This protein, often called importin α in higher
eukaryotes and Srp1p or Kap60p in S. cerevisiae, binds directly to the NLS
sequence within the cargo and then also interacts with a member of the
karyopherin family, generally referred to as importin β in higher eukaryotes
and Kap95p in S. cerevisiae [35, 82]. As described in more detail below, this
heterotrimeric complex is then imported into the nucleus. Thus, the
Kap60p/NLS cargo complex actually serves as a specialized cargo for
import by the karyopherin, Kap95p. Although the S. cerevisiae genome
encodes only a single Kap60 protein, there are at least six corresponding
importin α genes that have been identified in humans [62].
Table 1. Soluble nuclear transport factors

Common protein names Other names Function
(S. cerevisiae)
Ran GTPase Cycle
Ran (Gsp1p/Gsp2p) Cnr1p/Cnr2p (S. cerevisiae) Small GTPase that regulates

Cst17p (S. cerevisiae) cargo-receptor interactions
Spi1p (S. pombe)
Ran (metazoans)
RanGAP (Rna1p) Rna1p (S. pombe) GTPase activating protein for
Segregation distorter (D. Ran
melanogaster)
RanGAP1 (vertebrates)
RanGEF (Prp20p) Srm1p (S. cerevisiae) Guanine nucleotide exchange
Mtr1p (S. cerevisiae) factor for Ran
Pim1p (S. pombe)
BJ1 (D. melanogaster)
RCC1 (vertebrates)
Nuclear transport
receptors*
Importin/karyopherin α Srp1p (S. cerevisiae) NLS receptor

(Kap60p)
Importin/karyopherin 95 Rsl1p (S. cerevisiae) Complexes with Kap60p to
(Kap95p) import NLS cargo/import
receptor for ribosomal proteins
and other cargoes
CRM1 (Crm1p/Xpo1p) Kap124p (S. cerevisiae) NES receptor
Exportin 1 (vertebrates)
CAS (Cse1p) Kap109p (S. cervisiae) Kap60p recycling to the
cytoplasm
Transportin (Kap104p) hnRNP import
Kap121p Pse1p (S. cerevisiae) Ribosomal
protein/Pho4p/Spo12p import
Kap123p Yrb4p (S. cerevisiae) Ribosomal protein import
Kap108p Sxm1p (S. cerevisiae) Ribosomal protein import/La
protein import
Kap120p 60S ribosomal subunit export
Kap122p Pdr6p (S. cerevisiae) TFIIA import
Kap119p Nmd5p (S. cerevisiae) TFIIS transcription factor and
Hog1p import
Kap111p Mtr10p (S. cerevisiae) Npl3p import
Los1p Exportin-t (vertebrates) tRNA export
Kap114p HRC1004 (human) TATA-binding protein import
Kap142p Msn5p (S. cerevisiae) RP-A import and
Pho4p/Far1p/Mig1p/Ste5p
export
*There are a large number of importin/exportin/karyopherin receptors in yeast and

vertebrates. In some cases (as indicated) vertebrate homologues with the same apparent
function as the corresponding S. cerevisiae protein have been identified. However, in many
cases no obvious single functional vertebrate homologue corresponding to the S. cerevisiae
receptor has been assigned. For this reason the nuclear transport receptors listed focus on the
S. cerevisiae proteins where the family of receptors is most extensively characterized.
With the exception of the NLS adaptor protein, Kap60p, Kaps share a
common domain structure [34, 35]. Each has a conserved N-terminal domain
that mediates binding to the small GTPase Ran, which regulates cargo
binding to the Kap proteins as described in more detail in the following
section (2.3) on the Ran GTPase cycle. The central domain of the Kaps is an
FG binding domain that mediates interactions with the FG-repeat-containing
nucleoporins located in the nuclear pore. Finally, the least well understood
domain is the C-terminal domain, which mediates binding to the cargoes.
Some of the receptor/cargo interactions are starting to be defined at the
molecular level by structural studies of both yeast and vertebrate proteins.
The most structural information has been gathered on the heterodimeric
import receptor for classical NLS-containing cargoes. Structures of both
mouse and yeast importin α/Kap60p have provided important information
about how an NLS is recognized and how binding to the NLS cargo is
regulated [17, 61]. These studies also revealed that despite relatively low
sequence conservation between the yeast and vertebrate NLS receptor
proteins (~30% identity), the overall structure of the proteins is identical.
The structural basis for the interaction between vertebrate importin α and
importin β has also been defined [16]. In addition, crystallization of a
complex of vertebrate importin β with an FG-repeat peptide has revealed the
structural basis for the interaction of the transport receptor with the FG-
containing nucleoporins [5]. Although many of these structural studies have
examined vertebrate proteins, subsequent studies in yeast, which exploited
this structural information to generate rationally designed site-directed
mutants, strongly argue for functional conservation of these proteins
between yeast and higher eukaryotes [4, 82]. This lends confidence to the
assumption that studies of these nuclear transport proteins in the yeast model
system can provide important insight into their function in higher eukaryotes.
Alteration in several nuclear transport receptors have been implicated in
human cancer. The export receptor required for the recycling of the NLS
receptor, importin α, is a protein named CAS, which stands for cellular
apoptosis susceptibility [7]. The human CAS gene is amplified in several
transformed cell lines including breast, colon, and bladder cancer [12]. In
addition, its expression pattern and intracellular localization is altered in
70–90% of tumor cells in a population of invasive ductal and lobular breast

carcinomas [6]. The functional yeast homologue of CAS is a protein called
Cse1p, which was originally isolated in a screen for yeast mutants with
defects in chromosome segregation [113]. It is possible that CAS/Cse1p
serves as a critical regulator of growth control that when altered can cause
uncontrolled cell growth or cancer. It seems likely that other transport
receptors may also be implicated in tumorigenesis in the future. Consistent
with this notion, a recent study identified a translocation of a novel human
karyopherin, RanBP17, in acute lymphoblastic leukemia [39]. In addition, a
truncated form of the NLS receptor, importin α, has been identified in the
human breast cancer cell line, ZR-75-1 [59].
2.3 The Ran GTPase cycle
One of the major requirements for efficient transport of cargoes into and
out of the nucleus is that the system must have inherent directionality. For
example, in the case of nuclear protein import, the cargo protein must be
bound by the receptor in the cytoplasm, translocated into the nucleus, and
then dissociated from the receptor. There must be some mechanism to switch
the receptor from cargo pick up to cargo delivery. For most nuclear transport
processes, this switch is the Ran GTPase [22, 97, 107].
Work from a number of laboratories over the years has provided an
insight into how Ran imparts directionality on nuclear transport (For review
see [35]). The current model (Figure 1) relies on the compartmentalization of
RanGDP and RanGTP, where RanGDP is primarily cytoplasmic and RanGTP
is within the nucleus associated with chromatin [78]. The underlying
mechanism for this compartmentalization is the differential localization of
the proteins that regulate the GTPase activity of Ran. Like many other small
GTPases [70], isolated Ran hydrolyzes GTP very slowly. The rate of
hydrolysis is increased ~10,000-fold in the presence of the Ran GTPase
Activating Protein (RanGAP) [8], which is called Rna1p in S. cerevisiae [9].
The Ran Guanine nucleotide Exchange Factor (RanGEF) enhances the rate
of conversion of RanGDP to RanGTP [10]. The S. cerevisiae RanGEF is
Prp20p [30]. RanGAP is localized to the cytoplasm [46], which increases the
cytoplasmic pool of RanGDP. In contrast, RanGEF is enriched within the
nucleus where it is bound to chromatin at least in part through interactions
with histones [78, 79]. This enriches the nuclear pool of RanGTP.
Many of the studies that helped to elucidate the Ran cycle were carried
out in yeast. All proteins of the Ran cycle are highly conserved. For
example, the amino acid sequence of human Ran is 85% identical to that of
the S. cerevisiae Ran protein, Gsp1p. Human RanGEF, RanGAP, and NTF2
can all functionally replace their yeast counterparts [18, 30, 72]. This
conservation of function means that researchers can take advantage of the

numerous conditional alleles of these essential genes that have been
generated in yeast over the years [21]. These experimental tools have been
critical for the studies that have elucidated the mechanism of Ran action.
The mechanism by which the different nucleotide-bound states of Ran
regulate nuclear transport is illustrated in Figure 1 (Import and Export).
Import cargoes bind their karyopherin receptors in the cytoplasm. These
receptor/cargo complexes are imported into the nucleus where the complex
encounters RanGTP. RanGTP binds with high affinity to the N-terminus of
the karyopherin. This induces a conformational change that releases the
cargo into the nucleus. In contrast, export karyopherins bind their cargoes as
an obligate heterotrimeric complex that contains the receptor, the cargo, and
RanGTP. This heterotrimeric complex is exported to the cytoplasm where it
encounters RanGAP. Conversion of RanGTP to RanGDP in the presence of
RanGAP dissociates the export complex and delivers the cargo to the
cytoplasm. In both cases the receptors are recycled so that they can be used
for another round of transport. In addition it is necessary to replenish the
nuclear pools of RanGTP, which would be depleted by ongoing nuclear
export. This is accomplished by the small RanGDP-binding protein, NTF2,
which binds RanGDP in the cytoplasm and imports it into the nucleus where
it can be converted to RanGTP [84, 95]. How NTF2 is then recycled to the
cytoplasm has not yet been determined.
Much recent work on the Ran GTPase has revealed a more general role
for RanGTP in marking the position of the chromatin within the cell and
hence the position of the nucleus [107]. This more general function underlies
the well-characterized role of RanGTP in regulating import and export cargo
binding to the transport receptors. In order for import complexes to dissociate
in the proper cellular compartment, there must be a signal that they have
reached the nucleus. This signal is the contact with RanGTP, which is con-
centrated at the chromatin due to the chromatin association of the exchange
factor [78]. For nuclear protein export, the converse is true. Export complexes
are formed only in the presence of RanGTP, in the vicinity of chromatin, and
then disassembled in the cytoplasm where the RanGTP is hydrolyzed by the
action of the cytoplasmic RanGAP. The realization that RanGTP serves as a
general marker for the nuclear compartment and specifically the chromatin
explains several recent observations about additional roles for Ran. Work
from a number of laboratories suggests that RanGTP is required for mitotic
spindle function during cell division [55]. While this finding could suggest
that RanGTP binds directly to a component of the spindle apparatus, more
recent experiments suggest that RanGTP is required to release proteins that
are required for spindle function from the importin β family of transport
receptors [38, 75, 109]. A similar role for RanGTP in mediating nuclear
envelope assembly has also been identified both in vertebrates and in yeast
[88, 106, 122]. In this case, the RanGTP likely releases nuclear pore
proteins, which are required for the association and assembly of early NPCs
with the chromatin. In all three cases, nuclear transport, mitotic spindle
assembly, and nuclear envelope assembly, RanGTP serves to dissociate
proteins from the transport receptor in the vicinity of chromatin. This
concentrates proteins at the chromatin or within the nucleus where they
function. Thus, although RanGTP was originally identified as a critical
regulator of nuclear transport, this role is probably only one aspect of Ran
function.
Ran GTPase Cycle

cytoplasm nucleus
Ran
GDP
Ran
GDP
Ran Ran
GDP GDP
Ran
GEF
Ran
GAP
Ran Ran
GTP GTP
Ran
GTP
Ran
GTP
Import
Cargo
Cargo
Ran β β Ran
GDP Ran GTP
Ran GTP Ran
GDP GTP
Ran
GDP Cargo Ran
GTP
Cargo
Export β β
Ran
Ran GTP
GDP
Figure 1. The Ran GTPase cycle regulates the directionality of nuclear protein transport. The
RanGAP protein is localized to the cytoplasm, which leads to a high level of cytoplasmic
RanGDP. The RanGEF is localized to the nucleus, which leads to a high level of nuclear
RanGTP. This asymmetric distribution of RanGTP and RanGDP regulates the assembly of
receptor cargo complexes. For Import, the transport receptor (β) binds to the import cargo in
the cytoplasm. This complex is translocated through the nuclear pore into the nucleus. In the
nucleus RanGTP binds to the transport receptor, which causes a conformational change that
releases the cargo. For Export, the export receptor (β) binds to the export cargo in the nucleus
in an obligate trimeric complex with RanGTP. The trimeric complex is translocated through
the nuclear pore into the cytoplasm. In the cytoplasm, the complex encounters the RanGAP,
which hydrolyzes the RanGTP to RanGDP and dissociates the complex to release the cargo
into the cytoplasm.
Given the critical role of Ran in nuclear transport as well as other

important cellular processes, it may not be surprising that no mutations in the
Ran cycle components have yet been linked directly to human cancer. It
seems likely that the functions of these proteins are so critical to cellular
viability that mutations are incompatible with life. However, as we learn
more about the regulatory mechanisms, we may uncover changes in

regulators of the Ran cycle that do contribute to human disease.
2.4 Classical nuclear protein import/export
cytoplasm nucleus
Cargo Cargo Cargo

NLS NLS (4) NLS Cargo
α
NLS
(1)
Cargo (2) α
β NLS
β β
α (3) Ran
β GTP
α α
Ran α
α GAP α α Cse1
Cse1 Cse1 Cse1
Cse1 Ran Ran Ran
GTP (5) GTP GTP
Ran
GDP
Figure 2. The nuclear import pathway for cargoes that contain a classical NLS is the best
characterized nuclear transport pathway. This pathway can be dissected into at least five
distinct steps: (1) recognition and binding of the NLS cargo to the Kap60p(α)/Kap95p(β)
heterodimeric import receptor in the cytoplasm; (2) targeting to the nuclear pore through
interactions between Kap95p and the nuclear pore; (3) translocation through the pore
through transient interactions between Kap95p and the FG-repeat containing nucleoporins;
(4) delivery into the nucleus when RanGTP binds to Kap95p to cause a conformational
change that releases the Kap60p/NLS cargo; and (5) recycling of Kap60p to the cytoplasm in
a heterotrimeric complex with the export receptor, Cse1p, and RanGTP.
The best understood nuclear transport process is the import of protein

cargoes that contain a classical basic NLS (Figure 2). Thus, this process can
be used most readily to illustrate the steps that occur when a transport cargo
is moved into or out of the nucleus. Historically nuclear protein import was
divided into two steps, docking at the nuclear pore, an energy independent
step, and translocation into the nucleus, an energy-dependent step. Advances
in our understanding of the process and the players now lead us to define at
least five distinct steps for import of an S. cerevisiae cargo that contains a
classical NLS: (1) recognition and binding of the NLS cargo to the
Kap60p(α)/Kap95p(β) heterodimeric import receptor in the cytoplasm; (2)
targeting to the nuclear pore through interactions between Kap95p and the
nuclear pore; (3) translocation through the pore via transient interactions
between Kap95p and the FG-repeat containing nucleoporins; (4) delivery
into the nucleus where RanGTP binds to Kap95p to cause a conformational
change that releases the Kap60p/NLS cargo; and (5) recycling of Kap60p to
the cytoplasm in a heterotrimeric complex with Cse1p and RanGTP, and
Kap95 presumably in complex with RanGTP. Note that the only energy
expenditure in this process occurs when the karyopherin proteins are
recycled to the cytoplasm and the accompanying RanGTP is hydrolyzed.
Export of cargoes from the nucleus is very similar to the import process
except that the export complex forms in the presence of RanGTP (in the
nucleus) and is dissociated upon GTP hydrolysis (in the cytoplasm). Thus far
all export mechanisms seem to involve the direct recognition of the cargo
protein by the export Kap protein, but the molecular details of the
recognition have not been elucidated for the NES export receptor/cargo
complex. The export of cargoes that contain a leucine rich NES, which is
recognized by the export Kap, Crm1p/Xpo1p (CRM1 in vertebrates), serves
as a paradigm for transport receptor mediated nuclear export. The steps
involved in NES export are: (1) recognition and binding of the NES cargo in
a trimeric complex with Crm1p/Xpo1p; (2) targeting to the nuclear pore
through interactions between Crm1p/Xpo1p and the FG nucleoporins; (3)
translocation through the pore; (4) delivery of the cargo upon RanGTP
hydrolysis in the cytoplasm; and (5) recycling of the export receptor and
RanGDP to the nucleus. This cycle of export is virtually identical to the
recycling of Kap60p(α) by Cse1p that is illustrated in Figure 2. This
demonstrates the conservation of mechanism in the nuclear transport
process.
3 RNA EXPORT
Multiple classes of RNAs, including mRNAs, tRNAs, and U snRNAs, are

transcribed and processed within the nucleus and then transported to their
sites of action in the cytoplasm. Competition experiments have shown that
the export mechanisms of different classes of RNA are distinct from one
another [67, 76]. A great deal of information has emerged regarding factors
that are essential for RNA export [76], but like protein trafficking, the exact
mechanism of translocation through the NPC remains elusive. As compared
with proteins, RNAs require extensive processing before they reach their
mature form and are ready to exit the nucleus, and so it has sometimes been
difficult to separate activities required for RNA processing (such as splicing)
from bona fide mediators of export.
3.1 Export of RNA via classical nuclear transport

receptors
The export of tRNA, U snRNA, and rRNA follows pathways analogous to

nuclear protein export. For example, tRNA is recognized directly by the
importin-β family carrier exportin-t/Los1p [1, 42, 67, 90] and is exported as
a complex with RanGTP, which is disassembled in the cytoplasm when
RanGTP is hydrolyzed to RanGDP. Preferential export of mature tRNAs
seems to be achieved at least in part by the specificity of exportin-t for the
mature processed, modified, and appropriately aminoacylated tRNAs [94]. U
snRNAs are synthesized in the nucleus, transported to the cytoplasm where
they associate with protein components of mature snRNPs, and are then
reimported to the nucleus where they function in mRNA splicing [50]. The
monomethylated G cap of the initial export substrate is recognized by the
cap-binding complex (CBC) [50] and then exported from the nucleus in a
CRM1-dependent manner [31]. As there is no evidence that the CBC binds
directly to CRM1, it seems likely that an adaptor protein mediates this
interaction. rRNAs are exported in the context of large assembled RNP
complexes. Although export depends on Ran [48, 74], it is controversial
whether Ran plays a direct role in export or whether instead its activity may
be essential for the import of components required for RNP assembly [87].
3.2 mRNA export
Export of poly (A)+ RNA remains the least well understood of the RNA
export mechanisms. mRNAs are not exported to the cytoplasm as naked
nucleic acids, but rather as ribonucleoprotein complexes and it is generally
agreed that the export machinery recognizes signals within the proteins of
these complexes rather than the RNA itself [49, 77]. For example, export of
unspliced HIV transcripts from the nucleus is mediated by Rev through its
export by the NES receptor [20]. While this mechanism is exploited by HIV,
it seems that none of the karyopherin receptors, or even Ran itself, play a
central role in mRNA export.
Although the mechanistic details of mRNA export have not yet been fully
elucidated, it appears that there are two classes of proteins that are required
to achieve export of mature messages. First, there is a family of evolutionarily
conserved heterogeneous nuclear ribonucleoproteins (hnRNPs) that interact
with poly (A)+ RNA in vivo [25]. A number of these hnRNP proteins shuttle
between the cytoplasm and the nucleus and escort the poly (A)+ RNAs as
they are exported through the NPC [76, 93]. Current models suggest that at
least some of the hnRNP proteins may be involved in RNA processing steps
that occur co-transcriptionally. The hnRNPs that remain bound to the
maturing messages may serve as markers that the different processing steps
have been successfully accomplished. The second class of proteins consists
of those proteins that have been implicated more directly in the export
process, including the helicase, Sub2p (UAP56 in humans), and the
heterodimeric export receptor, Mex67p/Mtr2p (TAP/p15 in humans)
[20, 58, 92]. TAP was originally identified as a factor necessary for
the export of simian type D viral RNAs that contain a CTE (constitutive
transport element). Subsequent experiments showed that TAP/Mex67p is
also required for the export of endogenous mRNAs [57, 58] and that
mutations in the yeast MEX67 gene cause a rapid onset defect in the export
of poly (A)+ RNA [92]. TAP/Mex67p shuttles between the cytoplasm and
the nucleus [58], and in complex with p15/Mtr2p, binds both to mRNA and
to nucleoporins [2]. Thus, it could potentially target bound RNAs directly to
the NPC for export. Recent work has led to the identification of the TREX
(TRanscription/EXport) complex, which links mRNA transcription and
export from the nucleus [83]. This evolutionarily conserved complex
contains both factors required for transcription of mRNA and factors that
will be required for export of the mature message. A great deal of effort is
currently focused on understanding how the hnRNP proteins, the export
factors, and other accessory proteins cooperate to export mature mRNA from
the nucleus.
There are a number of studies that demonstrate increased expression of
several different hnRNP proteins in tumor cells as compared to normal
control cells. For example, Snead et al. [96] found that hnRNP B1 was
overexpressed in 84% of malignant tumors in lung disease as compared to
only 18% of benign tumors. This and other related studies have led to the
suggestion that hnRNP expression could be a useful marker for the early
detection of specific tumors [112].
4 REGULATION THROUGH NUCLEAR

TRANSPORT
We are only just beginning to reveal the role that nucleocytoplasmic

transport plays in cancer cell transformation and studies in S. cerevisiae and
S. pombe have been critical in advancing our knowledge. Studies of the cell
cycle regulatory proteins cyclin B1 (Clb2p in S. cerevisiae) and Cdc25C
(cdc25 in S. pombe), as well as the yeast transcription factor, Pho4p, have
helped to delineate the mechanisms whereby these growth regulatory
proteins traffic between the cytoplasm and the nucleus [37, 116]. Here we
discuss advances made using yeast, as well as other model systems to
understand the nuclear translocation of cyclin B1/Clb2p, Cdc25p, and
Pho4p. Specifically we will review how the cell has utilized this transport to
regulate the functions of these proteins and thereby the timing and accuracy
of cellular processes.
4.1 Cyclin B/Clb2p
Cyclin was originally identified as a protein that gradually increases in

concentration during the cell cycle and then is degraded at the onset of each
cellular division [26]. A family of proteins known as the cyclins has since
been identified, with homologues in all eukaryotes, that all contain a
conserved protein kinase-binding site called the cyclin box. These proteins
function as regulatory subunits of these kinases, known as cyclin-dependent
kinases [24]. Studies in yeast showed that these cyclin/cyclin-dependent
kinase complexes regulate the timing of cell cycle progression. Furthermore,
their regulated activity is necessary for spindle disassembly, cytokinesis, and
G1 transition [101].
Mitosis is triggered by the activity of the cyclin/cyclin-dependent kinase
complex in eukaryotes. To ensure that mitosis is not entered prematurely, the
activity of this complex is regulated through the steady state cytoplasmic
compartmentalization of the kinase and cyclin subunits [104], inactivating
phosphorylation of the complex subunits, as well as degradation of the
cyclin subunit, which occurs through the ubiquitin-mediated pathway [33].
These control mechanisms ensure that the cell does not prematurely enter the
next phase of the cell cycle.
At the beginning of S-phase, cyclin B/Clb2p begins to accumulate within
the cytoplasm of cells, but as the cell cycle progresses into prophase, cyclin
B/Clb2p is relocalized to the nucleus [81]. As shown in Figure 3 (Cyclin B
and Cdc25) this nuclear localization is due to import through the classical
import pathway of Kap60p(α) and Kap95p(β) in yeast [45] or through a
direct interaction with importin β in humans [73]. The protein is retained
within the nucleus because of the inability of the export receptor, CRM1, to
bind to the NES of a phosphorylated form of cyclin B/Clb2p [114]. Once
cyclin B/Clb2p is localized within the nucleus, cyclin-dependent kinase 1
(Cdc2 in mammalian cells/Cdc28p in S. cerevisiae) is activated to promote
mitosis. Studies using S. cerevisiae have shown that as the APC/cyclosome,
a ubiquitin ligase activity containing complex [60], regains activity during
mitosis, cyclin B/Clb2p is targeted for destruction [3, 117] and this
destruction is dependent on CRM1-mediated export [120] to the cytoplasmic
26S proteasome, allowing for transition into G1 of the next cell cycle. Thus,
the cell utilizes the intrinsic nuclear localization and export sequences
contained within cyclin B/Clb2p, in addition to the transport machinery, to
modulate the activity of the Cdc2–cyclin B complex.
Cyclin B and Cdc25 cytoplasm nucleus

14-3-3 NLS
α β
P P
P
NLS Cdc25 Ran
P P
NLS GTP
Cdc25
α
P
Cdc25
α
P
β
14-3-3
β Cdc2 Ran
GTP NLS
P P
NLS
P P
Cdc25 Cyclin B Cdc25

NLS
P
P
P P Cdc2
P
Cdc2 Cyclin B
α
Cyclin B Dephos- NLS β Cdc2
α P
NLS phorylation Ran Cyclin B
GTP NLS
β
P
Dephos-
CRM1 phorylation
Cdc2 Cdc2
Cyclin B CR Cyclin B CR
M
NES
S
S
M
NES
1 Cdc25 Cdc25
NE
NE
CRM1 CRM1 1
Pho4 P P Pho4
Pse1
transcription
low Pho4
Pho4 Pse1 Ran
P
Pho4
P
GTP
P phosphate Pse1 Ran
GTP
high Pho85
Ran Ran
GTP GTP phosphate Pho80
Msn5 Msn5
Msn5 P P P P P P
Ran Pho4 Pho4
P
Pho4
P
P
P
GDP P P
P
Figure 3. Cyclin B. Mitotic progression through the cell cycle depends on translocation of the
Cdc2–Cyclin B complex into the nucleus. The dimeric complex is dephosphorylated on
Cdc2p by Cdc25p, allowing access to the import–receptor complex consisting of importin
α/β. The quaternary complex translocates through the nuclear pore and dissociates upon
RanGTP binding to β in the nucleus. The Cdc2–Cyclin B complex is retained in the nucleus
by inhibitory phosphorylation of Cyclin B. At the end of mitosis, the complex is
dephosphorylated, allowing CRM1 to bind to the NES of Cyclin B. Upon binding, the
complex is exported from the nucleus and targeted for destruction by the 26S proteasome.
Cdc25. The 14-3-3 protein binds to and holds Cdc25p in an inactive state in the cytoplasm
during interphase by blocking access to its intrinsic NLS. Upon activating phosphorylation of
Cdc25p at prophase, importin α/β binds to the Cdc25p NLS and the trimeric complex is
translocated into the nucleus. The complex is dissociated by binding of RanGTP to β. Cdc25p
loses activity upon dephosphorylation, exposing its intrinsic NES, which is then bound by
CRM1. The dimeric complex is then translocated through the nuclear pore into the cytoplasm
where the complex is dissociated. Pho4. Under low phosphate conditions, the activity of the
Pho80p/Pho85p kinase complex is downregulated, leading to an underphosphorylated form of
Pho4p. Dephosphorylation reveals the NLS of Pho4p and allows binding by its import
receptor Pse1p. The dimeric complex is translocated into the nucleus where it is dissociated
by RanGTP binding to Pse1p. After Pho4p activates transcription of the phosphate responsive
genes, Pho4p becomes hyperphosphorylated, creating a binding site for its export receptor
Msn5p to form a trimeric complex with RanGTP. Following binding by Msn5p, Pho4p is
rapidly recycled to the cytoplasm where the trimeric complex is dissociated by hydrolysis of
RanGTP to RanGDP.
Cyclin B1 misexpression has been found in numerous cancers ranging

from prostate carcinomas [29] to esophageal squamous cell carcinomas
[103]. In normal esophageal squamous cells, cyclin B1 is localized in the
nucleus and expressed in only a small fraction of cells. However, in eso-
phageal squamous cell carcinomas, cyclin B1 is localized to the cytoplasm
and expressed in a large number of cells [103]. This mislocalization of cyclin
B1 has also been seen in other solid tumors as well as lymphomas and
leukemias [119]. These observations suggest that the proper localization of
cyclin B1 may be important in the maintenance of normal cell growth and
the prevention of tumor formation.
4.2 Cdc25
Evidence from S. pombe has provided much of the foundation for the
regulation of mitotic entry in eukaryotic cells (Figure 3). This entry is
triggered by the cyclin-dependent kinase, Cdc2/Cdc28p. As discussed above,
the activity of Cdc2/Cdc28p is dependent upon interaction with its regula-
tory subunit, cyclin B/Clb2p. Prior to mitosis, Cdc2–cyclin B complexes are
held in an inactive state by phosphorylation of Cdc2/Cdc28p. The cell
division cycle 25C protein phosphatase, Cdc25C/Cdc25p, acts on the Cdc2–
cyclin B complex to dephosphorylate Cdc2/Cdc28p, thus activating the
Cdc2–cyclin B complex to promote mitosis. In order to prevent early
activation, the cell must regulate the activity of Cdc25C/Cdc25p. This is
accomplished by regulating the nuclear transport of Cdc25C/Cdc25p.
Studies in both mammalian cells and fission yeast have shown that
Cdc25C/Cdc25p shuttles between the nucleus and cytoplasm throughout
the cell cycle, but has a steady state cytoplasmic localization during inter-
phase and a nuclear localization during prophase [41, 69]. Cdc25C/Cdc25p
contains both a classical NLS and an NES and studies using Xenopus and
S. pombe have shown that Cdc25C/Cdc25p is imported through interactions
with importin-α/Kap60p [65, 115] and importin-β/Kap95p [15]. Posttransla-
tional modifications near these targeting sequences control the steady state
localization of the protein by interfering with the recognition of these signals
by the receptors.
During interphase in both Xenopus [66] and S. pombe, [121] phos-
phorylation of Cdc25C/Cdc25p near its intrinsic NLS creates a binding
site for the phosphoserine-binding protein, 14-3-3. The 14-3-3 protein binds
to Cdc25C/Cdc25p and inhibits nuclear import probably by sterically
hindering the access of importin α to the NLS. This causes a redistribution
of Cdc25C/Cdc25p from the nucleus to the cytoplasm. This model is
supported by experiments from fission yeast, which show that in the absence
of the 14-3-3 protein, Rad24p, Cdc25C/Cdc25p is predominantly nuclear
[69]. Thus, 14-3-3 proteins regulate Cdc25C/Cdc25p function by inhibiting

its nuclear import.
In addition to 14-3-3 inhibition of nuclear import, Cdc25C/Cdc25p
contains a leucine-rich NES that is recognized by the export factor CRM1
[31, 69]. Experiments in both S. pombe and human cells show that mutation
or inhibition of CRM1 leads to nuclear accumulation of Cdc25C/Cdc25p
[36, 69]. This suggests that Cdc25C/Cdc25p is actively exported from the
nucleus throughout interphase and that this export is CRM1-dependent.
Cdc25C is involved in the formation of acute myeloid leukemia [47] and
cervical adenocarcinoma [37], and is the target of a number of anticancer
agents [37, 118]. Treatment of HeLa cells with the anticancer drug, UCN-01,
directly perturbs the normal cytoplasmic localization of Cdc25C causing a
nuclear accumulation [37]. In combination with DNA damage, this nuclear
accumulation of Cdc25C could potentially drive the cells to enter mitosis
prematurely by activating the Cdc2-cyclin B complex and bypassing the
normal G2/M checkpoint, leading to apoptosis. Thus, understanding the
regulation of Cdc25C nucleocytoplasmic transport may provide new
potential therapeutic targets for the treatments of leukemia and cervical
cancer.
4.3 Pho4p
Cellular response to environmental signaling allows for adaptation to

changes in growth conditions. This generally occurs through alterations in
gene expression by a signal-cascade event originating in the plasma
membrane, progressing through the cytoplasm, and ending with upregulation
of gene expression in the nucleus. Cells can be primed for rapid response to
extracellular signals by constitutively synthesizing the protein effectors
while at the same time inhibiting their function. These effectors are generally
transcription factors and one method for inhibiting their function is the
retention of these molecules in the cytoplasm until the signal needs to be
transduced into the nucleus.
Cellular signaling can be accomplished through phosphorylation of
the transcription factor to either activate or inactivate the protein. This
phosphorylation can occur through complexes such as cyclin–CDK. A
paradigm for signal transduction in S. cerevisiae is the cyclin–CDK
complex, Pho80p–Pho85p (Figure 3). This complex recognizes phosphate
depletion from the environment and transduces its signal through the
phosphorylation of the transcriptional activator Pho4p [52]. Under con-
ditions where phosphate levels are low, the Pho80p–Pho85p complex
activity is downregulated [91]. This results in the accumulation of an
unphosphorylated form of Pho4p [52] that is translocated into the nucleus by
its import receptor, Pse1p [54], a Kap protein that preferentially associates
with the unphosphorylated form of Pho4p [54]. Pho4p then activates the
transcription of the phosphate-responsive genes. When phosphate levels rise,
the Pho80p–Pho85p complex regains activity and hyper-phosphorylates
Pho4p. This phosphorylation causes Pho4p to be rapidly recycled to the
cytoplasm through its export receptor, Msn5p [53], an importin β family
member that specifically recognizes the phosphorylated form of Pho4p [53],
thus repressing the transcription of the phosphate-responsive genes.
The Pho80p–Pho85p complex phosphorylates Pho4p on multiple serine
resides [64]. Phosphorylation of Pho4p on two sites is necessary and
sufficient to cause nuclear export by Msn5p [64]. Additional phospho-
rylation of Pho4p on a third residue within the nuclear localization signal
inhibits its interaction with the import receptor, Pse1p [64]. Both of these
phosphorylation events serve to ensure that Pho4p remains cytoplasmic
under conditions of high phosphate levels. Thus, phosphorylation of these
residues serves to regulate the nucleocytoplasmic transport of Pho4p. By
examining nuclear transport of Pho4p, we can better understand how the
activity of transcription factors is regulated in response to environmental
signals.
5 CONCLUSIONS/IMPLICATIONS
FOR THE FUTURE
Obviously the use of yeast as a model system to study the nuclear

transport process has been extremely valuable in the past. The prediction is
that future studies of this highly conserved process in model organisms
including yeast will provide further insights. The goal is to define the
mechanisms that regulate nuclear transport of critical growth regulatory
molecules. These transport mechanisms could then be specifically targeted
for novel therapeutic regimes.
ACKNOWLEDGMENTS
We would like to acknowledge the members of the Corbett laboratory for

helpful discussions and comments. We thank Dr. Deanna Green for careful
reading of the manuscript. This work was supported by grants from the NIH
to AHC and ACB.
REFERENCES
1. Arts, G. J., M. Fornerod, and I. W. Mattaj. 1998. Identification of a nuclear export
receptor for tRNA. Curr. Biol. 12:305–314.
2. Bachi, A., I. C. Braun, J. P. Rodrigues, N. Pante, K. Ribbeck, C. von Kobbe,
U. Kutay, M. Wilm, D. Görlich, M. Carmo-Fonseca, and E. Izaurralde. 2000. The
C-terminal domain of TAP interacts with the nuclear pore complex and promotes export
of specific CTE-bearing RNA substrates. Rna 6:136–158.
3. Baumer, M., G. H. Braus, and S. Irniger. 2000. Two different modes of cyclin clb2
proteolysis during mitosis in Saccharomyces cerevisiae. FEBS Lett. 468:142–148.
4. Bayliss, R., S. W. Leung, R. P. Baker, B. B. Quimby, A. H. Corbett, and
M. Stewart. 2002. Structural basis for the interaction between NTF2 and nucleoporin
FxFG repeats. EMBO J. 21:2843–2853.
5. Bayliss, R., T. Littlewood, and M. Stewart. 2000. Structural basis for the interaction
between FxFG nucleoporin repeats and importin-beta in nuclear trafficking. Cell
102:99–108.
6. Behrens, P., U. Brinkmann, F. Fogt, N. Wernert, and A. Wellmann. 2001.
Implication of the proliferation and apoptosis associated CSE1L/CAS gene for breast
cancer development. Anticancer Res. 21:2413–2417.
7. Behrens, P., U. Brinkmann, and A. Wellmann. 2003. CSE1L/CAS: its role in
proliferation and apoptosis. Apoptosis 8:39–44.
8. Bischoff, F. R., C. Klebe, J. Kretschmer, A. Wittinghofer, and H. Ponstingl. 1994.
RanGAP1 induces GTPase activity of nuclear Ras-related Ran. Proc. Natl. Acad. Sci.
USA 91:2587–2591.
9. Bischoff, F. R., H. Krebber, T. Kempf, I. Hermes, and H. Ponstingl. 1995. Human
RanGTPase activating protein RanGap1 is a homologue of yeast Rna1p involved in
mRNA processing and transport. Proc. Natl. Acad. Sci. USA 92:1749–1753.
10. Bischoff, F. R., and H. Ponstingl. 1991. Catalysis of guanine nucleotide exchange on
Ran by the mitotic regulator RCC1. Nature 354:80–82.
11. Bogerd, H. P., R. A. Fridell, R. E. Benson, J. Hua, and B. R. Cullen. 1996. Protein
sequence requirements for function of the human T-cell leukemia virus type 1 Rex
nuclear export signal delineated by a novel in vivo randomization selection assay. Mol.
Cell. Biol. 16:4207–4214.
12. Brinkmann, U., E. Brinkmann, M. Gallo, U. Scherf, and I. Pastan. 1996. Role of
CAS, a human homologue to the yeast chromosome segregatin gene CSE1, in toxin and
tumor necrosis factor mediated apoptosis. Biochem. 35:6891–6899.
13. Chida, K., and P. K. Vogt. 1992. Nuclear translocation of viral Jun but not of cellular
Jun is cell cycle dependent. Proc. Natl. Acad. Sci. USA 89:4290–4294.
14. Chook, Y. M., and G. Blobel. 2001. Karyopherins and nuclear import. Curr. Opin.
Struct. Biol. 11:703–715.
15. Chua, G., C. Lingner, C. Frazer, and P. G. Young. 2002. The sal3(+) gene encodes
an importin-beta implicated in the nuclear import of Cdc25 in Schizosaccharomyces
pombe. Genetics 162:689–703.
16. Cingolani, G., C. Petosa, K. Weis, and C. W. Müller. 1999. Structure of importin-b
bound to the IBB domain of importin-a. Nature 399:221–229.
17. Conti, E., M. Uy, L. Leighton, G. Blobel, and J. Kuriyan. 1998. Crystallographic
analysis of the recognition of a nuclear localization signal by the nuclear import factor
karyopherin alpha. Cell 94:193–204.
18. Corbett, A. H., and P. A. Silver. 1996. The NTF2 gene encodes an essential, highly
conserved protein that functions in nuclear transport in vivo. J. Biol. Chem. 271:18477–
18484.
19. Cronshaw, J. M., A. N. Krutchinsky, W. Zhang, B. T. Chait, and M. J. Matunis.

2002. Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol.
158:915–927.
20. Cullen, B. R. 2000. Connections between the processing and nuclear export of mRNA:
evidence for an export license? Proc. Natl. Acad. Sci. USA 97:4–6.
21. Damelin, M., P. A. Silver, and A. H. Corbett. 2002. Nuclear protein transport. Meth.
Enzymol. 351:587–607.
22. Dasso, M. 2002. The Ran GTPase: theme and variations. Curr. Biol. 12:R502–508.
23. Dean, M., M. Park, and G. F. Vande Woude. 1987. Characterization of the rearranged
tpr-met oncogene breakpoint. Mol. Cell. Biol. 7:921–924.
24. Doree, M., and T. Hunt. 2002. From Cdc2 to Cdk1: when did the cell cycle kinase join
its cyclin partner? J. Cell Sci. 115:2461–2464.
25. Dreyfuss, G., M. J. Matunis, S. Pinol-Roma, and C. G. Burd. 1993. hnRNP proteins
and the biogenesis of mRNA. Annu. Rev. Biochem. 62:289–321.
26. Evans, T., E. T. Rosenthal, J. Youngblom, D. Distel, and T. Hunt. 1983. Cyclin: a
protein specified by maternal mRNA in sea urchin eggs that is destroyed at each
cleavage division. Cell 33:389–396.
27. Fabbro, M., and B. R. Henderson. 2003. Regulation of tumor suppressors by nuclear-
cytoplasmic shuttling. Exp. Cell Res. 282:59–69.
28. Fahrenkrog, B., and U. Aebi. 2002. The vertebrate nuclear pore complex: from
structure to function. Results Probl. Cell Differ. 35:25–48.
29. Farhana, L., M. Dawson, A. K. Rishi, Y. Zhang, E. Van Buren, C. Trivedi,
U. Reichert, G. Fang, M. W. Kirschner, and J. A. Fontana. 2002. Cyclin B and E2F-
1 expression in prostate carcinoma cells treated with the novel retinoid CD437 are
regulated by the ubiquitin-mediated pathway. Cancer Res. 62:3842–3849.
30. Fleischmann, M., M. W. Clark, W. Forrester, M. Wickens, T. Nishimoto, and
M. Aebi. 1991. Analysis of yeast prp20 mutations and functional complementation by
the human homolgue RCC1, a protein involved in the control of chromosome
condensation. Mol. Gen. Genet. 227:417–423.
31. Fornerod, M., M. Ohno, M. Yoshida, and I. W. Mattaj. 1997. CRM1 is an export
receptor for leucine-rich nuclear export signals. Cell 90:1051–1060.
32. Gerace, L. 1995. Nuclear export signals and the fast track to the cytoplasm. Cell
82:341–344.
33. Glotzer, M., A. W. Murray, and M. W. Kirschner. 1991. Cyclin is degraded by the
ubiquitin pathway. Nature 349:132–138.
34. Gorlich, D., S. Kostka, R. Kraft, C. Dingwall, R. A. Laskey, E. Hartmann, and
S. Prehn. 1995. Two different subunits of importin cooperate to recognize nuclear
localization signals and bind them to the nuclear envelope. Curr. Biol. 5:383–392.
35. Görlich, D., and U. Kutay. 1999. Transport between the cell nucleus and the
cytoplasm. Annu. Rev. Cell Dev. Biol. 15:607–660.
36. Graves, P. R., C. M. Lovly, G. L. Uy, and H. Piwnica-Worms. 2001. Localization of
human Cdc25C is regulated both by nuclear export and 14-3-3 protein binding.
Oncogene 20:1839–1851.
37. Graves, P. R., L. Yu, J. K. Schwarz, J. Gales, E. A. Sausville, P. M. O’Connor, and
H. Piwnica-Worms. 2000. The Chk1 protein kinase and the Cdc25C regulatory
pathways are targets of the anticancer agent UCN-01. J. Biol. Chem. 275:5600–5605.
38. Gruss, O. J., R. E. Carazo-Salas, C. A. Schatz, G. Guarguaglini, J. Kast, M. Wilm,
N. Le Bot, I. Vernos, E. Karsenti, and I. W. Mattaj. 2001. Ran induces spindle
assembly by reversing the inhibitory effect of importin alpha on TPX2 activity. Cell
104:83–93.
39. Hansen-Hagge, T. E., M. Schafer, H. Kiyoi, S. W. Morris, J. A. Whitlock, P. Koch,
I. Bohlmann, C. Mahotka, C. R. Bartram, and J. W. Janssen. 2002. Disruption of
the RanBP17/Hox11L2 region by recombination with the TCRdelta locus in acute

lymphoblastic leukemias with t(5;14)(q34;q11). Leukemia 16:2205–2212.
40. Harreman, M. T., T. M. Kline, M. R. Hodel, A. E. Hodel, and A. H. Corbett. 2004.
Regularion of nuclear import by phosphorylation adjacent to nuclear localization
signals. J. Biol. Chem. 279:20613–20621.
41. Heald, R., M. McLoughlin, and F. McKeon. 1993. Human wee1 maintains mitotic
timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase. Cell
74:463–474.
42. Hellmuth, K., D. M. Lau, F. R. Bischoff, M. Kunzler, E. Hurt, and G. Simos. 1998.
Yeast Los1p has properties of an exportin-like nucleocytoplasmic transport factor for
tRNA. Mol. Cell. Biol. 18:6374–6386.
43. Hodel, A. E., M. T. Harreman, M. E. Harben, J. Holmes, M. R. Hodel, Q. Shen, K.
Berland, and A. H. Corbett. 2005. Correlation between nuclear localization signal-
receptor affinity and in vivo localization. In preparation.
44. Hodel, M. R., A. H. Corbett, and A. E. Hodel. 2001. Dissection of a nuclear
localization signal. J. Biol. Chem. 276:1317–1325.
45. Hood, J. K., W. W. Hwang, and P. A. Silver. 2001. The Saccharomyces cerevisiae
cyclin Clb2p is targeted to multiple subcellular locations by cis- and trans-acting
determinants. J. Cell Sci. 114:589–97.
46. Hopper, A. K., H. M. Traglia, and R. W. Dunst. 1990. The yeast RNA1 gene product
necessary for RNA processing is located in the cytosol and apparently excluded from the
nucleus. J. Cell Biol. 111:309–321.
47. Horrigan, S. K., Z. H. Arbieva, H. Y. Xie, J. Kravarusic, N. C. Fulton, H. Naik,
T. T. Le, and C. A. Westbrook. 2000. Delineation of a minimal interval and
identification of 9 candidates for a tumor suppressor gene in malignant myeloid
disorders on 5q31. Blood 95:2372–2377.
48. Hurt, E., S. Hannus, B. Schmelzl, D. Lau, D. Tollervey, and G. Simos. 1999. A novel
in vivo assay reveals inhibition of ribosomal nuclear export in ran-cycle and nucleoporin
mutants. J. Cell Biol. 144:389–401.
49. Izaurralde, E. 2002. Nuclear export of messenger RNA. Results Probl. Cell Differ.
35:133–150.
50. Izaurralde, E., J. Lewis, C. Gamberi, A. Jarmalowski, C. McGuigan, and I. W.
Mattaj. 1995. A cap-binding protein complex mediating U snRNA export. Nature
376:709–712.
51. Jans, D. A., and S. Hubner. 1996. Regulation of protein transport to the nucleus:
central role of phosphorylation. Physiol. Rev. 76:651–685.
52. Kaffman, A., I. Herskowitz, R. Tjian, and E. K. O’Shea. 1994. Phosphorylation of
the transcription factor PHO4 by a cyclin-CDK complex, PHO80-PHO85. Science
263:1153–1156.
53. Kaffman, A., N. M. Rank, E. M. O’Neill, L. S. Huang, and E. K. O’Shea. 1998. The
receptor Msn5 exports the phosphorylated transcription factor Pho4 out of the nucleus.
Nature 396:482–486.
54. Kaffman, A., N. M. Rank, and E. K. O’Shea. 1998. Phosphorylation regulates
association of the transcription factor Pho4 with its import receptor Pse1/Kap121. Genes
Dev. 12:2673–2683.
55. Kahana, J. A., and D. W. Cleveland. 2001. Cell cycle. Some importin news about
spindle assembly. Science 291:1718–1719.
56. Kalderon, D., B. L. Roberts, W. D. Richardson, and A. E. Smith. 1984. A short
amino acid sequence able to specify nuclear location. Cell 39:499–509.
57. Kang, Y., and B. R. Cullen. 1999. The human Tap protein is a nuclear mRNA export
factor that contains novel RNA-binding and nucleocytoplasmic transport sequences.
Genes Dev. 13:1126–1139.
58. Katahira, J., K. Straßer, A. Podtelejnikov, M. Mann, J. U. Jung, and E. Hurt. 1999.
The Mex67p-mediated nuclear mRNA export pathway is conserved from yeast to
human. EMBO J. 18:2593–2609.
59. Kim, I. S., D. H. Kim, S. M. Han, M. U. Chin, H. J. Nam, H. P. Cho, S. Y. Choi,
B. J. Song, E. R. Kim, Y. S. Bae, and Y. H. Moon. 2000. Truncated form of importin
alpha identified in breast cancer cell inhibits nuclear import of p53. J. Biol. Chem.
275:23139–23145.
60. King, R. W., J.-M. Peters, S. Tugendreich, M. Rolfe, P. Heiter, and M. W.
Kirshcner. 1995. A 20s complex containing CDC27 and CDC16 catalyzes the mitosis-
specific conjugation of ubiquitin to cyclin B. Cell 81:279–288.
61. Kobe, B. 1999. Autoinhibition by an internal nuclear localization signal revealed by the
crystal strcuture of mammalian importin a. Nat. Struct. Biol. 6:301–304.
62. Kohler, M., S. Ansieau, S. Prehn, A. Leutz, H. Haller, and E. Hartmann. 1997.
Cloning of two novel human importin-alpha subunits and analysis of the expression
pattern of the importin-alpha protein family. FEBS Lett. 417:104–108.
63. Komeili, A., and E. K. O’Shea. 2000. Nuclear transport and transcription. Curr. Opin.
Cell Biol. 12:355–360.
64. Komeili, A., and E. K. O’Shea. 1999. Roles of phosphorylation sites in regulating
activity of the transcription factor Pho4. Science 284:977–980.
65. Kumagai, A., and W. G. Dunphy. 1999. Binding of 14-3-3 proteins and nuclear export
control the intracellular localization of the mitotic inducer Cdc25. Genes Dev. 13:1067–1072.
66. Kumagai, A., P. S. Yakowec, and W. G. Dunphy. 1998. 14-3-3 proteins act as
negative regulators of the mitotic inducer Cdc25 in Xenopus egg extracts. Mol. Biol.
Cell 9:345–354.
67. Kutay, U., G. Lipowsky, E. Izaurralde, F. R. Bischoff, P. Schwarzmaier,
E. Hartmann, and D. Görlich. 1998. Identification of a tRNA-specific nuclear export
receptor. Mol. Cell 1:359–369.
68. Lam, D. H., and P. D. Aplan. 2001. NUP98 gene fusions in hematologic malignancies.
Leukemia 15:1689–1695.
69. Lopez-Girona, A., B. Furnari, O. Mondersert, and P. Russell. 1999. Nuclear
localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein. Nature
397:172–175.
70. Manser, E. 2002. Small GTPases take the stage. Dev. Cell 3:323–328.
71. May, M. J., and S. Ghosh. 1997. Rel/NF-kappa B and I kappa B proteins: an overview.
Semin. Cancer Biol. 8:63–73.
72. Melchior, F., K. Weber, and V. Gerke. 1993. A functional homologue of the RNA1
gene product in Schizosaccharomyces pombe: purification, biochemical characterization,
and identification of leucine-rich repeat motif. Mol. Biol. Cell 4:569–581.
73. Moore, J. D., J. Yang, R. Truant, and S. Kornbluth. 1999. Nuclear import of
Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin
E and Cdc2/cyclin B1. J. Cell Biol. 144:213–224.
74. Moy, T. I., and P. A. Silver. 1999. Nuclear export of the small ribosomal subunit
requires the ran-GTPase cycle and certain nucleoporins. Genes Dev. 13:2118–2133.
75. Nachury, M. V., T. J. Maresca, W. C. Salmon, C. M. Waterman-Storer, R. Heald,
and K. Weis. 2001. Importin beta is a mitotic target of the small GTPase Ran in spindle
assembly. Cell 104:95–106.
76. Nakielny, S., and G. Dreyfuss. 1999. Transport of proteins and RNAs in and out of the
nucleus. Cell 99:677–690.
77. Nakielny, S., U. Fischer, W. M. Michael, and G. Dreyfuss. 1997. RNA transport.
Annu. Rev. Neurosci. 20:269–301.
78. Nemergut, M. E., C. A. Mizzen, T. Stukenberg, C. D. Allis, and I. G. Macara. 2001.
Chromatin docking and exchange activity enhancement of rcc1 by histones h2a and h2b.
Science 292:1540–1543.
79. Ohtsubo, M., H. Okazaki, and T. Nishimoto. 1989. The RCC1 protein, a regulator for
the onset of chromosome condensation locates in the nucleus and binds to DNA. J. Cell
Biol. 109:1389–1397.
80. Perkins, N. D. 2000. The Rel/NF-kappa B family: friend and foe. Trends Biochem. Sci.
25:434–440.
81. Pines, J., and T. Hunter. 1991. Human cyclins A and B1 are differentially located in
the cell and undergo cell cycle-dependent nuclear transport. J. Cell Biol. 115:1–17.
82. Quimby, B. B., and A. H. Corbett. 2001. Nuclear transport mechanisms. Cell. Mol.
Life Sci. 58:1766–1773.
83. Reed, R., and E. Hurt. 2002. A conserved mRNA export machinery coupled to pre-
mRNA splicing. Cell 108:523–531.
84. Ribbeck, K., G. Lippowsky, H. M. Kent, M. Stewart, and D. Görlich. 1998. NTF2
mediates nuclear import of Ran. EMBO J. 17:6587–6598.
85. Robbins, J., S. M. Dilworth, R. A. Laskey, and C. Dingwall. 1991. Two
interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of
a class of bipartite nuclear targeting sequence. Cell 64:615–623.
86. Rout, M. P., J. D. Aitchison, A. Suprapto, K.Hjertaas, Y. Zhao, and B. T. Chait.
2000. The yeast nuclear pore complex: composition, architecture, and transport
mechanism. J. Cell Biol. 148:635–652.
87. Rout, M. P., G. Blobel, and J. D. Aitchison. 1997. A distinct nuclear import pathway
used by ribosomal proteins. Cell 89:715–725.
88. Ryan, K. J., J. M. McCaffery, and S. R. Wente. 2003. The Ran GTPase cycle is
required for yeast nuclear pore complex assembly. J. Cell Biol. 160:1041–1053.
89. Ryan, K. M., A. C. Phillips, and K. H. Vousden. 2001. Regulation and function of the
p53 tumor suppressor protein. Curr. Opin. Cell Biol. 13:332–337.
90. Sarkar, S., and A. K. Hopper. 1998. tRNA nuclear export in saccharomyces
cerevisiae: in situ hybridization analysis. Mol. Biol. Cell. 9:3041–3055.
91. Schneider, K. R., R. L. Smith, and E. K. O’Shea. 1994. Phosphate-regulated
inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81. Science
266:122–126.
92. Segref, A., K. Sharma, V. Doye, A. Hellwig, J. Huber, R. Luhrman, and E. Hurt.
1997. Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA
and nuclear pores. EMBO J. 16:3256–3271.
93. Shyu, A. B., and M. F. Wilkinson. 2000. The double lives of shuttling mRNA binding
proteins. Cell 102:135–138.
94. Simos, G., and E. Hurt. 1999. Transfer RNA biogenesis: a visa to leave the nucleus.
Curr. Biol. 9:R238–241.
95. Smith, A., A. Brownawell, and I. G. Macara. 1998. Nuclear import of Ran is mediated
by the transport factor NTF2. Curr. Biol. 8:1403–1406.
96. Snead, D. R., B. Perunovic, N. Cullen, M. Needham, D. P. Dhillon, H. Satoh, and H.
Kamma. 2003. hnRNP B1 expression in benign and malignant lung disease. J. Pathol.
200:88–94.
97. Steggerda, S. M., and B. M. Paschal. 2002. Regulation of nuclear import and export
by the GTPase Ran. Int. Rev. Cytol. 217:41–91.
98. Strambio-de-Castillia, C., and M. P. Rout. 2002. The structure and composition of the
yeast NPC. Results Probl. Cell Differ. 35:1–23.
99. Strom, A. C., and K. Weis. 2001. Importin-beta-like nuclear transport receptors.
Genome Biol. 2:1–9.
100. Suntharalingam, M., and S. R. Wente. 2003. Peering through the pore: nuclear pore
complex structure, assembly, and function. Dev. Cell 4:775–789.
101. Surana, U., A. Amon, C. Dowzer, J. McGrew, B. Byers, and K. Nasmyth. 1993.
Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to
anaphase transition in budding yeast. EMBO J. 12:1969–1978.
102. Tagawa, T., T. Kuroki, P. K. Vogt, and K. Chida. 1995. The cell cycle-dependent
nuclear import of v-Jun is regulated by phosphorylation of a serine adjacent to the
nuclear localization signal. J. Cell Biol. 130:255–263.
103. Takeno, S., T. Noguchi, R. Kikuchi, Y. Uchida, S. Yokoyama, and W. Muller. 2002.
Prognostic value of cyclin B1 in patients with esophageal squamous cell carcinoma.
Cancer 94:2874–2881.
104. Toyoshima, F., T. Moriguchi, A. Wada, M. Fukada, and E. Nishida. 1998. Nuclear
export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.
EMBO J. 17:2728–2735.
105. Vigneri, P., and J. Y. Wang. 2001. Induction of apoptosis in chronic myelogenous
leukemia cells through nuclear entrapment of BCR-ABL tyrosine kinase. Nat. Med.
7:228–234.
106. Walther, T. C., P. Askjaer, M. Gentzel, A. Habermann, G. Griffiths, M. Wilm,
I. W. Mattaj, and M. Hetzer. 2003. RanGTP mediates nuclear pore complex assembly.
Nature 424:689–694.
107. Weis, K. 2003. Regulating access to the genome: nucleocytoplasmic transport
throughout the cell cycle. Cell 112:441–451.
108. Wen, W., J. L. Meinkoth, R. Y. Tsien, and S. S. Taylor. 1995. Identification of a
signal for rapid export of proteins from the nucleus. Cell 82:463–473.
109. Wiese, C., A. Wilde, M. S. Moore, S. A. Adam, A. Merdes, and Y. Zheng. 2001.
Role of importin-beta in coupling Ran to downstream targets in microtubule assembly.
Science 291:653–656.
110. Woods, Y. L., and D. P. Lane. 2003. Exploiting the p53 pathway for cancer diagnosis
and therapy. Hematol. J. 4:233–247.
111. Wozniak, R. W., M. P. Rout, and J. D. Aitchison. 1998. Karyopherins and kissing
cousins. Trends Cell Biol. 8:184–188.
112. Wu, S., M. Sato, C. Endo, A. Sakurada, B. Dong, H. Aikawa, Y. Chen, Y. Okada,
Y. Matsumura, E. Sueoka, and T. Kondo. 2003. hnRNP B1 protein may be a possible
prognostic factor in squamous cell carcinoma of the lung. Lung Cancer 41:179–186.
113. Xiao, Z., J. T. McGrew, A. J. Schroeder, and M. Fitzgerald-Hayes. 1993. CSE1 and
CSE2, two new genes required fro accurate mitotic chromosome segregation in
114. Yang, J., E. S. Bardes, J. D. Moore, J. Brennan, M. A. Powers, and S. Kornbluth.
1998. Control of cyclin B1 localization through regulated binding of the nuclear export
factor CRM1. Genes Dev. 12:2131–2143.
115. Yang, J., K. Winkler, M. Yoshida, and S. Kornbluth. 1999. Maintenance of G2 arrest
in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import.
EMBO J. 18:2174–2183.
116. Yasuda, M., F. Takesue, S. Inutsuka, M. Honda, T. Nozoe, and D. Korenaga. 2002.
Overexpression of cyclin B1 in gastric cancer and its clinicopathological significance:
an immunohistological study. J. Cancer Res. Clin. Oncol. 128:412–416.
117. Yeong, F. M., H. H. Lim, C. G. Padmashree, and U. Surana. 2000. Exit from mitosis
in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of
Cdc20. Mol. Cell 5:501–511.
118. Yin, M., G. Hapke, B. Guo, R. G. Azrak, C. Frank, and Y. M. Rustum. 2001. The
Chk1-Cdc25C regulation is involved in sensitizing A253 cells to a novel topoisomerase
I inhibitor BNP1350 by bax gene transfer. Oncogene 20:5249–5257.
119. Yu, M., Q. Zhan, and O. J. Finn. 2002. Immune recognition of cyclin B1 as a tumor
antigen is a result of its overexpression in human tumors that is caused by non-
functional p53. Mol. Immunol. 38:981–987.
120. Zachariae, W., and K. Nasmyth. 1996. TPR proteins required for anaphase
progression mediate ubiquitination of mitotic B-type cyclins of yeast. Mol. Biol. Cell
7:791–801.
121. Zeng, Y., and H. Piwnica-Worms. 1999. DNA damage and replication checkpoints in
fission yeast require nuclear exclusion of the Cdc25 phosphatase via 14-3-3 binding.
Mol. Cell. Biol. 19:7410–7419.
122. Zhang, C., M. W. Goldberg, W. J. Moore, T. D. Allen, and P. R. Clarke. 2002.
Concentration of Ran on chromatin induces decondensation, nuclear envelope formation
and nuclear pore complex assembly. Eur. J. Cell Biol. 81:623–633.
Chapter 4
STUDIES OF PROTEIN FARNESYLATION

IN YEAST
Nitika Thapar and Fuyuhiko Tamanoi

Department of Microbiology, Immunology & Molecular Genetics, Jonsson Comprehensive
Cancer Center, Molecular Biology Institute, University of California, Los Angeles, 405
Hilgard Ave., Los Angeles, CA 90095-1489
1 PROTEIN FARNESYLATION
Protein farnesylation has emerged as one of the important classes of

posttranslational modification of proteins [84, 109]. Farnesylation is the first
step in a three-step series of protein processing and involves the addition of a
farnesyl group to a cysteine located in a C-terminal motif called the CaaX
motif (C is cysteine, a is aliphatic amino acid, and X is the C-terminal amino
acid that is usually methionine, cysteine, glutamine, alanine, or serine).
These proteins subsequently undergo proteolytic cleavage and carbo-
xylmethylation (Figure 1). Significance of protein farnesylation in human
cancer has been underscored by the findings that many proteins undergoing
farnesylation play roles in signal transduction. Of these, Ras proteins are
particularly noteworthy.
101
© 2007 Springer.
102 N. Thapar and F. Tamanoi
CaaX
Farnesyl Pyrophosphate (FPP)

Protein Farnesyltransferase
Pyrophosphate (PP)
CaaX
aaX
CaaX Protease
Carboxyl Methyltransferase
C Me
Figure 1. Posttranslational modification of proteins containing the CaaX motif at their C-

terminus. Protein farnesyltransferase transfers a farnesyl group on the cysteine residue which
is followed by cleavage of the last three amino acids and subsequent methylation of the C-
terminal carboxyl group. (In the CaaX motif C is cysteine, a is an aliphatic amino acid and X
is the C-terminal amino acid.)
Protein farnesylation is catalyzed by protein farnesyltransferase (FTase)

that is conserved from yeast to human [84, 109]. This enzyme recognizes the
CaaX motif and transfers a farnesyl group from farnesyl pyrophosphate
(FPP), an intermediate in cholesterol biosynthesis, resulting in the formation
of a thioether bond. The catalytic mechanism of action of the FTase has been
elucidated [91]. The enzyme contains one molecule of a tightly bound zinc
ion (Zn2+) that participates in the catalytic reaction [51, 91, 97]. With yeast
FTase, the reaction proceeds by an ordered mechanism with FPP binding
first. In the case of mammalian enzyme, it has been suggested from kinetic
and structural studies that the bound FPP forms part of the binding surface of
the protein substrate [36]. Three-dimensional structures of the rat and human
enzymes have been determined with and without bound substrates [65, 82,
95]. The structure mainly consists of α-helices with the β-subunit forming a
barrel like structure and the α-subunit wrapping around the β-subunit from
one side of the barrel.
FTase belongs to a family of protein prenyltransferases which includes
protein geranylgeranyltransferase type I (GGTase I) and type II (GGTase II).
GGTase I catalyzes geranylgeranylation of proteins such as RhoA, Cdc42, or
Chapter 4: Studies of Protein Farnesylation in Yeast 103
Rac proteins [24, 39, 86]. The modification involves a 20-carbon geranyl-
geranyl group added to a cysteine in the CaaL motif (similar to the CaaX
motif except that the C-terminal amino acid is leucine or phenylalanine) [74,
108]. GGTase I shares a common α-subunit with FTase, while its β-subunit
shares about 30% homology with the β-subunit of FTase. GGTase II
catalyzes the addition of a geranylgeranyl group to both cysteines within the
CC or the CXC motif that are found in a number of Rab protein [88]. In
addition to α- and β-subunits that share homology with their counterparts in
FTase, GGTase II contains an additional component-Rep (Rab escort
protein) [96].
Recently, anticancer drugs based on the inhibition of protein farnesylation
have been developed. These small molecular weight compounds called
farnesyltransferase inhibitors (FTIs) selectively inhibit farnesyltransferase
[7, 38, 84, 94]. A variety of compounds including peptidomimetic inhibitors,
farnesyl pyrophosphate analogues, bisubstrate inhibitors, and natural
compounds have been identified. FTIs have been shown to reverse ras-
mediated phenotypes in ras-transformed cells. FTIs inhibit the growth of
tumors or even regress tumors in animal model systems [62]. Currently,
FTIs are being evaluated in a variety of clinical trials [57].
In this review, we discuss how yeast studies contributed to the overall
study of protein farnesylation. We will first describe yeast studies concerning
FTase as well as yeast-based assays that led to the identification of one of
the first generation FTI compounds. We will then focus on identification and
characterization of farnesylated proteins. Yeast has provided a compre-
hensive analysis of farnesylated proteins that was instrumental in the iden-
tification of a number of novel farnesylated proteins. Of particular interest
is Rheb, a novel family of the Ras-superfamily G-proteins. This protein is
highly conserved and plays a role in the regulation of cell cycle at the G1/S
phase. We will summarize the current understanding of Rheb in S. cerevisiae
and S. pombe.
2 CONTRIBUTION OF YEAST TO THE

DISCOVERY AND CHARACTERIZATION
OF PROTEIN FARNESYLTRANSFERASE
2.1 Identification of genes encoding subunits of protein

farnesyltransferase
DPR1/RAM1 and RAM2 encode β and α subunits of FTase in

S. cerevisiae, respectively [40, 41, 48]. These genes were identified from the
study to isolate mutants defective in membrane association of Ras proteins.
The isolation of the mutants involved reversion of ras activated phenotypes
in S. cerevisiae. Briefly, mutants were sought that reverse phenotypes due to
the activation of Ras2 such as heat shock sensitivity, decrease in glycogen
accumulation, and temperature sensitivity [34, 80]. DPR1/RAM1 and RAM2
encode proteins with 431aa and 316 aa, respectively. They share approxi-
mately 30% identity with corresponding subunits of mammalian FTase.
Sequence characterization of Ram2 and the α subunit led to the finding of
five tandem sequence repeats in these subunits [11]. Identification of these
genes was important in the study of FTase in mammalian cells. The presence
of two distinct genes affecting farnesylation provided hints for the idea that
the mammalian enzyme consists of two subunits. Sequence of DPR1/RAM1
determined in 1987 by our group provided the first information on one of the
FTase subunits [40]. Subsequent determination of mammalian FTase β-
subunit sequence in 1991 revealed sequence homology with Dpr1/Ram1
[21]. Sequence homology between Ram2 and the α-subunit was also
demonstrated [61].
Recently, we have identified genes encoding subunits of FTase in S. pombe.
The cwp1+ and cpp1+ genes encode α and β subunits of FTase, respectively
[4, 105]. Co-expression of Cpp1 and Cwp1 in Escherichia coli produces a
recombinant enzyme that is active in farnesylation [105]. The cwp1+ was
identified from the yeast two hybrid screen using cwg2+ [4]. cwg2+ encodes
the β-subunit of GGTase I, bearing similarity to its homologue in
S. cerevisiae-CAL1/CDC43 [73], and is involved in cell wall synthesis [30].
2.2 Mutational analysis of protein farnesyltransferase
One of the unique properties of FTase is its ability to recognize the CaaX
motif. This observation was critical in the development of peptidomimetic
FTIs, as they were derived from a tetrapeptide such as CVFM that inhibited
the activity of FTase. Thus, it was of interest to understand which amino
acid residues were involved in the CaaX recognition. We have employed

PCR-mediated random mutagenesis of S. cerevisiae FTase to gain insight
into this issue. The DPR1/RAM1 gene was randomly mutagenized and the
mutant library was screened for its ability to suppress temperature-sensitive
growth of cal1 mutant which is defective in GGTase I [28, 71]. The
intention here was to identify amino acid changes that converted the
recognition of the CaaX motif to the CaaL motif. This screen led to the
identification of amino acid changes at three residues-S159, Y362, and Y366
[28]. A change in either of these residues was sufficient to convert FTase to
mutants that had increased recognition of the CaaL motif. Y362 and Y366
are perfectly conserved in all farnesyltransferase subunits examined. The
Y362 mutant is well characterized but Y366 awaits further investigation.
The residues Y362 and Y366 correspond to residues Y361 and Y365 in the
mammalian enzyme. Alteration of Y361 to leucine in the human enzyme led
to an increase in the utilization of GST–CIIL substrate [29], suggesting that
this residue is important in the recognition of the CAAL motif. However, a
recent kinetic study suggests that the change in the CaaX motif recognition
by FTase by the alteration of Y361 reflects a kinetic effect due to the
complex mechanism of FTase [90]. In the crystal structure of mammalian
FTase [92, 95], Y361 is located in a pocket that would be occupied by the
second aliphatic amino acid in the CaaX motif. In addition, P152, which
corresponds to S159, is located close to the C-terminal amino acid of the
CaaX motif.
2.3 Identification of inhibitors of protein

farnesyltransferase by yeast-based assays
By using yeast-based assays, we have obtained one of the first generation

compounds of FTIs [46, 72]. This microbial screen was based on our
observation that Ste18 protein is farnesylated [33]. This γ-subunit of the
heterotrimeric G-protein is involved in yeast-mating functions to inhibit cell
growth by activating a MAP kinase cascade that consists of Ste11, 7, and
Kss/Fus [44, 49]. This growth inhibitory effect is normally blocked, as Ste18
is complexed with the α-subunit. Thus, the disruption of the α-subunit is
lethal. However, this lethality is suppressed when the farnesylation of Ste18
is inhibited. Culture media from a variety of microbial sources were
screened that led to the identification of manumycin as an inhibitor of FTase
[46]. Manumycin inhibited FTase by competing with FPP and inhibited ras-
activated phenotypes in yeast as well as in C. elegans [47, 72]. Manumycin
also inhibited proliferation of pancreatic cancer cells and the growth of
tumors in mice [54, 56].
3 YEAST STUDIES GREATLY EXPANDED OUR

UNDERSTANDING OF FARNESYLATED
PROTEINS
One of the current emphases of the farnesylation study is to determine

substrates of farnesylation. The first group of farnesylated proteins discovered
included fungal mating factors, nuclear lamins, and proteins involved in
visual signal transduction such as transducin [15, 24]. The significance of
farnesylation in cancer came into prominence when Ras was found to be
farnesylated [19, 45, 85]. Ras is the product of an oncogene and mutations of
Ras have been found in approximately 30% of human cancers [12].
Farnesylation is found to be critical for membrane localization of Ras and it
has also been demonstrated that mutations of the CaaX motif inhibit the
function of Ras [66]. A number of other proteins are also farnesylated. Thus,
identification and characterization of novel farnesylated proteins becomes
crucial for understanding how farnesylation regulates various cellular
mechanisms.
3.1 Farnesylated proteins in S. cerevisiae
The sequencing of the S. cerevisiae genome has greatly facilitated the

search for proteins which are possible substrates of FTase, based on the
presence of the CaaX motif at their C-termini. Table 1a lists the proteins in
S. cerevisiae which are farnesylated or predicted to be farnesylated.
Ras1 and 2 proteins are GTP-binding proteins and are the homologues of
the mammalian proto-oncogene product Ras [27, 79]. They are the major
players in the signal transduction pathway involving adenylyl cyclase (Cyr1)
[98]. Farnesylated Ras2–GTP complex was found to activate Cyr1 in vitro
more efficiently than either the unprenylated protein or a C-terminal deletion
mutant [63]. Invasive growth is regulated via the Cdc42–Ste20–MAPK
cascade as well as the cAMP-dependent protein kinase pathway [75]. The
critical role of farnesylation was studied by making CaaX box mutations as
well as deletions. The C319S mutant of Ras2 is defective in farnesylation
and all subsequent modifications i.e., proteolytic cleavage and methylation.
Spores containing this mutant fail to yield colonies in the absence of Ras1
[10].
Table 1a. S. cerevisiae CaaX motif-containing proteins which have been shown to be
farnesylated or are candidates for farnesylation based on sequence similarity
Protein CAAX motif Function/cellular role
Ras1 -CIIC G-protein, cAMP pathway
Ras2 -CIIS G-protein, cAMP pathway
Mfa1 -CVIA Mating pheromone a-factor
Mfa2 -CVIA Mating pheromone a-factor
Stel8 -CTLM Gγ involved in mating
Ydj1 -CASQ DnaJ homolog, protein transport
Xdj1j -CCIQ DnaJ homolog
Rho3 -CTIM Cell polarity
Rho4 -CIIM Cell polarity/Actin cytoskeleton organization
Rheb -CSIM GTP binding/Amino acid metabolism
Skt5 -CVIM Cell wall maintenance
Gis4 -CAIM Signal transduction/Cell stress
Rcy1 -CCIM Vesicular transport
Pex19 -CKQQ Peroxisome biogenesis
Atr1 -CTVA Small molecule transport
N1142 -CSIM Hemoprotein
Ykt6 -CIIM v-SNARE, vesicular transport
YCP4 -CTVM Unknown
YDL009C -CAVS Unknown
YPL191C -CVVM Unknown
YGL082W -CVIM Unknown
YMR265C -CSNA Unknown
YML133C -CCPS Unknown
YPR203W -CCPS Unknown
YFL065C -CCPS Unknown
YHL049C -CCPS Unknown
YDL151C -CYPA Unknown
YJL118W -CCCS Unknown
YIR007W -CVIS Unknown
Other Ras-superfamily G-proteins that are farnesylated are Rho3, 4, and

Rheb. Rho3 is involved in cell polarity [1], and we have shown that it
directly binds to Exo70, a subunit of the exocyst, a multiprotein complex
affecting fusion of secretory vesicles with the plasma membrane [83]. Rheb
is a recently discovered GTP, binding protein belonging to the Ras
superfamily of G proteins. It bears similarity to the Ras, Ral, and Rap
proteins. In S. cerevisiae, it has been shown to be involved in the uptake of
arginine and lysine. This appears to be due to the modulation of the activity
of the permeases Can1 and probably Lyp1 [101]. Mutation of the CaaX
motif (C206S) eliminates its function. The null mutant is viable but displays
hypersensitivity to canavanine and thialysine, the toxic analogues of arginine
and lysine respectively. Later in the chapter, we provide a more detailed
description of Rheb.
The Mfa1 and 2 proteins are mating pheromones (mating factor a)

exported from the cells by Ste6, which interact with α cells to produce cell
cycle arrest and mating responses [70]. Mutant forms of the proteins with
different substitutions in the CaaX motif are poor substrates for in vitro
farnesylation [99]. C-terminal farnesylation was shown to be required for the
production and export of a-factor-related peptide (AFRP) [22]. Ste18 is the γ
subunit of the guanine nucleotide-binding protein that mediates signal
transduction by pheromones during mating [9]. It is anchored to the plasma
membrane through hydrophobic interactions with its farnesylated C-
terminus. It activates the pheromone response pathway with Ste4 [81].
Mutation in the CaaX motif (C107S) prevents farnesylation and results in a
decrease of its steady state levels [33, 52, 103]. This mutant has decreased
response to pheromones [33, 103].
Ydj1 is a protein involved in protein import into mitochondria and
endoplasmic reticulum and is the homologue of E. coli DnaJ [17]. It belongs
to the Hsp40 family of co-chaperones and is required for the ubiquitination-
dependent degradation of short-lived and abnormal proteins [64, 67]. It
associates with the membrane through its farnesyl group, and C406S mutant
exhibits decreased membrane association [18, 55]. C-terminal farnesylation
is also required for growth at elevated temperatures [18]. Xdj1, a nonessential
homologue of DnaJ was identified as an ORF in the genomic DNA, however
no mRNA or protein for it has been detected [87]. Hdj2, a human
homologue of Ydj1, has recently been shown to provide a valuable tool to
follow inhibition of farnesylation in peripheral blood of patients treated with
FTI (2).
Pex19 is a protein involved in peroxisome membrane formation and
maintenance, which interacts with Pex3 only when it is farnesylated [42, 50].
Farnesylation at the cysteine in its CKQQ motif is essential for its function
in vivo [42]. Several other proteins predicted to be farnesylated include
known proteins such as Gis4 (protein implicated to be involved in the cAMP
pathway) [8], Skt5 (a killer toxin-resistant protein) [13, 14], Rcy1 (protein
involved in endocytic membrane traffic) [37], Ykt6 (synaptobrevin
homologue) [69, 100] as well as proteins with unknown function. There are
other proteins which have been predicted to be farnesylated but not yet
characterized.
3.2 Farnesylated proteins in S. pombe
The fission yeast S. pombe has a few proteins functionally related to the
S. cerevisiae proteins which have been shown to be farnesylated. As
summarized in Table 1b, these include Mfm1, 2 and 3, the functionally
redundant precursor polypeptides for the mating pheromone M factor
produced by h– cells which are structurally similar to the a-factor of

S. cerevisiae [60]. They are posttranslationally cleaved to yield the sequence
YTPKVPYMCVIA which is isoprenylated on the cysteine [26]. A synthetic
M-factor peptide lacking any modification is inactive. Ras1 is the
homologue of mammalian Ras and regulates cell morphogenesis and is
required for the nutrient starvation signal transduction pathway that leads to
mating [3, 35]. Rheb is a member of the Rheb GTPases but divergent from
the S. cerevisiae ortholog. It is known to perform functions distinct from
Ras1 [68]. The null mutant is lethal and the inhibition of Rheb farnesylation
leads to an arrest in the G0/G1 phase [68, 106]. Farnesylation-deficient
mutant of Rheb (SVIA) fails to rescue the rhb1– mutant suggesting that
farnesylation is important for its activity [106]. Rho2 and Rho 3 are
members of the Rho family of proteins involved in the control of cell
morphology and cell polarity [5]. Rho2 is involved in the regulation of cell
wall synthesis through the interaction with PKC homologues [16]. Lack of
Rho3 causes abnormal cell morphology and cytokinesis defects [105].
Table 1b. S. pombe CaaX motif-containing proteins which have been shown to be farnesylated
or are candidates for farnesylation based on sequence similarity
Protein CAAX motif Function/cellular role
SpRas -CVIC GTP binding/mating response
Mfm1 -CVIA Signal transduction/mating response
Rhb1 -CVIA Cell cycle regulator
Spj1 -CAQQ Protein folding/cell stress
Rho2 -CIIS Cell polarity/cell wall maintenance
Rho3 -CIIA Cell polarity/cell wall maintenance
Git11 -CTIS Signal transduction/mating response
SPBC405.06 -CQAQ Unknown (has DnaJ domain)
SPBC13G1.11 -CIIA Unknown (predicted SNARE)
SPCC417.05C -CIIS Unknown (predicted chitin biosynthesis)
SPAC24B11.10C -CVVM Unknown (predicted chitin biosynthesis)
SPAC607.09C -CALT Unknown (predicted cellular pH homeostasis)
SPAC17C9.14 -CPTQ Unknown (predicted peroxisome biogenesis)
4 RHEB, A NOVEL CLASS OF FARNESYLATED

PROTEINS INVOLVED IN THE REGULATION
OF CELL CYCLE PROGRESSION
Among many farnesylated proteins, Rheb has recently emerged as an

important protein. This protein belongs to the Ras superfamily of G- proteins.
There are features unique to the Rheb subfamily. Recent studies indicate the
need of Rheb to be farnesylated for its activity. In the following sections, we
describe characterization of this novel member of the Ras family.
4.1 Rheb binds GTP and is farnesylated
Rheb is a small GTP-binding protein belonging to the Ras superfamily. It

was first identified as the product of an inducible gene in the rat hippocampal
granule cells, which was induced in response to seizures and by NMDA-
dependent synaptic activity in the long-term potentiation paradigm [104]. It
is well conserved and homologues have been identified in human [43, 73],
Xenopus, zebrafish, Drosophila, sea squirt, Dictyostelium, Aspergillus [77],
S. cerevisiae, and S. pombe [101, 102] (Figure 2). In mammals, two genes
for Rheb exist – Rheb1 and Rheb2, unlike lower species which have a single
gene only [6, 78]. Its amino acid sequence shows the presence of highly
conserved GTP-binding regions as well as the CaaX motif. Interestingly,
Rheb proteins have an arginine residue at the third position in the G1-box
(corresponding to the 12th amino acid in Ras, which is involved in the
intrinsic GTPase activity of the protein) unlike most other Ras superfamily
proteins which have a glycine residue (Figure 3). The effector domain (F-V-E/
D-S-Y-Y/D-P-T-I-E-N-E/Q/T-F-T/S/N-R/K-x-x) is well conserved among
Rheb members. Rheb shares the highest amino acid homology with human
H-Ras (36%), human Rap2 (38%), and yeast Ras1 (43%). It is localized to
the plasma membrane like the Ras protein and found to be farnesylated
in vivo; treatment with farnesyltransferase inhibitors (FTIs) leads to
its mislocalization to the cytoplasm [23]. In humans it is ubiquitously
expressed, though high levels of expression were observed in the skeletal
G1
ScRheb MEYATMSSSNSTHNFQRKIALIGARNVGKTTLTVRFVESR 40
SpRheb M----------APIKSRRIAVLGSRSVGKSSLTVQYVENH 30
HsRheb M----------PQSKSRKIAILGYRSVGKSSLTIQFVEGQ 30
DmRheb M----------P-TKERHIAMMGYRSVGKSSLCIQFVEGQ 29
* . *.**..* *.***..* ...**..
Effector Domain G3
ScRheb FVESYYPTIENEFTRIIPYKSHDCTLEILDTAGQDEVSLL 80
SpRheb FVESYYPTIENTFSKNIKYKGQEFATEIIDTAGQDEYSIL 70
HsRheb FVDSYDPTIENTFTKLITVNGQEYHLQLVDTAGQDEYSIF 70
DmRheb FVDSYDPTIENTFTKIERVKSQDYIVKLIDTAGQDEYSIF 69
**.** *****.*.. .... ...******* *..
ScRheb NIKSLTGVRGIILCYSIINRASFDLIPILWDKLVDQLGKD 120

SpRheb NSKHSIGIHGYVLVYSITSKSSFEMVKIVRDKILNHTGTE 110
HsRheb PQTYSIDINGYILVYSVTSIKSFEVIKVIHGKLLDMVGKV 110
DmRheb PVQYSMDYHGYVLVYSITSQKSFEVVKIIYEKLLDVMGKK 109
. .* .* **... **... .. .*... *.
G4
ScRheb NLPVILVGTKADLGRSTKGVKRCVTKAEGEKLASTIGSQD 160
SpRheb WVPIVVVGNKSDLHM-----QRAVTAEEGKALANE----- 140
HsRheb QIPIMLVWNKKDLHM-----ERVISYEEGKALAES----- 140
DmRheb YVPVVLVGNKIDLHQ-----ERTVSTEEGKKLAES----- 139
.*...* .* ** .* .. .**. **..
G5
ScRheb KRNQAAFIECSAELDYNVEETFMLLLKQMERVEGTLGLDA 200
SpRheb --WKCAWTEASARHNENVARAFELIISEIEKQAN--PSPP 176
HsRheb --WNAAFLESSAKENQTAVDVFRRIILEAEKM-D--GAAS 175
DmRheb --WRAAFLETSAKQNESVGDIFHQLLILIENE-N--GNP- 173
. *. * ** . .. * .. *. .
CAAX
ScRheb ENNNKCSIM 209
SpRheb GDGKGCVIA 185
HsRheb QGKSSCSVM 184
DmRheb QEKSGCLVS 182
. . * .
Figure 2. Amino acid sequence alignment of Rheb protein from various species (Sc –
S. cerevisiae; Sp – S. pombe; Hs – Human; Dm – D. melanogaster). The G-boxes (G1–G5)
are indicated by overlining. The invariant arginine in the G1 box, the effector domain and the
CaaX motif are shown in bold. Asterisks represent identical residues and dots represent
similar residues.
1 G1 G2 G3 G4 G5 209
GARNVGKT S. cerevisiae Rheb CSIM
1 G1 G2 G3 G4 G5 185
GSRSVGKS S. pombe Rheb CVIA
Figure 3. Structure of S. cerevisiae and S. pombe Rheb proteins. The G-boxes are represented
as dark segments. The invariant arginine in the G1 box as well as the C-terminal CaaX motif
are indicated.
and cardiac muscle [43]. Rheb mRNA has been found to be induced in
cerebral-ischemia elicited events through NMDA receptor action in rat brain
[58] as well as in human fibroblasts exposed to UV radiation implying a
possible role in UV sensitization of cells [59].
Human Rheb was reported to be upregulated in several transformed cells
[43]. The growth regulatory activities of Rheb were explored by Clark et al.
[23] who found that neither the wild type nor the presumed constitutively
active (Q64L) Rheb protein could induce transformation in normal NIH3T3
cells. At the same time, a mutant Rheb, S20N that carries an amino acid
change analogous to the dominant negative form of Ras (S17N) did not
inhibit growth of the cells. Rheb was found to behave more like Rap1A [23],
which is a negative regulator of Ras function, as it was also found to
antagonize the oncogenic potential of Ras. It was also speculated that since
Rheb and Ras show strong identity in the effector domain region which is
crucial for Raf binding, Rheb may be binding to Raf in a nonproductive
manner and hence titrating it away from Ras and preventing the downstream
cascade [23]. On the other hand, Yee and Worley [107] found that Rheb
stimulates transformation of NIH3T3 fibroblasts when expressed in
conjunction with Raf-1. Thus, a synergistic interaction of Rheb with Raf-1
was suggested, as the transforming potential was much lower when Rheb or
Raf-1 alone was expressed singly. A report showing that Rheb inhibits
B-Raf has been published [53].
The Rheb protein became a prime focus of attention recently after
Drosophila and mammalian studies showed that it is a component of the
insulin/TOR/S6K pathway. Rheb activates S6K-mediated by TOR. Rheb
was found to be the direct target of the Tsc1/Tsc2 complex which serves as a
GAP for Rheb [6, 78]. Mutations in the Tsc1/Tsc2 genes have been shown to
be responsible for the development of tuberous sclerosis (TSC), a genetic
disease characterized by the presence of benign tumors known as hamar-
tomas in various organs and occurrence of seizures and mental retardation.
Studies involving Drosophilia and human Rheb indicate a potentially critical
role of Rheb in the pathogenesis of this disease. Thus, a further investigation
of the function of Rheb may provide insights into possible therapies for
TSC.
4.2 Genetic study of Rheb in S. cerevisiae revealed

the involvement of Rheb in arginine uptake
Rheb was first identified and characterized in S. cerevisiae (ScRheb) by

Urano et al. [101]. It shares an overall 26% sequence identity and 57%
similarity with human and fly Rheb proteins. The genetic study was
undertaken by creating a deficient strain and analyzing its phenotypes in the
total absence of endogenous Rheb or in the presence of mutant forms of the
protein. The disruption strain showed no loss in viability, and no growth,
temperature, and morphological defects were detected [101]. Mating,
sporulation, and secretion were found to be normal. Phenotypes observed by
the disruption of RAS1 or RAS2 genes were not observed in the ScRheb
deficient strain.
A unique phenotype exhibited by this strain was an increased sensitivity
to the arginine and lysine analogues – canavanine and thialysine, respectively
[101]. This phenotype was confirmed to be due to the deficiency in RHEB
by complementation studies involving wild-type ScRheb as well as the Rheb
from S. pombe (SpRheb), which were able to rescue the defect. The
increased sensitivity to the amino acid analogues appears to be due to an
enhanced uptake of arginine and lysine [3- to 3.5-fold and 1.5- to 2-fold
respectively). The strain specifically showed an increased uptake of the two
basic amino acids amongst all others tested [101]. The hypersensitivity to
canavanine appears to be due to a lack of regulation of Can1 – the permease
involved in arginine uptake. Chattopadhyay and Pearce [20] recently
reported that Rheb interacts with Btn2p, a yeast protein involved in the
regulation of vacuolar H+-ATPase. The interaction with Btn2p appears to

alter cellular localization of Rheb [20].
The roles of various conserved regions in the sequence were confirmed
by creating mutations in the effector domain, the G1 box and the CaaX motif
[101]. Effector domain mutants (Y46D and N51D) could not complement
the sensitivity to canavanine and thialysine, while the wild-type Rheb could.
Similarly, when the highly conserved arginine in the G1 box was mutated to
glycine, the mutant protein showed reduced ability to rescue the phenotype.
Farnesylation was found to be critical for ScRheb function since a change of
cysteine in the CaaX box to serine rendered the protein incapable of
complementing the hypersensitivity to canavanine as well as the increased
arginine uptake. The connection between arginine uptake and a signal
transduction pathway mediated by Rheb needs to be explored which could
highlight novel functions of this protein.
4.3 Genetic study of Rheb in S. pombe uncovered

the second function of Rheb-regulation
of cell cycle progression.
The S. pombe homologue of Rheb (SpRheb) was simultaneously

identified by Mach et al. [68] and Urano et al. [101]. Like the ScRheb, the
SpRheb shares a 26% sequence identity with the human and fly Rheb
proteins. The function of SpRheb in S. pombe was analyzed by creating a
disruption mutant as was done previously for ScRheb [106]. Unlike the
ScRheb disruption mutant which did not exhibit any growth defects, the
sprheb– strain did not grow. Use of a conditional mutant showed that the
inhibition of Rheb leads to the arrest of the cell cycle at the Go/G1 phase.
This phenotype could be rescued by the expression of the wild-type SpRheb
and human Rheb but not by the ScRheb.
In another study Mach et al. [68] made a similar conditional mutant of
Rheb (rhb1–) and showed that the cells arrested at the G0/G1 phase as small
round cells. The rhb1– allele could be complemented by wild-type rhb1+ and
rhb1Q64L (analogous to constitutively active H-Ras Q61L mutant).
Interestingly rhb1– cells arrested growth with a terminal phenotype similar to
that of nitrogen starved cells [25, 32, 93]. Cells depleted of nitrogen enter
stationary phase, are smaller than actively growing cells and remain viable
for several weeks. Nitrogen-starved cells are known to induce fnx1 mRNA, a
probable proton-driven plasma membrane transporter of the multidrug
resistance group [31]. Overexpression of fnx1 has also been shown to cause
growth arrest like nitrogen-starved cells [31]. A mutant of Rheb – rhb1–
D121A (a hypomorphic mutant analogous to S. cerevisiae tem1–3, a

temperature-sensitive allele [8]) was used to characterize the Rheb function
[68]. The involvement of SpRheb in cell cycle was implicated by studies of a
strain deficient in the β subunit of farnesyltransferase encoded by cpp1+
[106]. This farnesylation-deficient strain (cpp1–) was found to exhibit
enrichment of G0/G1 cells cycle as well as canavanine hypersensitivity.
Expression of SpRheb–CVIL, capable of being geranylgeranylated in the
cpp1– background could restore normal cell cycle profile. On the other hand,
the expression of SpRheb–SVIA which is incapable of being farnesylated
could not rescue this defect in both the cpp1– and rhb1– strains implying the
critical role of farnesylation of this protein in maintaining a normal cell
cycle.
Since previously it had been shown that ScRheb regulates arginine
uptake as well as canavanine sensitivity [101], a similar role was predicted
for SpRheb. In agreement with this idea, the cpp1– strain exhibited hyper-
sensitivity to canavanine. Furthermore, the increased ability to uptake
arginine could be rescued by the expression of SpRheb–CVIL but not by
wild-type Rheb since it is not capable of being farnesylated in that strain.
These observations suggest that SpRheb has dual functions, one to regulate
cell cycle and the other to regulate arginine uptake. The results again imply a
crucial role of farnesylation of the Rheb protein [105]. Figure 4 shows a
scheme for the function of S. cerevisiae and S. pombe Rheb.
5 SUMMARY
Yeast studies have significantly contributed to the understanding of

protein farnesylation. Identification and characterization of yeast mutants
defective in protein farnesyltransferase were critical for understanding the
structure and function of this enzyme in higher eukaryotes. Random
mutagenesis of yeast FTase β-subunit revealed the significance of three
residues, S159, Y362, and Y366 in the recognition of the CaaX motif. The
two tyrosine residues, perfectly conserved in all FTases so far examined,
appear to be of particular interest. Precise roles of the corresponding residues
in mammalian enzymes, Y361 and Y365, need to be investigated. Finally,
yeast provided an assay that was used to screen natural compounds to
identify inhibitors of FTase.
Rheb Cys Rheb Cys
Arginine uptake Arginine uptake Cell cycle progression
S. cerevisiae S. pombe
Figure 4. Schematic drawing showing the function of Rheb in S. cerevisiae and S. pombe.
Rheb is farnesylated at the C-terminal cysteine residue and localized to the plasma membrane.
In S. cerevisiae, Rheb regulates arginine uptake; in S. pombe, Rheb regulates cell cycle
progression as well as arginine uptake.
In addition, yeast studies have provided information on the physiological

function of a variety of farnesylated proteins. In this review, we focused on a
recently characterized novel member of the Ras superfamily G-proteins –
Rheb. Both S. cerevisiae and S. pombe have proven to be excellent systems
to study the function of Rheb. The availability of FTase-deficient mutants
has further been instrumental in defining the critical role of farnesylation of
Rheb. Although experiments with S. cerevisiae indicated a link between
Rheb and arginine uptake, the significance of this effect on cell physiology
is yet to be determined. On the other hand, experiments with S. pombe do
indicate a role of Rheb in regulating cell cycle. Simultaneously, the study
also revealed the critical role of farnesylation in maintaining cell cycle
progression. Thus, in both these systems, farnesylation was deemed
necessary for proper function of Rheb.
ACKNOWLEDGMENTS
We thank Angel Tabancay and Dr. Jun Urano for discussion. This work
is supported by NIH grant CA41996.
REFERENCES
1. Adamo, J. E., G. Rossi, and P. Brennwald. 1999. The Rho GTPase Rho3 has a direct
role in exocytosis that is distinct from its role in actin polarity. Mol. Biol. Cell. 10:4121–
4133.
2. Adjei, A. A., J. N. Davis, C. Erlichman, P. A. Svingen, and S. H. Kaufmann. 2000.
Comparison of potential markers of farnesyltransferase inhibition. Clin. Cancer Res.
6:2318–2325.
3. Ammerer, G. 1994. Sex, stress and integrity: the importance of MAP kinases in yeast.
Curr. Opin. Genet. Dev. 4:90–95.
4. Arellano, M., P. M. Coll, W. Yang, A. Duran, F. Tamanoi, and P. Perez. 1998.
Characterization of the geranylgeranyl transferase type I from Schizosaccharomyces
pombe. Mol. Microbiol. 29:1357–1367.
5. Arellano, M., P. M. Coll, and P. Perez. 1999. RHO GTPases in the control of cell
morphology, cell polarity, and actin localization in fission yeast. Microsc. Res. Tech.
47:51–60.
6. Aspuria, P-J., and F. Tamanoi. 2004. The Rheb family of GTP-binding proteins. Cell
Signal. 16:1105–1112.
7. Ayral-Kalowtian, S., and E.J. Salaski. 2002. Protein farnesyltransferase inhibitors.
Curr. Med. Chem. 9:1003–1032.
8. Balciunas, D., and H. Ronne. 1999. Yeast genes GIS1-4: multicopy suppressors of the
Gal- phenotype of snf1 mig1 srb8/10/11 cells. Mol. Gen. Genet. 262:589–599.
9. Bardwell, L., J. G. Cook, C. J. Inouye, and J. Thorner. 1994. Signal propagation and
regulation in the mating pheromone response pathway of the yeast Saccharomyces
cerevisiae. Dev. Biol. 166:363–379.
10. Bhattacharya, S., L. Chen, J. R. Broach, and S. Powers. 1995. Ras membrane
targeting is essential for glucose signaling but not for viability in yeast. Proc. Natl.
Acad. Sci. USA 92:2984–2988.
11. Boguski, M. S., A. W. Murray, and S. Powers. 1992. Novel repetitive sequence motifs
in the a and b subunits of prenyl-protein transferases and homology of the a subunit to
the MAD2 gene product of yeast. New Biol. 4:408–411.
12. Bos, J. L. 1989. ras oncogenes in human cancer:a review. Cancer Res. 49:4682–4689.
13. Bulawa, C. E. 1993. Genetics and molecular biology of chitin synthesis in fungi. Annu.
Rev. Microbiol. 47:505–534.
14. Cabib, E. 2000. On the zymogenic character of chitin synthase 3. Microbiology
146:1760–1761.
15. Caldwell, G. A., F. Naider, and J. M. Becker. 1995. Fungal lipopeptide mating
pheromones: a model system for the study of protein prenylation. Microbiol. Rev.
59:406–422.
16. Calonge, T. M., K. Nakano, M. Arellano, R. Arai, S. Katayama, T. Toda,
I. Mabuchi, and P. Perez. 2000. Schizosaccharomyces pombe rho2p GTPase regulates
cell wall a-glucan biosynthesis through the protein kinase pck2p. Mol. Biol. Cell.
11:4393–4401.
17. Caplan, A. J., and M. G. Douglas. 1991. Characterization of YDJ1: a yeast homologue
of the bacterial dnaJ protein. J. Cell. Biol. 114:609–621.
18. Caplan, A. J., J. Tsai, P. J. Casey, and M. G. Douglas. 1992. Farnesylation of YDJ1p
is required for function at elevated growth temperatures in Saccharomyces cerevisiae.
J. Biol. Chem. 267:18890–18895.
19. Casey, P. J., P. A. Solski, C. J. Der, and J. E. Buss. 1989. p21ras is modified by a
farnesyl isoprenoid. Proc. Natl. Acad. Sci. USA 86:8323–8327.
20. Chattopadhyay, S., and D. A. Pearce. 2002. Interaction with Btn2p is required for
localization of Rsg1p: Btn2p-mediated changes in arginine uptake in Saccharomyces
cerevisiae. Euk. Cell 1:606–612
21. Chen, W. J., D. A. Andres, J. L. Goldstein, D. W. Russell, and M. S. Brown. 1991.
cDNA cloning and expression of the peptide-binding β subunit of rat p21ras
farnesyltransferase, the counterpart of yeast DPR1/RAM1. Cell 66:327–334.
22. Chen, P., J. D. Choi, R. Wang, R. J. Cotter, and S. Michaelis. 1997. A novel a-
factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-
independent mechanism. Mol. Biol. Cell 8:1273–1291.
23. Clark, G. J., M. S. Kinch, K. Rogers-Graham, S. M. Sebti, A. D. Hamilton, and
C. J. Der. 1997. The Ras-related protein Rheb is farnesylated and antagonizes Ras
signaling and transformation. J. Biol. Chem. 272:10608–10615.
24. Clarke, S. 1992. Protein isoprenylation and methylation at carboxyl-terminal cysteine
residues. Annu. Rev. Biochem. 61:355–386.
25. Costello, G. L., L. Rodgers, and D. Beach. 1986. Fission yeast enters the stationary
phase G0 from either mitotic G1 or G2. Curr. Genet. 11:119–125.
26. Davey, J. 1992. Mating pheromones of the fission yeast Schizosaccharomyces pombe:
purification and structural characterization of M-factor and isolation and analysis of two
genes encoding the pheromone. EMBO J. 11:951–960.
27. DeFeo-Jones, D., E. M. Scolnick, R. Koller, and R. Dhar. 1983. ras-Related gene
sequences identified and isolated from Saccharomyces cerevisiae. Nature 306:707–709.
28. Del Villar, K., H. Mitsuzawa, W. Yang, I. Sattler, and F. Tamanoi. 1997. Amino
acid substitutions that convert the protein substrate specificity of farnesyltransferase to
that of geranylgeranyltransferase type I. J. Biol. Chem. 272:680–687.
29. Del Villar, K., J. Urano, L. Guo, and F. Tamanoi. 1999. A mutant form of human
protein farnesyltransferase exhibits increased resistance to farnesyltransferase inhibitors.
J. Biol. Chem. 274:27010–27017.
30. Diaz, M., Y. Sanchez, T. Bennett, C. R. Sun, C. Godoy, F. Tamanoi, A. Duran, and
P. Perez.1993. The Schizosaccharomyces pombe cwg2+ gene codes for the b subunit
of a geranylgeranyltransferase type I required for b-glucan synthesis. EMBO J.
12:5245–5254.
31. Dimitrov, K., and S. Sazer. 1998. The role of fnx1, a fission yeast multidrug resistance
protein, in the transition of cells to a quiescent G0 state. Mol. Cell. Biol. 18:5239–5246.
32. Fantes, P., and P. Nurse. 1977. Control of cell size at division in fission yeast by a
growth-modulated size control over nuclear division. Exp. Cell. Res. 107:377–386.
33. Finegold, A. A., W. R. Schafer, J. Rine, M. Whiteway, and F. Tamanoi. 1990.
Common modifications of trimeric G proteins and ras protein: involvement of
polyisoprenylation. Science 249:165–169.
34. Fujiyama, A., K. Matsumoto, and F. Tamanoi. 1987. A novel yeast mutant defective
in the processing of ras proteins: assessment of the effect of the mutation on processing
steps. EMBO J. 6:223–228.
35. Fukui, Y., T. Kozasa, Y. Kaziro, T. Takeda, and M. Yamamoto. 1986. Role of a ras
homolog in the life cycle of Schizosaccharomyces pombe. Cell 44:329–336.
36. Furfine, E. S., J. J. Leban, A. Landavazo, J. F. Moomaw, and P. J. Casey. 1995.
Protein farnesyltransferase: kinetics of farnesyl pyrophosphate binding and product
release. Biochemistry 34:6857–6862
37. Galan, J. M., A. Wiederkehr, J. H. Seol, R. Haguenauer-Tsapis, R. J. Deshaies,
H. Riezman, and M. Peter. 2001. Skp1p and the F-box protein Rcy1p form a non-SCF
complex involved in recycling of the SNARE Snc1p in yeast. Mol. Cell. Biol.
21:3105–3117.
38. Gibbs, J. B., and A. Oliff. 1997. The potential of farnesyltransferase inhibitors as
cancer chemotherapeutics. Annu. Rev. Pharmacol. Toxicol. 37:143–166.
39. Glomset, J. A., and C. C. Farnsworth. 1994. Role of protein modification reactions in
programming interactions between ras-related GTPases and cell membranes. Annu. Rev.
Cell. Biol. 10:181–205.
40. Goodman, L. E., C. M. Perou, A. Fujiyama, and F. Tamanoi. 1988. Structure and
expression of yeast DPR1, a gene essential for the processing and intracellular
localization of ras proteins. Yeast 4:271–281.
41. Goodman, L. E., S. R. Judd, C. C. Farnsworth, S. Powers, M. H. Gelb, J. A.
Glomset, and F. Tamanoi. 1990. Mutants of Saccharomyces cerevisiae defective in the
farnesylation of Ras proteins. Proc. Natl. Acad. Sci. USA 87:9665–9669.
42. Gotte, K., W. Girzalsky, M. Linkert, E. Baumgart, S. Kammerer, W. H. Kunau,
and R. Erdmann. 1998. Pex19p, a farnesylated protein essential for peroxisome
biogenesis. Mol. Cell. Biol. 18:616–628.
43. Gromov, P. S., P. Madsen, N. Tomerup, and J. E. Celis. 1995. A novel approach for
expression cloning of small GTPases: identification, tissue distribution and chromosome
mapping of the human homolog of rheb. FEBS Lett. 377:221–226.
44. Gustin, M. C., J. Albertyn, M. Alexander, and K. Davenport. 1998. MAP kinase
pathways in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62:1264–
1300.
45. Hancock, J. F., A. I. Magee, J. E. Childs, and C. J. Marshall. 1989. All ras proteins
are polyisoprenylated but only some are palmitoylated. Cell 57:1167–1177.
46. Hara, M., K. Akasaka, S. Akinaga, M. Okabe, H. Nakano, R. Gomez, D. Wood,
M. Uh, and F. Tamanoi. 1993. Identification of Ras farnesyltransferase inhibitors by
microbial screening. Proc. Natl. Acad. Sci. USA 90:2281–2285.
47. Hara, M., and M. Han. 1995. Ras farnesyltransferase inhibitors suppress the phenotype
resulting from an activated ras mutation in Caenorhabditis elegans. Proc. Natl. Acad.
Sci. USA 92:3333–3337.
48. He, B., P. Chen, S. Y. Chen, K. L. Vancura, S. Michaelis, and S. Powers. 1991.
RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components
of the farnesyltransferase that prenylates a-factor and Ras proteins. Proc. Natl. Acad.
Sci. USA 88:11373–11377.
49. Herskowitz, I. 1995. MAP kinase pathways in yeast: for mating and more. Cell 80:187–
197.
50. Hettema, E. H., W. Girzalsky, M. van Den Berg, R. Erdmann, and B. Distel. 2000.
Saccharomyces cerevisiae pex3p and pex19p are required for proper localization and
stability of peroxisomal membrane proteins. EMBO J. 19:223–233.
51. Hightower, K. E., and C. A. Fierke. 1999. Zinc-catalyzed sulfur alkyation:insights
from protein farnesyltransferase. Curr. Opin. Chem. Biol. 3:176–181.
52. Hirschman, J. E., and D. D. Jenness. 1999. Dual lipid modification of the yeast
Gv subunit Ste18p determines membrane localization of Gβv. Mol. Cell. Biol.
19:7705–7711.
53. Im, E., F. C. von Lintig, J. Chen, S. Zhuang, W. Qui, S. Chowdhury, P. F. Worley,
G. R. Boss, and R. B. Pilz. 2002. Rheb is in a high activation state and inhibits B-Raf
kinase in mammalian cells. Oncogene 21:6356–6365.
54. Ito, T., S. Kawata, S. Tamura, T. Igura, T. Nagase, J. I. Miyagawa, E. Yamazaki,
H. Ishiguro, and Y. Matasuzawa. 1996. Suppression of human pancreatic cancer
growth in BALB/c nude mice by manumycin, a farnesyl:protein transferase inhibitor.
Jpn. J. Cancer Res. 87:113–116.
55. Johnson, J. L., and E. A. Craig. 2001. An essential role for the substrate-binding
region of Hsp40s in Saccharomyces cerevisiae. J. Cell. Biol. 152:851–856.
56. Kainuma, O., T. Asano, M. Hasegawa, T. Kenmochi, T. Nakagohri, Y. Tokoro, and
K. Isono. 1997. Inhibition of growth and invasive activity of human pancreatic cancer
cells by a farnesyltransferase inhibitor, manumycin. Pancreas 15:379–383.
57. Karp, J. E., S. H. Kaufmann, A. A. Adjei, J. E. Lancet, J. J. Wright, and D. W.

End. 2001. Current status of clinical trials of farnesyltransferase inhibitors. Curr. Opin.
Oncol. 13:470–476.
58. Kinouchi, H., S. Arai, H. Kamii, K. Izaki, H. Kunizuka, K. Mizoi, and
T. Yoshimoto. 1999. Induction of Rheb mRNA following middle cerebral artery
occlusion in the rat. Neuroreport 10:1055–1059.
59. Kita, K., Y. P. Wu, S. Sugaya, T. Moriya, J. Nomura, S. Takahashi, H. Yamamori,
N. Nakajima, and N. Suzuki. 2000. Search for UV-responsive genes in human cells by
differential mRNA display: involvement of human ras-related GTP-binding protein,
Rheb, in UV susceptibility. Biochem. Biophys. Res. Commun. 274:859–864.
60. Kjaerulff, S., J. Davey, and O. Nielsen. 1994. Analysis of the structural genes
encoding M-factor in the fission yeast Schizosaccharomyces pombe: identification of a
third gene, mfm3. Mol. Cell. Biol. 14:3895–3905.
61. Kohl, N. E., R. E. Diehl, M. D. Schaber, E. Rands, D. D. Soderman, B. He, S. L.
Moores, D. L. Pompliano, S. Ferro-Novick, S. Powers, K. A. Thomas, and J. B.
Gibbs. 1991. Structural homology among mammalian and Saccharomyces cerevisiae
isoprenyl-protein transferases. J. Biol. Chem. 266:18884–18888.
62. Kohl, N. E., C. A. Omer, M. W. Conner, N. J. Anthony, J. P. Davide, S. J. deSolms,
E. A. Giuliani, R. P. Gomez, S. L. Graham, K. Hamilton, L. K. Handt, G. D.
hartman, K. S. Koblan, A. M. Kral, P. J. Miller, S. D. Mosser, T. J. O’Neill,
E. Rands, M. D. Schaber, J. B. Gibbs, and A. Oliff. 1995. Inhibition of
farnesyltransferase induces regression of mammary and salivary carcinomas in ras
transgenic mice. Nat. Med. 1:792–797.
63. Kuroda, Y., N. Suzuki, and T. Kataoka. 1993. The effect of posttranslational
modifications on the interaction of Ras2 with adenylyl cyclase. Science 259:683–686.
64. Lee, D. H., M. Y. Sherman, and A. L. Goldberg. 1996. Involvement of the molecular
chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal
proteins in Saccharomyces cerevisiae. Mol. Cell. Biol. 16:4773–4781.
65. Long, S. B., P. J. Casey, and L. S. Beese. 2002. Reaction path of protein farnesyl-
transferase at atomic resolution. Nature 419:645–650.
66. Lowy, D. R., and B. M. Willumsen. 1993. Function and regulation of ras. Annu. Rev.
Biochem. 62:851–891.
67. Lu, Z., and D. M. Cyr. 1998. Protein folding activity of Hsp70 is modified differentially
by the hsp40 co-chaperones Sis1 and Ydj1. J. Biol. Chem. 273:27824–27830.
68. Mach, K. E., K. A. Furge, and C. F. Albright. 2000. Loss of Rhb1, a Rheb-related
GTPase in fission yeast, causes growth arrest with a terminal phenotype similar to that
caused by nitrogen starvation. Genetics 155:611–622.
69. McNew, J. A., M. Sogaard, N. M. Lampen, S. Machida, R. R. Ye, L. Lacomis,
P. Tempst, J. E. Rothman, and T. H. Sollner. 1997. Ykt6p, a prenylated SNARE
essential for endoplasmic reticulum-Golgi transport. J. Biol. Chem. 272:17776–17783.
70. Michaelis, S., and I. Herskowitz. 1988. The a-factor pheromone of Saccharomyces
cerevisiae is essential for mating. Mol. Cell. Biol. 8:1309–1318.
71. Mitsuzawa, H., K. Esson, and F. Tamanoi. 1995. Mutant farnesyltransferase β subunit
of Saccharomyces cerevisiae that can substitute for geranylgeranyltransferase type I β
subunit. Proc. Natl. Acad. Sci. USA 92:1704–1708.
72. Mitsuzawa, H., and F. Tamanoi. 1995. In vivo assays for farnesyltransferase inhibitors
with Saccharomyces cerevisiae. Meth. Enzymol. 250:43–51.
73. Mizuki, N., M. Kimura, S. Ohno, S. Miyata, M. Sato, H. Ando, M. Ishihara,
K. Goto, S. Watanabe, M. Yamazaki, A. Ono, S. Taguchi, K. Okumura, M.
Nogami, T. Taguchi, A. Ando, and H. Inoko. 1996. Isolation of cDNA and genomic
clones of a human Ras-related GTP-binding protein gene and its chromosomal
localization to the long arm of chromosome 7, 7q36. Genomics 34:114–118.
74. Moomaw, J. F., and P. J. Casey. 1992. Mammalian protein geranylgeranyltransferase.

Subunit composition and metal requirements. J. Biol. Chem. 267:17438–17443.
75. Mosch, H. U., E. Kubler, S. Krappmann, G. R. Fink, and G. H. Braus. 1999.
Crosstalk between the Ras2p-controlled mitogen-activated protein kinase and cAMP
pathways during invasive growth of Saccharomyces cerevisiae. Mol. Biol. Cell.
10:1325–13235.
76. Ohya, Y., M. Goebl, L. E. Goodman, S. Petersen-Bjorn, J. D. Friesen, F. Tamanoi,
and Y. Anraku. 1991. Yeast CAL1 is a structural and functional homologue to the
DPR1(RAM) gene involved in ras processing. J. Biol. Chem. 266:12356–12360.
77. Panepinto, J. C., B. G. Oliver, T. W. Amlung, D. S. Askew, and J. C. Rhodes. 2002.
Expression of the Aspergillus fumigatus rheb homologue, rhbA, is induced by nitrogen
starvation. Fungal Genet. Biol. 36:207–214.
78. Patel, P. M., N. Thapar, L. Guo, M. Martinez, J. Maris, C-L. Gau, J. A. Legnyel,
and F. Tamanoi. 2003. Dresphilia Rheb GiPase is required for cell cycle progressin and
cell growth. J. Cell Sci. 116:3601–3610.
79. Powers, S., T. Kataoka, O. Fasano, M. Goldfarb, J. Strathern, J. Broach, and
M. Wigler. 1984. Genes in S. cerevisiae encoding proteins with domains homologous to
the mammalian ras proteins. Cell 36:607–612.
80. Powers, S., S. Michaelis, D. Broek, S. Santa Anna, J. Field, I. Herskowitz, and
M. Wigler. 1986. RAM, a gene of yeast required for a functional modification of RAS
proteins and for production of mating pheromone a-factor. Cell 47:413–422.
81. Pryciak, P. M., and F. A. Huntress. 1998. Membrane recruitment of the kinase
cascade scaffold protein Ste5 by the Gβv complex underlies activation of the yeast
pheromone response pathway. Genes Dev. 12:2684–2697.
82. Reid, T. S., K-L. Terry, P. J. Casey, and L. S. Beese. 2004. Crystallographic analysis
of Caax fenyltransferase complexed with substrates defines rules of protein substrate
selectivity. J. Mol. Biol. 343:417–433.
83. Robinson, N. G., L. Guo, J. Imai, E. A. Toh, Y. Matsui, and F. Tamanoi. 1999.
Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and
exocytosis, is a GTPase which interacts with Myo2 and Exo70. Mol. Cell. Biol.
19:3580–3587.
84. Sattler, I., and F. Tamanoi. 1996. Prenylation of RAS and inhibitors of
prenyltransferases, pp. 95–137. In H. Maruta and A. W. Burgess (eds.), Regulation of
the RAS Signalling Network. Wiley-Liss, New York.
85. Schafer, W. R., R. Kim, R. Sterne, J. Thorner, S. H. Kim, and J. Rine. 1989.
Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast
and humans. Science 245:379–385.
86. Schafer, W. R., and J. Rine. 1992. Protein prenylation: genes, enzymes, targets, and
functions. Annu. Rev. Genet. 26:209–237.
87. Schwarz, E., B. Westermann, A. J. Caplan, G. Ludwig, and W. Neupert. 1994.
XDJ1, a gene encoding a novel non-essential DnaJ homologue from Saccharomyces
cerevisiae. Gene 145:121–124.
88. Seabra, M. C. 2001. Biochemistry of Rab Geranylgeranyltransferase, p. 130–154. In
F. Tamanoi, and D. S. Sigman (eds.), The Enzymes, vol.21. Academic Press, San Diego.
89. Shirayama, M., Y. Matsui, and E. A. Toh. 1994. The yeast TEM1 gene, which
encodes a GTP-binding protein, is involved in termination of M phase. Mol. Cell. Biol.
14:7476–7482.
90. Spence, R. A., K. E. Hightower, K. L. Terry, L. S. Beese, C. A. Fierke, and P. J.
Casey. 2000. Conversion of Tyr361β to Leu in mammalian protein farnesyltransferase
impairs product release but not substrate recognition. Biochemistry 39: 13651–13659.
91. Spence, R. A., and P. J. Casey. 2001. Mechanism of catalysis by protein
farnesyltransferase, p. 1–18. In F. Tamanoi and D. S. Sigman (eds.), The Enzymes,
vol.21. Academic Press, San Diego.
92. Strickland, C. L., W. T. Windsor, R. Syto, L. Wang, R. Bond, Z. Wu, J. Schwartz,

H. V. Le, L. S. Beese, and P. C. Weber. 1998. Crystal structure of farnesyl protein
transferase complexed with a CaaX peptide and farnesyl diphosphate analogue.
Biochemistry. 37:16601–16611.
93. Su, S. S., Y. Tanaka, I. Samejima, K. Tanaka, and M. Yanagida. 1996. A nitrogen
starvation-induced dormant G0 state in fission yeast: the establishment from uncommitted
G1 state and its delay for return to proliferation. J. Cell. Sci. 109:1347–1357.
94. Tamanoi, F., C. L. Gau, C. Jiang, H. Edamatsu, and J. Kato-Stankiewicz. 2001.
Protein farnesylation in mammalian cells: effects of farnesyltransferase inhibitors on
cancer cells. Cell. Mol. Life. Sci. 58:1636–1649.
95. Terry, K. L., S. B. Long, and L. S. Beese. 2001. Structure of protein
farnesyltransferase, p. 19–46. In F. Tamanoi, and D. S. Sigman (eds.), The Enzymes,
vol.21. Academic Press, San Diego.
96. Thoma, N. H., A. Iakovenko, R. S. Goody, and K. Alexander. 2001.
Phosphoisoprenoids modulate association of Rab geranylgeranyltransferase with REP-1.
J. Biol. Chem. 276:48637–48643.
97. Tobin, D. A., J. S. Pickett, H. L. Hartman, C. A. Fierke, and J. C. Penner-Hahn.
2003. Structural characterization of the zinc cite in protein farnesyltransferase. J. Am.
Chem. Soc. 125:9962–9969.
98. Toda, T., I. Uno, T. Ishikawa, S. Powers, T. Kataoka, D. Broek, S. Cameron,
J. Broach, K. Matsumoto, and M. Wigler. 1985. In yeast, RAS proteins are
controlling elements of adenylate cyclase. Cell 40:27–36.
99. Trueblood, C. E., V. L. Boyartchuk, E. A. Picologlou, D. Rozema, C. D. Poulter,
and J. Rine. 2000. The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct
substrate specificities. Mol. Cell. Biol. 20:4381–4392.
100. Tsui, M. M., W. C. Tai, and D. K. Banfield. 2001. Selective formation of Sed5p-
containing SNARE complexes is mediated by combinatorial binding interactions. Mol.
Biol. Cell. 12:521–538.
101. Urano, J., A. P. Tabancay, W. Yang, and F. Tamanoi. 2000. The Saccharomyces
cerevisiae Rheb G-protein is involved in regulating canavanine resistance and arginine
uptake. J. Biol. Chem. 275:11198–11206.
102. Urano, J., C. Ellis, G. J. Clark, and F. Tamanoi. 2001. Characterization of Rheb
functions using yeast and mammalian systems. Meth. Enzymol. 333:217–231.
103. Whiteway, M. S., and D. Y. Thomas. 1994. Site-directed mutations altering the CAAX box
of Ste18, the yeast pheromone-response pathway Gv subunit. Genetics 137:967–976.
104. Yamagata, K., L. K. Sanders, W. E. Kaufmann, W. Yee, C. A. Barnes, D. Nathans,
and P. F. Worley. 1994. rheb, a growth factor- and synaptic activity-regulated gene,
encodes a novel Ras-related protein. J. Biol. Chem. 269:16333–16339.
105. Yang, W., J. Urano, and F. Tamanoi. 2000. Protein farnesylation is critical for
maintaining normal cell morphology and canavanine resistance in Schizosaccharomyces
pombe. J. Biol. Chem. 275:429–438.
106. Yang, W., A. P. Tabancay, Jr., J. Urano, and F. Tamanoi. 2001. Failure to
farnesylate Rheb protein contributes to the enrichment of G0/G1 phase cells in
the Schizosaccharomyces pombe farnesyltransferase mutant. Mol. Microbiol.
41:1339–1347.
107. Yee, W. M., and P. F. Worley. 1997. Rheb interacts with Raf-1 kinase and may
function to integrate growth factor- and protein kinase A-dependent signals. Mol. Cell.
Biol. 17:921–933.
108. Yokoyama, K., and M. H. Gelb. 2001. Protein geranylgeranyltransferase type I,
pp. 105–130. In F. Tamanoi and D. S. Sigman (eds.), The Enzymes, vol.21. Academic
Press, San Diego.
109. Zhang, F. L., and P. J. Casey. 1996. Protein prenylation: molecular mechanisms and
functional consequences. Annu. Rev. Biochem. 65:241–269.
Chapter 5
FROM BREAD TO BEDSIDE: WHAT BUDDING
YEAST HAS TAUGHT US ABOUT THE
IMMORTALIZATION OF CANCER CELLS
Soma S.R. Banik and Christopher M. Counter

Departments of Pharmacology and Cancer Biology and Radiation Oncology, Duke University
Medical Center, Box 3813, Durham, NC, USA 27710
1 INTRODUCTION
The budding yeast Saccharomyces cerevisiae is a formidable model

system indeed. With the entire genome sequenced, unparalleled genetic
malleability, and an eukaryotic background, this system is virtually beyond
compare for studying the multitude of biological pathways that are
conserved amongst eukaryotes. Importantly with regards to human cancer,
many of the cellular processes of the mammalian cell can be found,
admittedly in a stripped-down version, in yeast. In this regard, yeasts are
fertile ground for elucidating mechanisms and identifying the key players in
cellular processes. This invaluable information can then act as a guide for
the cancer cell biologist attempting to navigate the analogous pathway in the
far more complex and elaborate system of the mammalian cell. One example
of where yeast has been used in this regard is in understanding how cancer
cells acquire the ability to divide indefinitely. Here we highlight the
enormous contributions made by studies performed in the model system of
S. cerevisiae to our understanding of this tumourigenic process.
2 CELLULAR IMMORTALIZATION
It has long been appreciated that normal human somatic cells adapted to
grow outside the body, or in culture, divide a limited number of times [29,
123
© 2007 Springer.
124 S. S. R. Banik and C. M. Counter
40]. In contrast, cancer cells have the potential to divide indefinitely,

displaying immortality [6]. HeLa cells are perhaps the best example of this.
First isolated in 1951 from a cervical cancer specimen, these cells have been
cultured to this day in one form or another in laboratories around the world
[56]. The aberrantly long lifespan unique to cancer cells in culture suggests
that the immortalization process may represent a key event in tumouri-
genesis. Indeed, the clonal nature of tumour progression supports the notion
that cancer cells undergo numerous cycles of cell division in the process of
giving rise to a tumour [12, 25, 26, 79]. This basic and fundamental difference
between normal cells and cancer cells suggests the tantalizing possibility that
understanding the cellular mechanisms of cell immortalization might reveal
novel molecular targets that might be exploited in cancer therapy. Importantly,
as is the case for other therapeutic targets, much of the groundwork for under-
standing these processes was first established by studying the analogous
system in yeast.
3 TELOMERES
Remarkably, one of the key determinants of cellular immortalization is

found in non-coding regions at the extreme ends of chromosomes. These
terminal telomere structures are complex with a host of specialized cellular
proteins to cap and protect the ends of eukaryotic chromosomes from
illegitimate recombination and degradation [9]. The telomere itself ends in a
3′ single-stranded overhang that invades the double-stranded telomeric tract,
forming a t-loop configuration that is thought to constitute an essential
structure for chromosome protection [33]. The proteins that complex with
telomeric DNA do so in an organized and structured fashion. For example,
in humans, hTRF1 is known to bind to the double-stranded region [11, 85],
whereas the protein hTRF2 is found to foster the formation of a triplex DNA
structure where the terminal 3′ overhang is tucked into the double-stranded
telomere tract [33]. Lastly, it has been suggested that the telomeric overhang
may be bound by the protein hPot1 [8]. To add an additional layer of
complexity to this structure, many other proteins then associate with these
core telomere-binding proteins [9]. Together, the DNA and associated
proteins form a functional telomere. In the absence of functional telomeres,
mammalian chromosomes become highly unstable and recombinogenic,
resulting in the formation of dicentric and ring chromosomes [16, 41].
Indeed, the protective nature of telomeric DNA was characterized in yeast
where cloned telomeric repeats were shown to have the property of
stabilizing linear DNA [74]. In fact, telomeric sequences provided a final
essential structure for the formation of artificial yeast chromosomes (YACs),
now an integral tool in mammalian genetics [60].
Chapter 5: From Bread to Bedside 125
Complete replication of telomeric DNA must contend with two hurdles.

First, the most terminal primer for lagging strand replication leaves a gap
following removal of the RNA primer but that cannot be filled in by any
conventional DNA polymerase, resulting in telomere shortening [64, 80].
Second, leading strand synthesis forms a flush end that must be processed to
produce the G-strand overhang necessary to produce the telomeric loop
structure, presumably by the partial degradation of the C-strand, again
resulting in a net loss of telomeric DNA [82]. Thus, another feature of
telomeres must be a unique mode of replication, as continual erosion of
chromosomes clearly cannot be tolerated over time, from one generation to
the next. In fact, this process of telomere shortening also limits the
maximum lifespan of normal human somatic cells [10, 37, 76].
4 TELOMERE LENGTH DICATES THE LIFESPAN

OF HUMAN CELLS
The inability to completely replicate the ends of chromosomes was first

postulated to account for the finite lifespan of human somatic cells in 1971
[64]. Twenty years after this hypothesis was put forth, it was demonstrated
that telomeres in normal somatic cells were far shorter than those found in
germ cells, which are considered to be “biologically immortal” [15]. The actual
process of telomere shortening during DNA replication was subsequently
documented in cell culture and in vivo. Specifically, it was shown that
telomere length decreased with increasing cell division in cultured human
fibroblasts [37], or with increasing donor age in hematopoetic cells [39].
Indeed, telomere shortening with cell division or donor age has now been
documented in a variety of other human somatic cells [7, 13, 22, 45, 46, 58,
73, 77, 78, 83]. In the case of cells growing in culture, it is now apparent that
the proliferative capacity of the culture is directly proportional to the
telomere length of the founding cells and the number of chromosome
replications, as opposed to time, further supporting the model that telomere
shortening dictates cell lifespan [1–3].
Although these observations made in mammalian cells were telling, it was
in yeast that the most direct and compelling evidence was found to suggest
that continual telomeric erosion would ultimately limit cellular lifespan. In
an elegant yeast genetic screen undertaken by Lundblad and Szostak, a yeast
mutant was identified that lost the ability to maintain telomere length [55].
Since this mutation caused ever shortening telomeres, the gene harbouring
this mutation was named EST1. The specific importance of the EST1 protein
was ascertained by the direct genetic disruption of the EST1 gene, which
lead to telomere shortening and eventually a severe compromise in cellular
viability. Thus, the ability to easily disrupt genes in yeast provided the first
direct evidence for a relationship between telomere length and cellular

lifespan in eukaryotic cells [55].
5 TELOMERE SHORTENING IS ARRESTED

IN IMMORTAL CANCER CELLS
As per the model predicting that telomere shortening limits cellular

lifespan, it follows that immortal cancer cells must employ a specialized
mechanism to overcome the problem associated with telomere shortening.
This phenomenon of cellular immortalization and the pathways involved can
be recapitulated in transformed cells growing in culture. Here, somatic cells
are driven to divide beyond their normally allotted lifespan by perturbing the
function of cell cycle regulatory molecules such as p53, p21, and Rb [67, 71].
As telomeres in these cells nonetheless do continue to shorten, a period of
severe genomic instability ensues eventually leading to massive cell death.
This cycle of instability and cell death selects for rare cells that have
acquired genetic alterations that allow for indefinite proliferation. Telomere
length analysis of these cells revealed that the mortal cells in the population
perished with extremely short and unstable telomeres, whereas telomere
shortening and gross genetic instability were suspended in the immortal cell
clones [16]. This phenomenon of telomere maintenance appeared to suggest
a general mechanism, as it was independent of both cell type, and the
mechanism used to derail normal cellular growth regulatory mechanisms
[16, 17, 47, 69, 75]. Like the immortalized cells, cancer cells have also been
shown to maintain telomere length in culture, presumably reflecting a
similar selection of mutations that stabilizes telomere length in vivo [6, 18].
Taken together, it was proposed that if unchecked, loss of telomeric DNA
would be lethal, as already noted in yeast; however cancer cells can
surmount this proliferative blockade through the illegitimate activation of a
telomere maintenance mechanism [16].
6 TELOMERASE
In 1985 Greider and Blackburn published a seminal paper in which they

identified a biochemical activity in extracts isolated from the single-celled
ciliate Tetrahymena. This activity could elongate a single-stranded telo-
meric oligonucleotide by the addition of telomeric repeats [31]. The
discovery of this “telomere terminal transferase” or “telomerase” activity in
Tetrahymena established a foundation for studying telomere biology in
simple-model organisms such as ciliates and yeast. The telomerase RNA
subunit was subsequently cloned and shown to contain a telomeric template

[32], which was copied by a protease-sensitive subunit onto telomeres as
DNA, suggesting that the catalytic subunit functions as a reverse trans-
criptase. Later, it was shown that telomerase was present in a human cell line
[59], and that the enzyme was activated when cells became immortal [16].
Importantly, telomerase activity was not detectable in extracts prepared from
normal somatic cells but was found in extracts prepared from actual cancer
cells [18, 44, 68]. We now know that telomerase activity counteracts
telomere shortening in aberrantly proliferating cancer cells and allows them
to escape the “telomere barrier” to cell division. In fact, telomerase activity
has been detected in thousands of tumours from every cancer type, making
activation of this enzyme one of the most common changes in human
cancers [68].
Understanding how telomerase is activated during cancer, and what
components comprise the enzyme is critical in elucidating its role in cancer,
and in developing ways to target the immortalization process in cancer cells.
As mentioned, the enzyme was known to be composed of at least two
subunits, an RNA and a protease-sensitive subunit. To account for the
activation of telomerase activity in cancer cells, it was hypothesized that one
or both of the genes encoding these subunits were transcriptionally silent in
normal cells, but upregulated during tumourigenesis. The first subunit
identified in humans was the RNA, whose expression was found to be
ubiquitous [27]. This left the possibility that the protease-sensitive subunit(s)
were normally repressed in somatic cells, but upregulated in cancers. To test
this hypothesis, it was of utmost importance to clone the catalytic subunit of
telomerase.
7 YEAST TO THE RESCUE
The cloning of the catalytic subunit of telomerase proved to be a far more

formidable task than the cloning of the RNA subunit. A number of putative
protein catalytic subunits proved to be red herrings as telomerase activity
could not be attenuated upon protein ablation. The difficulty proved to be
related to the exceedingly low level of protein expression in most sys
tems. Comprehensive gene expression studies estimate that only 1 to 2 tran-
scripts encoding this subunit are found in cancer cells [49]. Such low levels
made biochemical purification of human telomerase or transcript-based
comparisons to identify the catalytic subunit transcript challenging. Such
low expression levels, however, do not pose a problem for genetic-based
searches in yeast. Furthermore, the identification of the yeast protein could
then be used to seek out the human homologue from human genome or
expressed sequence tag sequence databases, or as a probe to screen libraries

for homologues from progressively more complex eukaryotes.
Four features of the budding yeast cerevisiae made it an ideal system to
search for the catalytic subunit. First, sequencing of the yeast genome was
under full steam at the time that the catalytic subunit was cloned, providing a
seamless union between genetic screens and gene identification. Second, an
assay had been developed to detect telomerase activity in cerevisiae, which
would be essential to test if a candidate gene actually encoded the catalytic
subunit of telomerase [14]. Third, the unparalleled genetic malleability of
cerevisiae, allowed genetic screens to be done that would be impossible in
other organisms. Fourth, the cloning of the cerevisiae telomerase RNA
subunit allowed for the characterization a phenotype associated with a loss
of telomerase activity. Loss of this RNA-encoding gene, termed TLC1, gave
rise to telomere shortening and a decrease in cell viability, a phenotype
easily scored [72]. All of these features proved to be invaluable in the
cloning of the catalytic subunit of telomerase.
8 GENETIC AND BIOCHEMICAL APPROACHES

IDENTIFY THE YEAST CATALYTIC SUBUNIT
OF TELOMERASE
To definitively clone the catalytic subunit of telomerase, two parallel

approaches were employed. In both experimental schemes, the use of yeast
as a model system was paramount to the identification of this elusive
protein. One of these experimental paradigms involved the initial
biochemical purification of telomerase from the ciliate Euplotes crassus –
yet another simple model organism specifically used for its high level of
telomerase activity. Here, the telomerase complex was purified using an
oligonucleotide complementary to the telomerase RNA template [51].
Intriguingly, upon microsequence analysis, one of the proteins purified
through this approach proved to show sequence homology with the Est2
protein [53] previously shown to lie in the telomere maintenance pathway in
S. cerevisiae [50]. This suggested that the Euplotes protein p123 as well as
Est2 may in fact function as the catalytic protein of the telomerase enzyme
[53].
The second approach used to clone the catalytic subunit of telomerase was
based completely on a genetic screen performed in yeast. This screen was
designed in consideration of the two mechanisms used by yeast to maintain
telomeres, namely telomerase-mediated addition of telomeric repeats via
copying of a telomeric template, and the RAD52-mediated recombination of
telomeric elements. Importantly, the genetic ablation of both of these
telomere-maintaining pathways was known to induce yeast to undergo

senescence and cell death [54, 72]. This known phenomenon was exploited
in order to reveal the catalytic subunit of telomerase. As such, yeast as were
randomly mutated in hopes of disrupting the gene encoding the telomerase
catalytic subunit. The mutant yeasts were then induced to spontaneously
extrude the RAD52 gene involved in telomere recombination. Those yeast as
that did not survive in the absence of this second telomere-maintaining gene,
were presumed to harbour a telomere defect, and thus were screened for the
presence of short telomeres and an absence of the in vitro primer-elongating
activity characteristic of the telomerase enzyme. Two populations of mutant
yeasts were found to satisfy the telomerase-defining criteria outlined above,
thus identifying genes-encoding components of the core telomerase enzyme.
The first of these genes was TLC1, encoding the previously identified
telomerase RNA. The second was the gene encoding the Est2 protein [20].
Thus, through an entirely independent, experimental approach, EST2 was
predicted to encode the protein catalytic subunit of telomerase.
To prove that the Est2 protein was the catalytic subunit of telomerase,
both studies relied heavily upon genetic manipulations that were easily
accomplished in yeast, but terribly difficult in other eukaryotes. As EST2
was shown to contain weak sequence homology with reverse transcriptases,
invariant amino acids in the putative catalytic core of Est2p were specifically
mutated to decipher possible telomeric effects [20, 53]. Indeed, mutations of
these conserved amino acids resulted in senescence, telomere shortening,
and importantly, loss of telomerase catalytic activity. These experiments
conclusively demonstrated that Est2p was not only essential for telomerase
activity, but actually encoded the catalytic subunit of telomerase.
9 TELOMERASE IS ESSENTIAL FOR

IMMORTALIZATION AND TUMOURIGENESIS
Having first identified the telomerase catalytic subunit from model

eukaryotic organisms, the cloning of human TERT followed almost
immediately. As is the case for numerous proteins that show sequence
homology between lower eukaryotes and vertebrates, telomerase subunits
from Euplotes and yeast were successfully used to identify the human
hTERT homologue through databases searches of human gene sequences
[38, 43, 57, 61]. Clearly, in the absence of such information about the yeast
and ciliate proteins, the cloning of hTERT would have been an incredibly
tedious endeavour, slowed by the inability to make rapid genetic
manipulations in mammalian cells. Instead, once again, the characterization
of yeast and ciliate proteins was able to catapult cancer researchers into the
realm of elucidating uncharted cancer processes in humans. Accordingly,
upon the cloning of hTERT, the pace of research in cancer and cellular
immortalization hastened dramatically. First, it was shown that the hTERT
mRNA was indeed upregulated during the immortalization process in
cultured cells, and present in cancer cells but generally not in normal
somatic cells [43, 48, 57, 61]. More importantly, ectopic expression of the
hTERT cDNA in telomerase-negative mortal cells was shown to restore
telomerase activity, arrest telomere shortening and immortalize cells,
resembling all of the characteristics of telomerase function that had already
been delineated in yeast [10, 19, 38, 62, 76, 81].
The ability to immortalize normal human cells has now given rise to a
plethora of opportunities with respect to both research and therapeutics. For
example, isogenic normal cells from patients can now be immortalized to
provide isogenic controls for cancer cell lines isolated from tumours from
the same patient, thus representing an invaluable resource for assessing a
normal baseline in studies of drug action and gene activation [28]. In
addition, the ability to immortalize normal cells has widespread applications
beyond the realm of cancer research. In the case of often rare genetic
disorders, fibroblasts can be isolated and stably immortalized to provide an
unlimited supply of cells that can be used for disease characterization and to
develop possible therapeutic interventions [65]. With respect to tissue
engineering, activation of telomerase in normal cells may prove to be
important for increasing the proliferative lifespan necessary for making
genetic manipulations in vitro [70].
While the role of telomerase had been documented in the process of cell
immortalization, its function in tumourigenesis was conclusively established
in the first study that converted normal cells into cancer cells through the
introduction of defined genetic elements. Human kidney or mammary
epithelial, fibroblast, or astrocyte cells were made to be tumourigenic upon
infection with the SV40 early region to derail cell cycle checkpoints,
oncogenic ras to provide proliferative signals, and hTERT to allow for
cellular immortalization [23, 35, 66]. These cells clearly showed hallmarks
of cancer, namely, having the capability to form colonies in a semi-solid
medium and giving rise to tumours upon injection into nude mice; however,
these phenotypes were markedly absent in cells not infected with hTERT.
Thus, the link between the processes of hTERT expression, cellular immor-
talization, and tumourigenesis was firmly established.
10 CHARACTERIZATION OF YEAST TELOMERASE

PROVIDES THE FIRST METHOD TO INHIBIT
THE HUMAN ENZYME IN CANCER CELLS
Telomerase is activated in approximately 85% of human cancers [68] in a

wide variety of tumour types where its function is critical for cellular
immortalization. In contrast, most normal cells lack telomerase activity.
Thus, it stands to reason that the inhibition of telomerase could represent a
novel and selective strategy of killing tumour cells via induction of telomere
shortening and cellular crisis, whilst having minimal detrimental effects on
normal tissue. To test this possibility, researchers were again guided by
experiments done first in yeast. Specifically, expression of a catalytically
inactive Est2p protein in otherwise normal telomerase-positive yeast, led to
the shortening of telomeres. This phenotype suggested that the catalytically
dead protein could act as a dominant-negative by sequestering factors away
from the active enzyme [53]. To test this, the same mutation that inactivated
telomerase catalytic activity in yeast was made in hTERT, and was found to
abrogate the catalytic activity of the enzyme [62, 81]. Ectopic expression of
this mutant did in fact have a dominant-negative effect in telomerase-
positive transformed or tumour cell lines, where it reduced or even abolished
measurable telomerase activity. Successful expression of the dominant-
negative in human cells also led to telomere shortening and even cell death
[30, 36, 84]. Notably, when injected into nude mice, cancer cells expressing
this dominant-negative hTERT, did not produce tumours, or formed only
small tumours that regressed [34, 36]. Importantly, telomerase-negative cells
were unaffected by the expression of this dominant-negative protein [36].
These experiments, which find their origins in yeast, have now spawned
efforts to generate small molecule inhibitors of telomerase.
In an effort to recapitulate the phenotype of the dominant-negative version
of hTERT, several groups have attempted to inhibit telomerase enzyme
activity through a variety of experimental approaches. Perhaps the most
promising of these has been the use of a small-molecule telomerase inhibitor
[21]. Administration of this inhibitor to cancer cells in culture led to telo-
mere shortening and cellular senescence. Furthermore, drug-treated cells
also had a reduced tumourigenic potential when injected into nude mice. The
pursuit of telomerase inhibitors is an excellent example of how a phenotype
first documented in yeast has laid the foundation for the development of
compounds that might be clinically useful in the treatment of human disease.
11 BACK TO BASICS
Having identified the core components of human telomerase and the basic
mechanism through which telomerase functions, we are now poised to
dissect telomerase function and the mechanisms governing cancer cell
immortalization in more precise detail. In particular, to further understand
telomerase function, it will be necessary to elucidate the mechanism by
which telomerase-dependent elongation of telomeres occurs in immortal
human cells. Once again, studies done in yeast are leading the way.
Much work has been accomplished in yeast towards understanding one
mode of telomerase-dependent elongation of telomeres; namely to regulation
of telomerase access to telomeres. Specifically, several of the proteins now
known to be involved in telomere-targetting were in fact discovered in initial
genetic screens that identified the proteins Est1, Est2, Est3, and Cdc13
which function in the telomerase pathway [50, 55]. While Est1p has been
shown to associate with telomerase, it does not appear to directly target
telomerase to the telomere end. Cdc13p on the other hand, binds the extreme
end of the telomere, having high affinity for single-stranded telomeric DNA
[63]. This implies that telomerase may be recruited to telomere ends through
these two intermediate proteins. This model was further substantiated in
experiments where the fusion of Est1 and Cdc13 led to greatly elongated
telomeres [24], and fusion of the telomerase enzyme to Cdc13 was able to
bypass the requirement for a functional Est1 protein within the cell [24].
As the mechanism of telomerase recruitment may also be conserved
between yeast and humans, considerable effort is now being aimed at
understanding this process in human cells. Support for the existence of this
mode of telomerase regulation in humans has come from telomerase fusion
experiments done in human cells. Specifically, we identified a mutant of
hTERT that retained telomerase activity yet was unable to elongate
telomeres [4] – a phenotype reminiscent of yeast mutants defective in
telomerase recruitment [24, 42, 52]. To test if this mutant of hTERT was
indeed defective in telomere targetting, it was fused to the telomere-binding
protein TRF2. While the mutant hTERT protein alone was unable to
elongate telomeres or immortalize cells, the function of the mutant protein
was rescued upon artificially targetting it to telomeres via the TRF2 protein
[5], indicating that human telomerase is regulated by its access to telomeres.
Presumably such regulation is mediated by protein–protein interactions.
In this regard we note that the human protein hPot1 [8] may be a functional
homologue of the yeast protein Cdc13, which is involved in the recruitment
of telomerase to telomeres. While a possible telomerase recruitment function
for Pot1 analogous to that of Cdc13 remains speculative at this time, future
studies guided by the yeast literature will certainly help in defining this
function.
12 CONCLUSION
Cellular immortalization is one of the fundamental and defining

characteristics of cancer. In the majority of human cancers, immortalization
is also conferred by the telomere-maintaining activities of the enzyme
telomerase. As the telomerase pathway is highly conserved amongst almost
all eukaryotic organisms, many studies that have elucidated the function of
human telomerase have relied heavily on studies previously done in yeast.
As yeast constitute an excellent model system highly amenable to
elucidating genes responsible for a variety of cellular functions, many
proteins in the telomerase pathway including the catalytic subunit of
telomerase were first identified in yeast. Indeed, many proteins in the
telomerase pathway, including the catalytic subunit of telomerase, were first
identified in yeast. As new functions of telomerase are characterized and
new members of the telomerase pathway are identified, the yeast telomerase
pathway will certainly continue to be an invaluable system to aid in
elucidating the contribution of telomerase to cellular immortalization and
tumourigenesis.
ACKNOWLEDGMENTS
We would like to thank Dr. Raymund Wellinger for reviewing this

manuscript, and providing helpful comments.
REFERENCES
1. Allsopp, R. C., E. Chang, M. Kashefi-Aazam, E. I. Rogaev, M. A. Piatyszek, J.
W. Shay, and C. B. Harley. 1995. Telomere shortening is associated with cell
division in vitro and in vivo. Exp. Cell Res. 220:194–200.
2. Allsopp, R. C., and C. B. Harley. 1995. Evidence for a critical telomere length in
senescent human fibroblasts. Exp. Cell Res. 219:130–136.
3. Allsopp, R. C., H. Vaziri, C. Patterson, S. Goldstein, E. V. Younglai, A. B.

Futcher, C. W. Greider, and C. B. Harley. 1992. Telomere length predicts
replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA 89:10114–
10118.
4. Armbruster, B. N., S. S. Banik, C. Guo, A. C. Smith, and C. M. Counter. 2001.
N-terminal domains of the human telomerase catalytic subunit required for enzyme
activity in vivo. Mol. Cell. Biol. 21:7775–7786.
5. Armbruster, B. N., K. T. Etheridge, D. Broccoli, and C. M. Counter. 2002.
A putative telomere recruiting domain in the catalytic subunit of human telomerase.
submitted.
6. Bacchetti, S., and C. M. Counter. 1995. Telomeres and telomerase in human
cancer (Review). Int. J. Oncol. 7:423–432.
7. Bandyopadhyay, D., N. Timchenko, T. Suwa, P. J. Hornsby, J. Campisi, and E.
E. Medrano. 2001. The human melanocyte: a model system to study the
complexity of cellular aging and transformation in non– fibroblastic cells. Exp.
Gerontol. 36:1265–75.
8. Baumann, P., and T. R. Cech. 2001. Pot1, the putative telomere end-binding
protein in fission yeast and humans. Science 292:1171–1175.
9. Blackburn, E. H. 2001. Switching and signaling at the telomere. Cell 106:661–
673.
10. Bodnar, A. G., M. Ouellette, M. Frolkis, S. E. Holt, C. P. Chiu, G. B. Morin, C.
B. Harley, J. W. Shay, S. Lichtsteiner, and W. E. Wright. 1998. Extension of
life-span by introduction of telomerase into normal human cells. Science 279:349–
352.
11. Broccoli, D., A. Smogorzewska, L. Chong, and T. de Lange. 1997. Human
telomeres contain two distinct Myb-related proteins, TRF1 and TRF2. Nat. Genet.
17:231–235.
12. Cairns, J. 1975. Mutation selection and the natural history of cancer. Nature
255:197–200.
13. Chang, E., and C. B. Harley. 1995. Telomere length and replicative aging in
human vascular tissues. Proc. Natl. Acad. Sci. USA 92:11190–11194.
14. Cohn, M., and E. H. Blackburn. 1995. Telomerase in yeast. Science 269:396–
400.
15. Cooke, H. J., and B. A. Smith. 1986. Variability at the telomeres of the human
X/Y pseudoautosomal region. Cold Spring Harb. Symp. Quant. Biol. 1:213–219.
16. Counter, C. M., A. A. Avilion, C. E. Le Feuvre, N. G. Stewart, C. W. Greider,
C. B. Harley, and S. Bacchetti. 1992. Telomere shortening associated with
chromosome instability is arrested in immortal cells which express telomerase
activity. EMBO J. 11:1921–1929.
17. Counter, C. M., F. M. Botelho, P. Wang, C. B. Harley, and S. Bacchetti. 1994.
Stabilization of short telomeres and telomerase activity accompany immortalization
of Epstein-Barr virus-transformed human B lymphocytes. J. Virol. 68:3410–3414.
18. Counter, C. M., H. W. Hirte, S. Bacchetti, and C. B. Harley. 1994. Telomerase
activity in human ovarian carcinoma. Proc. Natl. Acad. Sci. USA 91:2900–2904.
19. Counter, C. M., M. Meyerson, E. N. Eaton, L. W. Ellisen, S. D. Caddle, D. A.
Haber, and R. A. Weinberg. 1998. Telomerase activity is restored in human cells
by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase.
Oncogene 16:1217–1222.
20. Counter, C. M., M. Meyerson, E. N. Eaton, and R. A. Weinberg. 1997. The

catalytic subunit of yeast telomerase. Proc. Natl. Acad. Sci. USA 94: 9202–9207.
21. Damm, K., U. Hemmann, P. Garin-Chesa, N. Hauel, I. Kauffmann, H.
Priepke, C. Niestroj, C. Daiber, B. Enenkel, B. Guilliard, I. Lauritsch, E.
Muller, E. Pascolo, G. Sauter, M. Pantic, U. M. Martens, C. Wenz, J. Lingner,
N. Kraut, W. J. Rettig, and A. Schnapp. 2001. A highly selective telomerase
inhibitor limiting human cancer cell proliferation. EMBO J. 20:6958–6968.
22. Decary, S., V. Mouly, C. B. Hamida, A. Sautet, J. P. Barbet, and G. S. Butler-
Browne. 1997. Replicative potential and telomere length in human skeletal muscle:
implications for satellite cell-mediated gene therapy. Hum. Gene. Ther. 8:1429–
1438.
23. Elenbaas, B., L. Spirio, F. Koerner, M. D. Fleming, D. B. Zimonjic, J. L.
Donaher, N. C. Popescu, W. C. Hahn, and R. A. Weinberg. 2001. Human breast
cancer cells generated by oncogenic transformation of primary mammary epithelial
cells. Genes Dev. 15:50–65.
24. Evans, S. K., and V. Lundblad. 1999. Est1 and Cdc13 as comediators of
telomerase access. Science 286:117–120.
25. Fairweather, D. S., M. Fox, and G. P. Margison. 1987. The in vitro lifespan of
MRC-5 cells is shortened by 5-azacytidine-induced demethylation. Exp. Cell. Res.
168:153–159.
26. Fearon, E. R., and B. Vogelstein. 1990. A genetic model for colorectal
tumorigenesis. Cell 61:759–767.
27. Feng, J., W. D. Funk, S. S. Wang, S. L. Weinrich, A. A. Avilion, C. P. Chiu, R.
R. Adams, E. Chang, R. C. Allsopp, J. Yu, S. Le, M. D. West, C. B. Harley, W.
H. Andrews, C. W. Greider, and B. Villeponteau. 1995. The RNA component of
human telomerase. Science 269:1236–1241.
28. Gazdar, A. F., V. Kurvari, A. Virmani, L. Gollahon, M. Sakaguchi, M.
Westerfield, D. Kodagoda, V. Stasny, H. T. Cunningham, Wistuba, II, G.
Tomlinson, V. Tonk, R. Ashfaq, A. M. Leitch, J. D. Minna, and J. W. Shay.
1998. Characterization of paired tumor and non-tumor cell lines established from
patients with breast cancer. Int. J. Cancer 78:766–774.
29. Goldstein, S. 1990. Replicative senescence: the human fibroblast comes of age.
Science 249:1129–1133.
30. Gou, C., D. Geverd, R. Liao, N. Hamad, C. M. Counter, and D. T. Price. 2001.
Inhibition of telomerase is related to the lifespan and tumorigenicity of human
prostate cancer cells. J. Urol. 166:694–698.
31. Greider, C. W., and E. H. Blackburn. 1985. Identification of a specific telomere
terminal transferase activity in Tetrahymena extracts. Cell 43:405–413.
32. Greider, C. W., and E. H. Blackburn. 1989. A telomeric sequence in the RNA of
Tetrahymena telomerase required for telomere repeat synthesis. Nature 337:331–
337.
33. Griffith, J. D., L. Comeau, S. Rosenfield, R. M. Stansel, A. Bianchi, H. Moss,
and T. de Lange. 1999. Mammalian telomeres end in a large duplex loop. Cell
97:503–514.
34. Guo, C., D. Geverd, R. Liao, N. Hamad, C. M. Counter, and D. T. Price. 2001.
Inhibition of telomerase is related to the life span and tumorigenicity of human
prostate cancer cells. J. Urol. 166:694–698.
35. Hahn, W. C., C. M. Counter, A. S. Lundberg, R. L. Beijersbergen, M. W.

Brooks, and R. A. Weinberg. 1999. Creation of human tumour cells with defined
genetic elements. Nature 400:464–468.
36. Hahn, W. C., S. A. Stewart, M. W. Brooks, S. G. York, E. Eaton, A. Kurachi,
R. L. Beijersbergen, J. H. Knoll, M. Meyerson, and R. A. Weinberg. 1999.
Inhibition of telomerase limits the growth of human cancer cells. Nat. Med.
5:1164–1170.
37. Harley, C. B., A. B. Futcher, and C. W. Greider. 1990. Telomeres shorten during
ageing of human fibroblasts. Nature 345:458–460.
38. Harrington, L., W. Zhou, T. McPhail, R. Oulton, D. S. Yeung, V. Mar, M. B.
Bass, and M. O. Robinson. 1997. Human telomerase contains evolutionarily
conserved catalytic and structural subunits. Genes Dev. 11:3109–3115.
39. Hastie, N. D., M. Dempster, M. G. Dunlop, A. M. Thompson, D. K. Green, and
R. C. Allshire. 1990. Telomere reduction in human colorectal carcinoma and with
ageing. Nature 346:866–868.
40. Hayflick, L., and P. S. Moorhead. 1961. The serial cultivation of human diploid
cell strains. Exp. Cell Res. 25:585–621.
41. Hemann, M. T., M. A. Strong, L. Y. Hao, and C. W. Greider. 2001. The shortest
telomere, not average telomere length, is critical for cell viability and chromosome
stability. Cell 107:67–77.
42. Hughes, T. R., S. K. Evans, R. G. Weilbaecher, and V. Lundblad. 2000. The
Est3 protein is a subunit of yeast telomerase. Curr. Biol. 10:809–812.
43. Kilian, A., D. D. L. Bowtell, H. E. Abud, G. R. Hime, D. J. Venter, P. K. Keese,
E. L. Duncan, R. R. Reddel, and R. A. Jefferson. 1997. Isolation of a candidate
human telomerase catalytic subunit gene, which reveals complex splicing patterns
in different cell types. Hum. Mol. Genet. 6:2011–2019.
44. Kim, N. W., M. A. Piatyszek, K. R. Prowse, C. B. Harley, M. D. West, P. L. Ho,
G. M. Coviello, W. E. Wright, S. L. Weinrich, and J. W. Shay. 1994. Specific
association of human telomerase activity with immortal cells and cancer. Science
266:2011–2015.
45. Kiyono, T., S. A. Foster, J. I. Koop, J. K. McDougall, D. A. Galloway, and A. J.
Klingelhutz. 1998. Both Rb/p16INK4a inactivation and telomerase activity are
required to immortalize human epithelial cells. Nature 396:84–88.
46. Klingelhutz, A. J., S. A. Barber, P. P. Smith, K. Dyer, and J. K. McDougall.
1994. Restoration of telomeres in human papillomavirus-immortalized human
anogenital epithelial cells. Mol. Cell. Biol. 14:961–969.
47. Klingelhutz, A. J., S. A. Barber, P. P. Smith, K. Dyer, and J. K. McDougall.
1994. Restoration of telomeres in human papillomavirus-immortalized human
anogenital epithelial cells. Mol. Cell. Biol. 14:961–969.
48. Kolquist, K. A., L. W. Ellisen, C. M. Counter, M. Meyerson, L. K. Tan, R. A.
Weinberg, D. A. Haber, and W. L. Gerald. 1998. Expression of TERT in early
premalignant lesions and a subset of cells in normal tissues. Nat. Genet. 19:182–
186.
49. Lal, A., A. E. Lash, S. F. Altschul, V. Velculescu, L. Zhang, R. E. McLendon,
M. A. Marra, C. Prange, P. J. Morin, K. Polyak, N. Papadopoulos, B.
Vogelstein, K. W. Kinzler, R. L. Strausberg, and G. J. Riggins. 1999. A public
database for gene expression in human cancers. Cancer Res. 59:5403–5407.
50. Lendvay, T. S., D. K. Morris, J. Sah, B. Balasubramanian, and V. Lundblad.

1996. Senescence mutants of Saccharomyces cerevisiae with a defect in telomere
replication identify three additional EST genes. Genetics 144:1399–1412.
51. Lingner, J., and T. R. Cech. 1996. Purification of telomerase from Euplotes
aediculatus: requirement of a primer 3' overhang. Proc. Natl. Acad. Sci. USA
93:10712–10717.
52. Lingner, J., T. R. Cech, T. R. Hughes, and V. Lundblad. 1997. Three ever
shorter telomere (EST) genes are dispensable for in vitro yeast telomerase activity.
53. Lingner, J., T. R. Hughes, A. Shevchenko, M. Mann, V. Lundblad, and T. R.
Cech. 1997. Reverse transcriptase motifs in the catalytic subunit of telomerase.
Science 276:561–567.
54. Lundblad, V., and E. H. Blackburn. 1993. An alternative pathway for yeast
telomere maintenance rescues est1 – senescence. Cell 73:347–360.
55. Lundblad, V., and J. W. Szostak. 1989. A mutant with a defect in telomere
elongation leads to senescence in yeast. Cell 57:633–643.
56. Masters, J. R. 2002. HeLa cells 50 years on: the good, the bad and the ugly. Nat.
Rev. Cancer 2:315–319.
57. Meyerson, M., C. M. Counter, E. N. Eaton, L. W. Ellisen, P. Steiner, S. D.
Caddle, L. Ziaugra, R. L. Beijersbergen, M. J. Davidoff, Q. Liu, S. Bacchetti,
D. A. Haber, and R. A. Weinberg. 1997. hEST2, the putative human telomerase
catalytic subunit gene, is upregulated in tumor cells and during immortalization.
Cell 90:785–795.
58. Miura, N., I. Horikawa, A. Nishimoto, H. Ohmura, H. Ito, S. Hirohashi, J. W.
Shay, and M. Oshimura. 1997. Progressive telomere shortening and telomerase
reactivation during hepatocellular carcinogenesis. Cancer Genet. Cytogenet. 93:56–
62.
59. Morin, G. B. 1989. The human telomere terminal transferase enzyme is a
ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59:521–529.
60. Murray, A. W., and J. W. Szostak. 1983. Construction of artificial chromosomes
in yeast. Nature 305:189–193.
61. Nakamura, T. M., G. B. Morin, K. B. Chapman, S. L. Weinrich, W. H. Andrews,
J. Lingner, C. B. Harley, and T. R. Cech. 1997. Telomerase catalytic subunit
homologs from fission yeast and human. Science 277:955–959.
62. Nakayama, J., H. Tahara, E. Tahara, M. Saito, K. Ito, H. Nakamura, T.
Nakanishi, T. Ide, and F. Ishikawa. 1998. Telomerase activation by hTRT in
human normal fibroblasts and hepatocellular carcinomas. Nat. Genet. 18:65–68.
63. Nugent, C. I., T. R. Hughes, N. F. Lue, and V. Lundblad. 1996. Cdc13p: a
single-strand telomeric DNA-binding protein with a dual role in yeast telomere
maintenance. Science 274:249–252.
64. Olovnikov, A. M. 1971. [Principle of marginotomy in template synthesis of
polynucleotides]. Dokl. Akad. Nauk SSSR 201:1496–1499.
65. Ouellette, M. M., L. D. McDaniel, W. E. Wright, J. W. Shay, and R. A.
Schultz. 2000. The establishment of telomerase-immortalized cell lines
representing human chromosome instability syndrome. Hum. Mol. Genet. 9:403–
411.
66. Rich, J. N., C. Guo, R. E. McLendon, D. D. Bigner, X. F. Wang, and C. M.

Counter. 2001. A genetically tractable model of human glioma formation. Cancer
Res. 61:3556–3560.
67. Sedivy, J. M. 1998. Can ends justify the means? Telomeres and the mechanisms of
replicative senescence and immortalization in mammalian cells. Proc. Natl. Acad.
Sci. USA 95:9078–9081.
68. Shay, J. W., and S. Bacchetti. 1997. A survey of telomerase activity in human
cancer. Eur. J. Cancer 33:787–791.
69. Shay, J. W., G. Tomlinson, M. A. Piatyszek, and L. S. Gollahon. 1995.
Spontaneous in vitro immortalization of breast epithelial cells from a patient with
Li-Fraumeni syndrome. Mol. Cell. Biol. 15:425–432.
70. Shay, J. W., and W. E. Wright. 2000. The use of telomerized cells for tissue
engineering. Nat. Biotechnol. 18:22–23.
71. Shay, J. W., W. E. Wright, and H. Werbin. 1991. Defining the molecular
mechanisms of human cell immortalization. Biochim. Biophys. Acta. 1072:1–7.
72. Singer, M. S., and D. E. Gottschling. 1994. TLC1: template RNA component of
Saccharomyces cerevisiae telomerase. Science 266:404–409.
73. Stampfer, M. R., A. Bodnar, J. Garbe, M. Wong, A. Pan, B. Villeponteau, and
P. Yaswen. 1997. Gradual phenotypic conversion associated with immortalization
of cultured human mammary epithelial cells. Mol. Biol. Cell 8:2391–2405.
74. Szostak, J. W., and E. H. Blackburn. 1982. Cloning yeast telomeres on linear
plasmid vectors. Cell 29:245–255.
75. Toouli, C. D., L. I. Huschtscha, A. A. Neumann, J. R. Noble, L. M. Colgin,
B. Hukku, and R. R. Reddel. 2002. Comparison of human mammary epithelial
cells immortalized by simian virus 40 T-antigen or by the telomerase catalytic
subunit. Oncogene 21:128–139.
76. Vaziri, H., and S. Benchimol. 1998. Reconstitution of telomerase activity in
normal human cells leads to elongation of telomeres and extended replicative life
span. Curr. Biol. 8:279–282.
77. Vaziri, H., W. Dragowska, R. C. Allsopp, T. E. Thomas, C. B. Harley, and P.
M. Lansdorp. 1994. Evidence for a mitotic clock in human hematopoietic stem
cells: loss of telomeric DNA with age. Proc. Natl. Acad. Sci. USA 91:9857–9860.
78. Vaziri, H., F. Schachter, I. Uchida, L. Wei, X. Zhu, R. Effros, D. Cohen, and C.
B. Harley. 1993. Loss of telomeric DNA during aging of normal and trisomy 21
human lymphocytes. Am. J. Hum. Genet. 52:661–667.
79. Wainstein, M. A., F. He, D. Robinson, H. J. Kung, S. Schwartz, J. M. Giaconia,
N. L. Edgehouse, T. P. Pretlow, D. R. Bodner, E. D. Kursh, and et al. 1994.
CWR22: androgen-dependent xenograft model derived from a primary human
prostatic carcinoma. Cancer Res. 54:6049–6052.
80. Watson, J. D. 1972. Origin of concatemeric T7 DNA. Nature New Biol. 239:197–
201.
81. Weinrich, S. L., R. Pruzan, L. Ma, M. Ouellette, V. M. Tesmer, S. E. Holt, A.
G. Bodnar, S. Lichtsteiner, N. W. Kim, J. B. Trager, R. D. Taylor, R. Carlos,
W. H. Andrews, W. E. Wright, J. W. Shay, C. B. Harley, and G. B. Morin.
1997. Reconstitution of human telomerase with the template RNA component hTR
and the catalytic protein subunit hTRT. Nat. Genet. 17:498–503.
82. Wellinger, R. J., K. Ethier, P. Labrecque, and V. A. Zakian. 1996. Evidence for
a new step in telomere maintenance. Cell 85:423–433.
83. Yang, L., T. Suwa, W. E. Wright, J. W. Shay, and P. J. Hornsby. 2001.

Telomere shortening and decline in replicative potential as a function of donor age
in human adrenocortical cells. Mech. Ageing Dev. 122:1685–1694.
84. Zhang, X., V. Mar, W. Zhou, L. Harrington, and M. O. Robinson. 1999.
Telomere shortening and apoptosis in telomerase-inhibited human tumor cells.
Genes Dev. 13:2388–2399.
85. Zhong, Z., L. Shiue, S. Kaplan, and T. de Lange. 1992. A mammalian factor that
binds telomeric TTAGGG repeats in vitro. Mol. Cell. Biol. 12: 4834–4843.
Chapter 6
HSP90 CO-CHAPERONES IN SACCHAROMYCES
CEREVISIAE
Marija Tesic and Richard F. Gaber

Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University,
Evanston, Illinois 60208
Heat-shock response is a general mechanism through which cells cope

with external, as well as internal, stresses. Although the heat-shock response
is elicited under conditions of stress, proteins involved in this set of
pathways are also necessary for basic maintenance of cellular constituents,
which involves regulation of proper levels, folded states, and conformations
of proteins. Heat shock proteins play key roles in ensuring the fine-tuning
and integrity of the signal transduction pathways, including those involved in
cellular proliferation and cell cycle regulation. Understanding the function of
the heat shock proteins is, therefore, central to our knowledge of proper cell
regulation and how it might go awry; it is also essential for developing
productive interventions to prevent the loss of this regulation.
Hsp90 is an essential, abundant, and highly conserved heat shock protein.
It interacts with a large number and variety of wild type and mutant proteins
that are partially folded, or adopt unstable or inactive conformations.
Because of its ability to buffer the morphologic variability within the cell
and allow mutant or partially folded proteins to perform wild-type functions
Hsp90 is considered a molecular “evolutionary capacitor” [142]. Hsp90 is
also a global signal transduction regulator that can modulate responses of
numerous signaling molecules to intra- and extracellular stimuli. A subset of
Hsp90 substrate proteins are involved in the regulation of cellular
proliferation and transformation. Deeper understanding of the regulation of
these substrates by Hsp90 will further our knowledge of tumorigenesis in
general, and thus lead to approaches through which it can be prevented.
Hsp90 is a molecular chaperone that performs its roles in concert with
other proteins, collectively referred to as Hsp90 co-chaperones. Most
mammalian Hsp90 co-chaperones are conserved in Saccharomyces
141
© 2007 Springer.
142 M. Tesic and R. F. Gaber
cerevisiae (see Table 1). On the one hand, this facilitates (the) analysis of
Hsp90 co-chaperones in vivo with a facile and genetically tractable system.
On the other hand, the absence of certain mammalian homologs of the
Hsp90 complex in yeast affords an opportunity to use yeast as a “bag of
enzymes” to study in vivo precisely those components that are missing.
Table 1. Conservation of Hsp90 co-chaperones in mammalian and S. cerevisiae cells. The

third column lists relevant structural domains of the listed co-chaperones. The fourth column
lists selected Hsp90 substrates known to interact with either mammalian or S. cerevisiae co-
chaperones. Applicable references are provided in the text.
Mammalian S. cerevisiae Domains Substrates
Hsp90α, Hsp90β Hsp82, Hsc82 ATPase N/A
Hsp70 Ssal-4 ATPase N/A
2+
Hsp40/Hdj1, Hdj2 Ydj1, Sis1 J domain, Zn GR, v-Src, Hap1
finger
Hop/p60 Sti1 TPR GR, PR, v-Src, HSF,
Gen2, p53
Hip/p48 ___ TPR, GGMP repeat GR, PR
p23 Sbal1 ___ GR, PR, AR, MR,
ER, TR, PKR,
telomerase, p53,
HSF, Gen2, AhR
CyP40 Cpr6, Cpr7 TPR, PPIase GR, v-Src, Hfl1, p53
FKBP51, FKBP52 ___ TPR- PPIase GR, PR
PP5 Ppt1 TPR, phosphatase GR, p53
p50/Cdc37 Cdc37 Kinase binding v-Src, Ste11, Raf1,
Cdk4, Cdk6, AR,
Gen2
ARA9/XAP2/AIP ___ TPR, FKBP domain AhR
TTC4 Cns1 TPR GR, Hsf1
AKT1,2,3, kinases? Sch9 S/T kinase, C2 GR, Hap1, Ste11
domain
Hsp110 Sse1 Calmodulin binding GR, HSF
Bag1 ? Ub-like, Hsp 70- GR, p53
binding
CHIP ___ TPR, U-Box GR, CFTR
? Hch1 ___ ___
Chapter 6: Hsp90 Co-Chaperones in S. cerevisiae 143
The high degree of conservation of most Hsp90 complex components

suggests that the mechanisms of function of Hsp90 are conserved. Since
Hsp90 is capable of playing multiple roles in the regulation of its substrates,
the co-chaperones might aid Hsp90 at a number of points of regulation,
which may differ considerably among different substrates. Additionally,
some of the co-chaperones may have regulatory functions in their own right,
independent of their roles in the Hsp90 complex. This discussion is focused
on the role that Hsp90 co-chaperones play in helping Hsp90 to control
proper expression levels, activity, and localization of client proteins. Another
essential and highly conserved heat shock protein, Hsp70, plays a prominent
role in the regulation of some Hsp90 substrates, but the discussion of this
chaperone and its co-chaperones is beyond the scope of this article (for
review, see [104]).
1 HSP90 AS A FINELY TUNED CHAPERONE

MACHINE
Hsp90 is a highly conserved molecular chaperone. Eukaryotic Hsp90 is

essential (for recent reviews, see [123, 181]) and abundant, constituting up to
1–2% total cellular protein [75]. Although the number of proteins with
which it is known to interact is large and still growing, Hsp90 does not
interact with substrates and co-chaperones indiscriminately, but apparently
in a highly specific and regulated manner. It is of interest to consider the
question of how one molecule can provide at the same time such a wide
scope of productive interactions, and the specificity required for control.
While part of the answer is provided by the versatile nature of the Hsp90
molecule itself, the full answer to this question requires understanding of the
interacting partners of Hsp90, its co-chaperones.
Hsp90 consists of three domains: the amino terminal domain, the middle
region, and the carboxy terminal domain. The amino terminus contains an
ATPase domain of about 23 kDa; in yeast, it is approximately 210 amino
acids in length [145]. Hsp90 homologs in S. cerevisiae have a considerably
higher ATPase activity [0.1–0.2 1/min) [122, 135] than human Hsp90 (0.02
1/min [119]), but the biological significance of this difference is still unclear.
Although the ATPase activity of Hsp90 as compared with other ATPases is
low, it is essential for its in vivo functions [117, 122]. Geldanamycin (GA), a
potential antitumor agent that mimicks the structure of ATP, inhibits Hsp90
ATPase activity by blocking the ATP-binding pocket [134].
In S. cerevisiae, the middle region of Hsp90 extends approximately from
residues 210 to 272. It enhances the affinity of the amino terminus for
unfolded substrates [145], but is dispensable for viability [94].
The carboxy terminus of S. cerevisiae Hsp90 binds some of the Hsp90

substrates, for example steroid receptors [145]. The carboxy terminus also
binds those co-chaperones that contain the tetratricopeptide (TPR) domain,
including Hop, CyP40, FKBP51 and FKBP52, and PP5 (see Table 1). TPRs
are structural motifs involved in protein–protein interactions of a variety of
proteins (for review, see [9]). The principal interaction between co-
chaperones containing TPR domains and Hsp90 is mediated by the carboxy-
terminal sequence of Hsp90, MEEVD. The carboxy terminus may also be
the location of a second, cryptic ATP-binding site [96], that is exposed upon
binding of the nucleotide at the N-terminus [159].
Hsp90 exists in solution as a dimer, and it is the dimeric form that binds
substrates and co-chaperones. Although the primary, stable dimerization is
mediated by the carboxy terminus [107], both amino and carboxy termini of
Hsp90 are involved in dimerization. The amino terminus mediates transient
dimerization of Hsp90 in response to ATP binding [20, 133, 172].
Dimerization may not be required for interaction with substrates, or viability.
A short deletion in the carboxy terminus of chicken Hsp90 leads to loss of
dimerization, but has no effect on the interaction of the chaperone with
estrogen receptor in vivo; additionally, the mutant chicken protein confers
viability to S. cerevisiae cells lacking endogenous Hsp90 [101].
Although both amino and carboxy termini of Hsp90 can bind substrates
and ATP in vitro [145, 182], suggesting that the domains might function
independently, work from several laboratories reveals that there is a finely
tuned communication between the two termini of the molecule that enable it
to undergo activation cycles with its clients and co-chaperones [20, 133, 140,
146, 159, 172]. Through conformational changes involving the entire Hsp90
molecule, the ATPase activity of the amino terminus is tightly coupled to the
chaperoning of substrates, which is most likely performed by the carboxy
terminus.
1.1 Hsp90 in S. cerevisiae
In budding yeast, two genes, HSP82 and HSC82, encode Hsp90 proteins
that are 97% identical. HSP82 was initially identified using differential
plaque filter hybridization performed to find genes expressed specifically
in heat shocked cells [54]. HSC82 was subsequently identified by hybri-
dization of HSP82 to yeast genomic library [12]. Cells harboring deletions
of either HSP82 or HSC82 are viable, but the double deletion is lethal
[12]. Under normal conditions, Hsc82 is present at approximately 20-fold
greater levels than Hsp82, and is induced only about twofold upon heat
shock. In contrast, when cells are heat shocked, HSP82 is induced some 20-
fold, to a level approximately the same as Hsc82 levels [12]. Deletions of
HSC82 or HSP82 do not exhibit dosage compensation and have no

discernible effects on the expression of other genes [12]. Cells in which
either gene is deleted exhibit weak temperature sensitivity (ts) at 37°C.
Underscoring the abundance of Hsp90, cells remain viable even when its
levels are decreased to 10% of wild type, although this decrease leads to
partial loss of some Hsp90 functions. Overexpression of Hsp90 does not
offer increased protection from heat shock, nor does it enhance
thermotolerance [30]. A number of mutant alleles in HSP82 and HSC82
have been generated [11, 84, 113] and have proven useful in studying the
function of this protein, especially in investigating the effects of loss of
Hsp90 function on the client proteins in vivo.
2 HSP90 SUBSTRATES IN S. CEREVISIAE
Most Hsp90 substrates identified to date are signaling molecules and fall
largely into two groups: transcription factors and protein kinases (for review,
see [130]), although there is a growing list of substrates not involved in
signal transduction. Common to several Hsp90 substrates is their propensity
to adopt multiple conformations. The requirement for the Hsp90 complex
arises from the need to chaperone each client into its proper conformation, in
which the molecule is poised to receive a signal and perform its cellular role.
Table 2 outlines heterologous Hsp90 substrates that have been expressed in
budding yeast cells and known endogenous substrates. In this section,
selected substrates are treated in detail according to the following criteria:
historical importance and widespread use in the studies of Hsp90 (steroid
receptors), exemplification of regulation by a co-chaperone (aryl hydro-
carbon receptor), and involvement in cellular proliferation and tumorigenesis
(p53, Ste11, and telomerase).
2.1 Heterologous substrates
Some of mammalian Hsp90 client proteins that are not conserved in

S. cerevisiae, but were known from biochemical studies to bind the Hsp90
machinery, have been expressed in S. cerevisiae cells in order to study their
in vivo regulation by the Hsp90 complex.
2.1.1 Steroid receptors
When rat glucocorticoid receptor (GR) is expressed in S. cerevisiae, it is

capable of enhancing transcription driven by a glucocorticoid response
element (GRE) in a ligand-dependent fashion [147]. Genetically decreasing
yeast Hsp90 levels to about 5% of wild type leads to reduction in GR

signaling [126]. This was the first evidence that Hsp90 plays a functional
role in steroid receptor complexes with which it was known to physically
interact. Maturation of steroid receptors in yeast has since become a
commonly used assay for general Hsp90 function. For example, a number of
Hsp90 mutants have been isolated in a screen for decreased GR signaling
[11]. To date, several steroid receptors have been expressed in yeast (see
Table 6.2) [11, 53]. Although a functional dependence of androgen receptor
(AR) on Hsp90 was not demonstrable using in vitro approaches [115, 118],
expression of AR in S. cerevisiae cells carrying mutant Hsp90 allowed
demonstration of the requirement for a functional Hsp90 complex [53].
Table 2. Hsp90 substrates that have been expressed or identified in S. cerevisiae. The right
column lists known S. cerevisiae co-chaperones that have been experimentally demonstrated
to interact with the listed substrates either physically, genetically, or in functional assays. The
applicable references are given in the text.
Hsp90 substrates in S. cerevisiae S. cerevisiae co-chaperones
interacting with substrates
Ydj1, Sti1, Sba1, Cpr7, Cns1,
Steroid receptors
Glucocorticoid receptor (GR)

Sch9, Sse1
Progesterone receptor (PR) Sti1, Sba1
Heterologous substrates
Estrogen receptor (ER) Sba1

Mineralocorticoid receptor (MR) Sba1
Androgen receptor (AR) Sba1, Cdc37
Aryl hydrocarbon (dioxin) receptor (AhR) ⎯
Retinoic acid receptor (RXR) ⎯
v-Src Ydj1, Sti1, Cpr7, Cdc37
c-Src ⎯
p53 ⎯
PKR Sba1, Cdc37
Heat Shock Factor (Hsf1) Sti1, Cpr7, Cns1, Sch9, Sse1
Hap1 Ydj1, Sch9
Endogenous
substrates
Ste11 Cdc37, Sch9

Gcn2 Sti1, Sba1, Cdc37
Cna2 ⎯
Telomerase ⎯
2.1.2 Aryl hydrocarbon receptor (AhR)
AhR is a nuclear receptor that, like steroid receptors, binds its ligands
(mostly planar aromatic molecules such as dioxin) inside the cell. Upon
ligand binding, AhR heterodimerizes with ARNT (AhR nuclear
translocator). Both AhR and ARNT contain bHLH domain for DNA
binding. AhR was initially expressed in yeast cells as a LexA fusion in order
to eliminate the need for ARNT coexpression, demonstrating for the first
time the need for Hsp90 in the signaling of this nuclear receptor [18].
Another fusion, with the DNA-binding domain of human GR to eliminate
the need for the dioxin-responsive elements, has also been expressed in yeast
[175]. In order to more faithfully replicate the mammalian system, AhR and
ARNT were coexpressed in yeast cells [106] where they exhibited depen-
dence on components of the Hsp90 complex [105].
An AhR co-chaperone, ARA9/XAP2/AIP, was identified in a yeast two-
hybrid screen for proteins that interact with AhR [17]. Its expression in
S. cerevisiae, which does not harbor a homolog, enhanced the ligand respon-
siveness of AhR [17]. It is evident from the work of several laboratories that
ARA9/XAP2/AIP determines the specificity of the Hsp90 complex for AhR
[6, 19, 82, 83, 90, 102, 103, 125].
Several important lessons can be learned from the studies of ARA9/
XAP2/AIP that might also apply to other Hsp90 co-chaperones. First,
ARA9/XAP2/AIP interacts directly both with Hsp90 and AhR [102], and the
interaction with AhR is mediated by the TPR domain of ARA9/XAP2/AIP, a
domain that is primarily implicated in binding to Hsp90. This suggests a
possibility that the TPR domains of other TPR-containing Hsp90 co-
chaperones direct the interaction between the co-chaperones and various
substrates, in addition to mediating the interaction between co-chaperones
and Hsp90. Second, ARA9/XAP2/AIP regulates AhR at least in part by
increasing the proportion of the receptor in the cytoplasm [82, 90, 125].
Other co-chaperones may also play roles in the proper localization of Hsp90
substrates, possibly even bringing together the substrates and the Hsp90
complex. Third, it has been shown that ARA9/XAP2/AIP protects AhR
against proteasome-dependent degradation [82]. ARA9/XAP2/AIP may have
a chaperone activity on its own, possibly mediated by the FKBP-like
immunophilin domain situated in the amino terminus of the protein. Several
co-chaperones (p23, CyP40, FKBPs) have in vitro chaperoning activities
[13, 58], which may be important in the maintenance of specific Hsp90
substrates in proper folded states.
2.1.3 v-Src and c-Src
It has been known for some time that the oncogenic protein kinase v-Src
can be co-immunoprecipitated with Hsp90 [14]. Expression of v-Src in
S. cerevisiae cells is toxic, presumably due to nonspecific phosphorylation of
yeast proteins [15, 88]. Hsp90 maintains normal levels and specificity of the
kinase [177]. It was initially thought that only v-Src, but not c-Src depends
on the presence of the functional Hsp90 complex. However, use of a
particularly stringent Hsp90 mutant in S. cerevisiae revealed that c-Src also
requires Hsp90 for full activity [178]. The regulation of v-Src by Hsp90
complex reveals aspects of both positive and negative control: the first step,
in which Hsp90 helps the kinase adopt the proper conformation, is followed
by a second step in which Hsp90 prevents the kinase from being activated.
The intrinsic structural differences between the c-Src and v-Src presumably
contribute to the varying extent of dependence on Hsp90 [178]. The
comparison between the extent of dependence of the two kinases on Hsp90
could provide useful insights into the nature of the Hsp90 substrate
recognition.
2.1.4 p53
p53 is a transcription factor with a well-studied role as a tumor

suppressor. In rabbit reticulocyte lysates, a mutant form of the p53 protein
(p53V143A) interacts with Hsp90 whereas the wild type does not [8]. Addition
of Hsp90 inhibitors, geldanamycin (GA) and macbecin I, impaired the
ability of Hsp90 to interact with several p53 mutant forms [8, 176]. Expres-
sion of wild type and mutant p53 in S. cerevisiae showed that the mutant
Hsp90G313N is unable to bind mutant p53V143A, demonstrating authenticity of
the interaction [8]. In contrast, purified Hsp90 interacts with the purified
wild type p53, but not a mutant, p53R175H [87]. A co-chaperone, Bag-1, is
capable of dissociating wild-type p53–Hsp90 complexes in vitro [87].
Overall, the fact that p53 (both in wild type and mutant forms) interacts with
the components of the Hsp90 complex provides a direct connection between
tumorigenic signaling in cells and chaperone complexes that might regulate
this signaling cascade.
2.2 Endogenous substrates
Relatively few endogenous yeast substrates of Hsp90 are known. Most of

them were initially discovered through their similarity to the binding
partners of Hsp90 in mammalian cells.
2.2.1 Ste11
Ste11 occupies a role in MAPK pathways analogous to that of the

mammalian Raf serine/threonine kinase. Several groups have shown that
Raf forms GA-sensitive complexes with Hsp90 [60, 149, 150, 160, 161,
171]. One of the signaling pathways involving Ste11 is the yeast phero-
mone response, a MAPK cascade activated by the binding of a mating
pheromone to a cell surface receptor, leading ultimately to cell cycle arrest.
S. cerevisiae cells in which Hsp90 is mutated are unable to undergo arrest in
the presence of a mating pheromone [93]. In Hsp90 mutant cells, both the
accumulation of Ste11 and its activity are decreased. A screen for multicopy
suppressors of this phenotype led to the isolation of Ste5, a component of the
MAPK-signaling module that functions as a scaffold to maintain association
of the other components of the cascade (Ste11, Ste7, Fus3/Kss1) [93].
It is possible that the specificity of Hsp90 interaction with Ste11 is
determined by the Hsp90 co-chaperone Cdc37. Cdc37 has been dubbed a
“kinase-targeting subunit” of the Hsp90 complex [165], and cdc37 mutant
cells have reduced Ste11 function [2]. Cdc37, Ste11, and Hsp90 form a
heterocomplex in yeast cells [2]. Since such complexes have also been
identified in mammalian cells [60, 160], there appears to be strong
conservation in the way components of the MAPK cascade are regulated by
the Hsp90 complex, thus allowing researchers to extend studies of yeast cell
cycle to mammalian systems.
2.2.2 Telomerase
Hsp90 was identified as a multicopy suppressor of mutant Stn1 [61], a

protein that interacts with a telomerase regulator Cdc13. Overexpression of
Hsp90 leads to telomere shortening in both stn1 mutant and wild-type cells,
but genetically decreasing Hsp90 levels to 5% wild-type has no effect on
telomere length [61]. Interestingly, hTERT, a human catalytic subunit of
telomerase, was identified in a yeast two-hybrid screen for proteins that
interact with a Hsp90 co-chaperone, p23 [70]. Both Hsp90 and p23 enhance
telomerase activity in vitro and in cell culture. Future studies should reveal
whether the regulation of telomerase by the Hsp90 complex resembles that
of other client proteins. It has been proposed that telomerase expression is
necessary for the immortalization of cancer cells [35, 65]. Therefore,
inhibition of Hsp90 function might lead to the reversal of cellular
transformation, at least partly, through the telomerase-based mechanism and
contribute to the antitumorigenic effect of Hsp90 inhibitor drugs, such as
GA.
3 HSP90 CO-CHAPERONES IN BUDDING YEAST
Mechanism of Hsp90 function has primarily been studied using in vitro

approaches, especially reconstitution of the assembly of Hsp90
heterocomplexes, where progesterone receptor (PR) or glucocorticoid
receptor (GR) served as model substrates (for review, see [31] and [131]).
Other studies have, in one way or another, corroborated the model of overall
Hsp90 function that has emerged from the in vitro reconstitution
experiments. The picture of Hsp90 that arises from these studies is that of a
highly dynamic molecule that joins and departs from complexes containing
its substrates and co-chaperones (Figure 1). During the course of activation
by the Hsp90 complex, the substrate is transformed from an inactive and
possibly partially unfolded state, to a conformation in which it is poised to
respond to appropriate cellular signals.
Figure 1. Sequence of events leading to the activation of a model substrate by Hsp90 and
Hsp70 and their co-chaperones. Although steps in this sequence have been elucidated using
mammalian cells, protein names indicated here are those of S. cerevisiae (co-chaperones).
Details of dynamic interactions between the proteins are described in the text.
Steroid receptors have been used as model substrates that are chaperoned
by Hsp90 from a conformation incapable of binding the hormone ligand to a
hormone-binding form [31, 131]. A highly conserved and essential cha-
perone, Hsp70, is thought to be the first to bind the substrate, thus defining
the early phase of the steroid receptor maturation cycle [74, 132, 155]. A
recent report suggests, however, that Hsp40/Hdj1, a co-chaperone of Hsp70,
may, in fact, be the first to bind, at least to PR [66]. Another Hsp70
regulator, Hip, is also associated with progesterone receptors [129], and
might aid the formation of this early complex by stabilizing the interaction
between Hsp70 and the substrate [59]. Regardless of their relative order, it is
by now well established that Hsp70 and its co-chaperones Hip and
Hsp40/Hdj1 participate in the early stages of substrate activation. Hsp90 is
the next component to enter the complex. Its initial binding may be mediated
by the co-chaperone, Hop, which is capable of binding both Hsp90 and
Hsp70 concurrently [27, 28, 43, 76, 156]. The presence of Hop defines the
intermediate complex. Hop is replaced by another set of co-chaperones,
which bind to the same acceptor site on Hsp90 as Hop. These are most often
large immunophilins of the cyclophilin (CyP40) or FKBP class (FKBP51

and FKBP52) [68, 120, 138, 143, 144, 168]. The late or mature complex is
defined by the presence of immunophilins and is thought to be stabilized by
another co-chaperone, p23 [42, 79, 157]. In the mature complex, the steroid
receptor has been activated and is, therefore, capable of binding ligand. Most
likely, the Hsp90 machinery disassembles upon ligand binding [132]. At this
point, the continued interaction of client proteins with the Hsp90 machinery
prevents them from being activated without proper signaling. Therefore,
Hsp90, and the proteins that assist it, can regulate their targets both
positively and negatively. This dual role of the Hsp90 complex in substrate
control is an important aspect of its cellular function. To understand the
involvement of Hsp90 co-chaperones in the regulation of Hsp90 substrates,
it will be necessary to delineate different points of the Hsp90 cycle at which
co-chaperones are thought to play a role.
The exact make up of the Hsp90 machinery that is necessary and
sufficient for the transformation of steroid receptors is still unclear. For
example, while purified Hsp70, Hsp90, and Hop can transform GR, p23 is
still necessary for the stabilization of complexes [42]. Additionally, it has
been reported that, under some conditions, Hsp90 and Hsp70 alone can
mediate steroid receptor maturation [136]. However, the in vivo mechanism
is likely to be far more complex and the yeast system has been of great use
in addressing the question of the requirement for the various co-chaperones
in Hsp90 functions in the cell.
3.1 TRP-containing co-chaperones
3.1.1 Hop/Sti1
The homolog of mammalian Hop, STI1 was first identified in S.

cerevisiae as a stress-inducible mRNA [116]. sti1∆ cells are viable at 30°C,
but exhibit a slow-growth phenotype at both higher and lower temperatures.
In mammalian cells, Hop has been established as a common co-chaperone
for Hsp90 and Hsp70 that brings the two chaperones together and thus
facilitates formation of the intermediate complexes in the steroid receptor
maturation cycle [27, 28, 43, 76, 156]. The structures of human Hop TPR
domains corroborate this view [148]. Hop/Sti1 has nine TPRs. The three
amino terminal TPRs bind to Hsp70. Of the other six TPRs, the three central
repeats are sufficient to bind Hsp90. The crystal structures of two TPR
domains of Hop bound to their appropriate Hsp90 and Hsp70 peptides
explain how Hop might serve as a bridge between Hsp90 and Hsp70. It is of
interest that the carboxy-terminal motif of Hsp90, MEEVD, binds to the TPR
domain of Hop with a very similar dissociation constant (Kd = 11 µM) to the
entire C-terminal domain of Hsp90 (Kd = 6 µM). This indicates that the main
(and possibly sole) structural determinants for binding to TPR domains lie
within this small region of Hsp90.
Reconstitution of steroid receptor complexes in reticulocyte lysates and
formation of complexes from purified components defined the minimal
chaperone machinery required to transform steroid receptors into an active
state: Hsp90, Hsp70, Hop, Hsp40, and p23 [130, 131]. Because of the
enhancement in the rate of steroid receptor activation in the presence of Hop
[109], it can be concluded that this co-chaperone at least enhances the
assembly of the complex and its activity on substrates, although there may
not be an absolute requirement for Hop in Hsp90 complexes.
Combined genetic and biochemical data from a number of laboratories
suggest that Hop/Sti1 helps Hsp90 in the formation of productive complexes
i.e., those capable of chaperoning substrates. In sti1∆ cells, signaling through
eIF2α kinase Gcn2 is decreased [46]. Deletion of STI1 in S. cerevisiae leads
to at least a threefold decrease in the activation of a GRE-LacZ reporter
gene, and significant loss of v-Src activity [21]. While sti1∆ affects only the
activity and not the level of v-Src protein, mutations in hsp90 decrease both.
It is therefore possible that Sti1 is involved specifically with one aspect of v-
Src chaperoning by Hsp90, aimed at maintaining its activity. However, a
closer look at the Hsp90 and Sti1 complexes in S. cerevisiae reveals a more
puzzling picture. When Hsp90 and GR complexes are precipitated from wild
type and sti1∆ cells, there is no observable difference in the association
between Hsp90 and Hsp70 [21]. Therefore, either there is a considerable
difference between the yeast and mammalian complexes with respect to the
requirement for Hop/Sti1 to link Hsp90 and Hsp70, or Hop/Sti1 is not
required to bring together the two chaperones in vivo. Indeed, because Hsp70
and Hsp90 can interact independently with substrate molecules, Hop/Sti1
may be required only under some circumstances.
How Hop/Sti1 exerts the observed stimulatory influence on the
maturation of Hsp90 substrates remains an important question. In addition to
its bridging role, which may or may not be important in vivo, Hop/Sti1
protein is apparently involved in the regulation of Hsp90 ATPase activity. In
the presence of purified Sti1, the ATPase activity of purified yeast Hsp90 is
reduced [135]. Sti1 also elicits a significant conformational change in Hsp90
upon binding to the chaperone in vitro [135]. Since the ATPase and
chaperoning activities of Hsp90 are tightly coupled, Sti1 may regulate the
chaperoning of the Hsp90 substrates through its effect on the ATPase
activity.
3.1.2 CyP40/Cpr6, Cpr7
CyP40 is a large immunophilin of the cyclophilin family, named thus

because of binding to the immunosuppressive drug cyclosporin A [63].
CyP40 was discovered as a component of estrogen-receptor complexes
[138]. The two yeast homologs of CyP40, Cpr6 and Cpr7, were first
identified in a yeast two-hybrid screen by their interaction with Rpd3, a
transcriptional regulator [49]. Cpr6 and Cpr7 are 41% identical, and share
the organization of CyP40: amino-terminal peptidyl-prolyl cis-trans
isomerase (PPIase) domain and a carboxy-terminal domain consisting of
three TPR units, ending with a putative calmodulin-binding domain.
Whereas CPR6 is heat-shock inducible, CPR7 is not [44]. cpr6∆ has no
discernable phenotype, while cpr7∆ cells grow slowly at all temperatures.
Overexpression of bovine CyP40, or overexpression of Cpr6, does not
alleviate the slow growth of cpr7∆ cells [44].
Both Cpr6 and Cpr7 bind directly to Hsp90 through their TPR domains
[47]. Although Cpr6 shares a higher degree of homology with human CyP40
(47%) than does Cpr7 [35%), cpr6∆ has no effect on the signaling of steroid
receptors in S. cerevisiae [170]. A requirement for a cyclophilin in two
Hsp90 functions was demonstrated by deleting CPR7, which leads to a
decrease in both GR maturation and v-Src activity [47].
Cpr6 and Cpr7 are peptidyl-prolyl isomerases, enzymes that catalyze the
conversion of peptide bonds adjacent to a proline from cis- to trans-. Since
Hsp90 is involved in the folding of substrates into proper conformations,
isomerase activities of immunophilins (both CyP40 and FKBPs) may help
Hsp90 in this aspect of its cellular role. The isomerase activities of Cpr6 and
Cpr7 are substantially different [99]. The PPIase activity of Cpr6 is 100-fold
higher than that of Cpr7 when RCM-RNAse T1 is used as a substrate, and
sixfold higher when two short synthetic peptides were assayed [99]. It has
been proposed that Cpr7 might have evolved to isomerize a specific, still
unidentified, substrate, and thus provide specificity for a subset of Hsp90
complexes [99]. However, the Cpr7 TPR domain alone is sufficient both for
the normal growth of yeast cells, and apparently for full Hsp90 chaperone
action [50, 44]. It is possible that, by binding to Hsp90, the TPR domains of
co-chaperones modify the conformation Hsp90 itself is adopting, and
thereby affect the physical and functional state of Hsp90. The two models of
cyclophilin function in the Hsp90 complex are not mutually exclusive, and
the regulation involving the isomerization of substrates could exist in
addition to the TPR-mediated regulation of Hsp90 itself.
CyP40 and its homologs may be partly regulated by localization. In
mammalian cells, CyP40 is mainly nucleolar at normal conditions [97, 120],
but is redistributed throughout the nucleus upon heat shock [97]. One
possibility regarding the function of Hsp90 co-chaperones is that they serve

to properly localize Hsp90 in the cell. For example, FKBP52 interacts with
dynein through the isomerase domain [152]. The binding of hormone to the
steroid receptor leads to the exchange of FKBP51 for FKBP52 in concert
with the latter’s recruitment to dynein; transport of the steroid receptor-
Hsp90–FKBP52–dynein complex from the cytosol to the nucleus is most
likely mediated by the interaction of dynein with microtubules [39].
Therefore, the cyclophilin class of immunophilins might also be involved in
the regulation of Hsp90 by cellular localization, in a manner similar to
FKBPs.
In vivo functions of CyP40 homologs are also known in other organisms.
An Arabidopsis thaliana CyP40 homolog, SQN, is required for the
vegetative growth of the plant shoot [7]. A Schizosaccharomyces pombe
CyP40 homolog, Wis2, was found as a multicopy suppressor of mutations in
cell cycle regulation [173], suggesting another possible connection between
Hsp90 and the regulation of cell proliferation.
3.1.3 TTC4/Cns1
Cns1 was identified as a multicopy suppressor of the slow growth

phenotype of cpr7∆ cells [44, 98] and in a multicopy screen for suppressors
of the temperature sensitive phenotype of some hsp90ts– mutants [114]. Cns1
is essential in S. cerevisiae. It has three TPRs in the amino terminus, while
the carboxy terminus contains a domain of unknown function. Cns1 binds to
Hsp90 through the TPR domain, and appears to share complexes with Cpr7,
but not with Cpr6 [44, 98]. In cpr7∆ cells, overexpression of Cns1 can
restore both normal growth and Hsp90-related functions, including
maturation of GR, and negative regulation of HSF [98]. Like CPR7, CNS1 is
not heat-inducible. In contrast with Sti1/Hop, there is no evidence that Cns1
can bind directly to Hsp70. CNS1 also shows strong genetic interactions with
CPR7 [188 – added in proof ]. This suggests that Cns1 and Cpr7 either share
a common function, or are involved in the same complex, which may or may
not contain Hsp90.
The possibility that Hsp90 complexes exist which contain multiple TPR-
domain co-chaperones has interesting implications for our understanding of
the functional role of these co-chaperones. Immunophilins such as CyP40
and FKBP52 that bind to Hsp90 through their TPR domain were initially
shown to exist in distinct Hsp90 complexes [79, 121]. This led to a
hypothesis that Hsp90 contains only one TPR-accepting pocket, for which
different TPR domain-containing co-chaperones compete. However, Sti1,
like Hsp90 is a dimer in solution and binds to Hsp90 in a 1:1 molar ratio
[135]. It has been shown that purified Cpr6 also binds to a Hsp90 dimer in a
1:1 molar ratio, although Cpr6 is a monomer in solution [99, 135]. These
findings suggest that two TPR proteins can bind to the Hsp90 dimer
simultaneously and that TPRs may not necessarily compete for binding to
Hsp90; instead, their binding may be independent of each other, or even
cooperative. Consistent with this, others and we have shown that Cns1 and
Cpr7 can be found in the same Hsp90 complexes in vivo [44, 98]. This
model of binding corresponds well with our current understanding of the
interactions between TPR proteins and Hsp90, mediated by the MEEVD
motif. Since each of the two Hsp90 monomers contains this motif, it is easy
to envision how two TPR domains can be accommodated by the Hsp90
dimer. Interestingly, the observation of at least one mixed TPR hetero-
complex, containing Cns1 and Cpr7, suggests that other combinations may
exist and that they may play a role in Hsp90 substrate specificity. By
combining even a small set of different TPR co-chaperones, a large number
of distinct Hsp90 complexes can be achieved, perhaps providing the
specificity of the Hsp90 complexes for their substrates.
Cns1 belongs to a protein family with homologs in S. pombe, Caenor-
habditis elegans, Drosophila melanogaster, and humans. The Drosophila
Dpit47 interacts with the DNA polymerase alpha subunit [36]. The human
Cns1 homolog, TTC4, is of particular interest as a potential tumor
suppressor. The TTC4 gene maps to the region of chromosome 1p31 which
undergoes loss of heterozygosity (LOH) in up to 50% of breast cancers [69,
167]. In addition, in samples from patients with malignant melanoma, six
different point mutations in TTC4 were found [128]. We have found that
TTC4 can interact with yeast Hsp82 in vivo (Tesic, M. and R. Gaber,
unpublished data), providing a possible connection between the Hsp90
chaperone machinery and human tumorigenesis.
3.1.4 PP5/Ppt1
Both human protein phosphatase 5 (PP5), and its S. cerevisiae homolog,

Ppt1, were identified as TPR domain containing phosphatases [26]. The two
comprise a unique phosphatase subfamily [162]. They share 46% identity in
the phosphatase domain, and 36% identity in the TPR domain. The
interaction of PP5 with Hsp90 in COS cells is mediated by its TPR domain
[24]. Interestingly, when the TPR domain alone of PP5 is overexpressed, it
lowers the transcriptional activity of GR in CV-1 cells, suggesting a stimu-
latory role for PP5 in steroid receptor maturation [24]. However, the use of
antisense oligonucleotides leads to an increase in GR-dependent signaling
[187]. Therefore, it is not clear whether PP5 antagonizes or enhances steroid
receptor-mediated transcriptional activation [32]. So far, possible effects
on cellular Hsp90 functions of deleting PPT1 in S. cerevisiae have not
been investigated and may provide interesting insights into the intricacies of
the mechanism of action of this phosphatase in Hsp90 complexes.
The analysis of PP5-containing Hsp90 complexes demonstrates that this
phosphatase competes with other TPR domain co-chaperones for binding to
the Hsp90 [151]. Interestingly, PP5 is also able to compete with the binding
of Hop, which could not be out-competed by the immunophilins [151]. This
lends credence to the hypothesis that there is a hierarchy of binding to the
TPR-accepting pocket of Hsp90, which is determined by the relative-binding
constants, concentration, and availability of particular chaperones.
The crystal structure of the amino terminus of PP5 was the first structure
of a Hsp90-binding TPR domain to be solved [38], providing insight into the
structure-function relationships of this highly versatile protein–protein
interaction motif [89]. Each individual TPR consists of two antiparallel α
helices at a 24° angle, which are tandemly repeated. Eight conserved
residues, mostly small and large hydrophobic amino acids, determine the
highly degenerate signature of any TPR motif, and are buried within the
structure. Certain exposed charged residues likely to be involved in protein–
protein interactions were mutated in PP5 and shown to be important for the
binding of PP5 to Hsp90 [141]. These lysines and arginines are highly
conserved among all TPR-domain containing Hsp90-binding co-chaperones.
A structure of the TPR domain of Hop co-crystallized with the MEEVD
peptide from Hsp90 corroborated these findings by showing that the very
same charged residues made crucial interactions with Hsp90 [148].
Interestingly, mutations in the MEEVD sequence of Hsp90 significantly
reduce, but do not abrogate, the binding of PP5 TPRs to Hsp90 [141],
suggesting that there may be additional binding determinants between Hsp90
and its TPR co-chaperones.
It is still unclear whether the phosphatase domain of PP5 is involved in
the regulation of Hsp90-dependent events. The simplest model would
suggest a role for PP5 in targeting the Hsp90 multichaperone complex to a
substrate to be dephosphorylated. However, it is possible, as was shown for
Cpr7 in S. cerevisiae [50], that the phosphatase domain of PP5 is dispensable
for its Hsp90-related role. In support of the idea that TPR domains regulate
Hsp90 function, the TPR domain of PP5 is emerging as a highly versatile
functional unit. It can negatively regulate the phosphatase activity of PP5 by
steric hindrance, which is released by the addition of polyunsaturated fatty
acids [25, 154]. A study of a series of mutations in PP5 made in order to
investigate how the TPR domain accommodates the interactions both with
Hsp90 and the phosphatase domain of PP5 itself reveals that the residues
involved in binding to Hsp90 are distinct from those involved in the
autoregulatory role [81]. In general, multiple functions may exist for TPR
domains of other Hsp90 binding proteins.
Although ppt1∆ cells grows normally, treatment with antisense PP5
oligonucleotides leads to G1 cell cycle arrest in S. cerevisiae cells [186].
Investigation of possible downstream effects of PP5 loss of function reveals
a strong increase in the level of p53 phosphorylation, and increased affinity
for its genomic response elements; PP5 is also able to dephosphorylate p53
in vitro. Given these results, and the observed negative regulation of the GR,
it is possible that PP5 is involved in cancers through effects on both GR and
p53 regulation [187]. Since both GR and p53 interact with Hsp90, PP5 may
be one of the co-chaperones dedicated to these Hsp90 substrates.
3.2 Other co-chaperones
3.2.1 p23/Sba1
Although it has been known for some time that steroid receptor
complexes contain a protein of 23 kDa in size termed p23 [158], the
S. cerevisiae homolog, Sba1, was identified only recently [10, 52]. SBA1 is
constitutively expressed, and sba1∆ cells are viable at 30°C and 37°C, but
grow slowly at 16°C. In S. pombe, a p23 homolog Wos2 interacts with the
Hsp90 substrate Wee1, which is involved in cell cycle control [111]. In
L cell cytosol and rabbit reticulocyte lysate reconstitution assays, p23
stabilizes Hsp90-steroid receptor complexes [42].
There are at least two requirements for the high-affinity binding of p23 to
the amino terminus of Hsp90 [29]: dimerization of Hsp90 [62] and ATP
binding (but not ATP hydrolysis) [20, 133]. p23 seems to play a role in the
“coupling” of the ATPase activity and the substrate release by Hsp90 [180].
p23 may accomplish this, at least in part, by inhibiting the ATPase activity
of Hsp90, thus extending the duration of the interaction between the client
protein and the chaperone, allowing for the productive folding to occur
[100]. This observation is in contrast with a previous report in which no
effect on the ATPase activity of purified yeast Hsp90 was observed in the
presence of purified Sba1 [117].
It has been somewhat difficult to establish whether Sba1 in S. cerevisiae
is truly a functional homolog of p23. sba1∆ cells are sensitive to
benzoquinoid ansamycins, a class of molecules such as GA, a potent
inhibitor of Hsp90 ATPase activity. In general, sensitivity to GA of yeast
cells carrying mutations in certain genes points to involvement of those
genes in Hsp90-related pathways. Sba1 was found in Hsp90 complexes,
along with Cpr6 and a small amount of Sti1; this interaction was inhibited by
GA and another benzoquinoid ansamycin, macbecin I (Mb I) [52]. However,

deletion of SBA1 does not suppress the lethality of cells in which v-Src is
overexpressed. Similarly, AR and GR signaling is only marginally affected
by sba1∆ [52]. On the other hand, the hypersensitivity of sba1∆ cells to
benzoquinone ansamycins can be rescued by expression of human p23 [10].
A recent report, however, finds strong differential effects of both p23 and
Sba1 on a number of steroid receptors [57]. It is notable that, in these assays,
the effects of p23 and Sba1 are virtually indistinguishable. While the role of
Hsp90 may be to control hormone-binding affinity of the steroid receptors,
p23 could be affecting their transactivational activity, which may take place
significantly later [57]. In agreement with this is the observation that the
interaction between Sba1 and two different steroid receptors (GR and MR)
persists even in the absence of Hsp90 [57]. Two other groups also observed
Hsp90-independent binding of p23 to endogenous yeast Hsp90 substrates,
Gcn2 and Hap1 [45, 72]. p23 is likely involved in the regulation of Hsp90
substrates in two ways: by binding the substrate proteins directly, and by
controlling the coordination between the ATPase cycle and client refolding
performed by Hsp90.
3.2.2 Hsp40 (Hdj)/Ydj1
Ydj1 is a S. cerevisiae homolog of bacterial DnaK co-chaperone, DnaJ, a

founding member of a highly conserved family of Hsp40 chaperones [16].
Hsp40 enhances the ATPase activity of Hsp70, as well as the release of ATP
from Hsp70 [23, 183]. Therefore, it is primarily an Hsp70 co-chaperone.
However, it is present in Hsp90 complexes and affects Hsp90 functions.
Hsp40 homologs in all organisms have a similar domain structure: an amino
terminal J-domain stimulates ATP hydrolysis by Hsp70 and is flanked by a
centrally located glycine and phenylalanine rich region; the carboxy
terminus contains a domain capable of binding unfolded substrates. ydj1∆
S. cerevisiae cells grow slowly at 23°C, and are inviable at 37°C. This
phenotype can be suppressed by the expression of human Hdj2, but not Hdj1
[56]. S. cerevisiae contains another Hsp40 homolog, Sis1, which is essential
and involved in some Hsp70 functions [95]. The substrate-binding domain of
either Sis1 or Ydj1 performs an essential function in yeast cells, and acts in
cis with the J domain [77].
The involvement of Ydj1 in Hsp90 complexes was revealed by synthetic
lethality of Ydj1G135D in combination with Hsp82G170D temperature-sensitive
mutant [86]. By itself, Ydj1G315D increases both basal and hormone-induced
activities of two steroid receptors (GR and ER) expressed in yeast. Ydj1G135D
can also rescue from lethality S. cerevisiae cells in which v-Src is
overexpressed. Although v-Src activity, but protein level is not reduced in
ydj1∆ cells, mutations in the J-domain can reduce both mRNA and protein
levels of v-Src [40]. Interestingly, mutations in Ydj1 have differential effects
on various Hsp90 substrates. In ydj1∆ cells, androgen receptor-mediated
signaling is compromised [56]. In contrast, in most ydj1 mutant cells,
signaling mediated by GR and ER was enhanced, yet the levels and the
activity of v-Src were mostly decreased [78]. Together with Hsp70, Ydj1
represses the activation of an endogenous yeast substrate, Hap1, in the
absence of heme [71]. The negative effects of Ydj1 on the activation of
Hsp90 substrates appear to be mediated by its J domain [71, 78].
Collectively, these findings suggest that Ydj1 is involved in multiple steps of
client protein regulation by the Hsp90 machinery, including direct
interactions with both the chaperones as well as the substrates themselves.
3.2.3 Sch9
Sch9 was discovered as a multicopy suppressor of a cdc25ts mutant [169],

and subsequently found in a transposon mutagenesis screen as a suppressor
of the G2/M cell cycle arrest phenotype exhibited by a HSF truncation
mutant [1–583] [108]. It has homology with the serine/threonine class of
protein kinases. S. cerevisiae cells carrying the HSF[1–583] mutant also
show loss of some Hsp90-dependent activities: GR maturation, Hap1-
dependent transcription, and Ste11-mediated pheromone responsiveness.
Loss of Sch9 activity restores normal function to these cells, suggesting that
Sch9 can act as a negative regulator of Hsp90. Interestingly, deletion of
SCH9 has no effect on pheromone response upon addition of the pheromone
ligand; however, the basal level of activity is strongly enhanced [108].
Therefore, Sch9 probably acts to modulate Hsp90 function at the point of
control of client proteins in the absence of their activating signals. Sch9 was
also found to regulate stress responses and chronological life span in S.
cerevisiae cells [51]. Although it is known that a decrease in Hsp90 function
can slow down chronological aging of yeast cells [64], it is not clear whether
Hsp90 plays a role in aging processes through Sch9.
3.2.4 Hsp110/Sse1
SSE1 and its homolog, SSE2, were identified in a calmodulin-binding

fraction of a yeast expression library [110]. The two are 76% identical, and
have homology to Hsp70 family members. sse1∆, but not sse2∆, causes
slow growth at all temperatures; the combination of two deletions does not
show synthetic effects. Overexpression of human Hsp110 does not rescue
sse1∆ cells from slow growth. sse1∆ exhibits genetic interaction with sti1∆,
and is hypersensitive to benzoquinone ansamycins; Sse1 physically
associates with Hsp82 [92]. Loss of Sse1 function compromises GR

maturation, and decreased basal HSF repression [92]. It is clear that Sse1
plays a role in the Hsp90 complexes, but the details of its involvement
remain to be determined.
3.2.5 Cdc37
It has been known for a long time that Cdc37 is involved in the regulation
of cell cycle at start [139]. Both Cdc37 and Hsp90 are required for the
signaling by the sevenless receptor tyrosine kinase in D. melanogaster [37].
Like Hsp90, Cdc37 in S. cerevisiae is an essential protein and a dimer, but
unlike Hsp90, its cellular concentration is low (about 0.01% total protein)
and is not increased by heat shock [85]. Cdc37 was first seen in steroid
receptor complexes as a protein of about 50 kDa size [124], and virtually all
cytosolic Cdc37 is associated with Hsp90 [174].
Cdc37 has been dubbed the “protein kinase targeting subunit” of Hsp90
[164, 165]. It interacts with a number of kinases that also bind to Hsp90,
including cell cycle regulator Cdk4 [165], Raf-1 [160], and v-Src [41].
However, it is now clear that its cellular role in general, and its role as an
Hsp90 co-chaperone in particular, is more versatile than just as a
determinator of specificity for Hsp90.
Cdc37 is also a molecular chaperone in its own right. Like Hsp90, it is
capable of maintaining unfolded β-galactosidase in a folding-competent state
[85]. Genetic interactions between HSP90 and CDC37 in both budding yeast
and fruit flies indicated that their roles overlap, but are not identical.
Consistent with the idea that Cdc37 interacts solely with Hsp90 kinase
substrates, overexpression of Cdc37 in the Hsp82G170D mutant restored
normal v-Src, but not GR, activity [85]. However, signaling by a different
steroid receptor, AR, was strongly affected by deletion of CDC37 [55].
Extending these findings, AR, but not GR, interacted with recombinant
Cdc37 in a GA-sensitive manner [137]. Therefore, Cdc37 is found
predominantly, but not exclusively, in Hsp90-protein kinase complexes.
Apparently, even if the substrates of Hsp90 belong to the same functional
class, like AR and GR, and share some structural similarities, they can have
distinct requirements from the Hsp90 complex. It is possible that the
structural differences between these substrates are significant enough to
require the use of a different set of chaperones and co-chaperones by the
cell. Binding to the Hsp90 complex may be determined by the folded state of
the substrate, rather than simply by the structure of folded domains. It is
conceivable that, in a partially folded state, AR resembles protein kinases
more closely than does GR, and is therefore recruited by Cdc37–Hsp90
complexes.
Cdc37 binds to Hsp90 at a site adjacent to the PP5 binding site. Full
length PP5, but not the TPR domain alone, competes with Cdc37 for the
binding to Hsp90, suggesting steric obstruction between Cdc37 and the
phosphatase domain of PP5 [153].
A direct role for Cdc37 in neoplastic processes is beginning to emerge.
Mouse mammary tumor virus promoter driven MMTV-Cdc37 transgenic
mice develop tumors at a similar rate as MMTV-cyclin D1 mice [163]. In
addition, human prostate cancer cells show increased expression levels of
CDC37 [166]. How CDC37 realizes its oncogenic property is not clear. A
possible mechanism involves its known interaction with oncogenes such as
Cdk4 and Raf1. In addition, both Cdc37 and Hsp90 have been found to
associate with oncogenic protein-tyrosine kinases, v-Yes, v-Fps, and v-Fgr
[73]. Cdc37 may regulate on its own, and help Hsp90 in regulating, a
number of proteins involved in cellular proliferation. Understanding the
mechanism and selectivity of this co-chaperone will remain particularly
important in the attempts to prevent cellular transformation by targeting
Hsp90 and its substrates.
4 S. CEREVISIAE AS A SYSTEM TO STUDY HSP90

CO-CHAPERONES
Key to the use of S. cerevisiae as a model system to study conserved

cellular processes are the similarities between yeast and higher eukaryotes
with respect to that particular set of processes. As mentioned earlier, most
components of the Hsp90 machinery, and the Hsp90 complexes themselves,
are conserved in S. cerevisiae (see Table 1 and [22]). In addition, the
mechanism of function of this machinery is similar to that in mammalian
cells sufficiently to enable proper regulation of nonconserved substrates,
such as steroid receptors and Src kinases.
Some important co-chaperones, however, are not found in S. cerevisiae. A
class of TPR co-chaperones that bind to Hsp90, consisting of large
immunophilins FKBP51 and FKBP52, have no S. cerevisiae homologs. Hip,
a co-chaperone for Hsp70, is also not conserved in S. cerevisiae. Two other
co-chaperones missing in budding yeast are the regulators of Hsp90 and
Hsp70, Bag-1 [80] and CHIP [5, 33]. It is possible that the functions these
proteins play are conserved, but are performed by evolutionarily divergent
homologs. Conversely, some co-chaperones of Hsp90 that have been
identified in S. cerevisiae apparently do not have functional homologs in
higher organisms. Hch1, a protein with no apparent homologs to any
proteins in higher eukaryotes [34, 67], has been identified as a multicopy
suppressor of the Hsp82E381K ts mutant [114]. Genetic analysis predicts that
this protein is a general regulator of Hsp90, and it is surprising that it is not

conserved. Therefore, although most components of Hsp90 complexes are
conserved, some may be specific to S. cerevisiae, probably because they
supply functions unique to the needs of a yeast cell.
The absence of some Hsp90 co-chaperones in S. cerevisiae may prove to
be advantageous in the study of nonconserved Hsp90 complex components.
In the same way that expression of heterologous substrates provides a tool
for investigating the role of Hsp90 complex components in vivo, expression
in S. cerevisiae of mammalian-specific Hsp90 co-chaperones, for example
FKBPs, may shed light on their in vivo roles.
There are considerable differences in the intrinsic ATPase activity of
Hsp90 and the way it is regulated between S. cerevisiae and metazoa. The
ATPase activity of purified yeast Hsp90 is at least several fold higher than
the ATPase activity of human Hsp90 [100, 112, 119, 135]. Furthermore,
effects of yeast co-chaperones on the ATPase activity of yeast Hsp90 and
effects of mammalian co-chaperones on the ATPase activity of mammalian
Hsp90 are significantly different. While Sti1 decreases the ATPase activity
of Hsp90 [135], Hop has no effect on the basal rate of ATP hydrolysis, but
inhibits the substrate protein-stimulated rate [100]. In addition, while Sba1
apparently has no effect on the ATP hydrolysis by yeast Hsp90 [117], p23
stimulates basal and substrate-stimulated ATPase activity [100]. If these are
bona fide differences between S. cerevisiae and human Hsp90, and not just
discrepancies in experimental approaches, then findings from the yeast
system may not be quite so readily extrapolated to mammalian cells, at least
regarding the regulation of the ATPase activity.
To compare the degree of conservation between fungal and mammalian
Hsp90 complexes, it is instructive to consider those substrates that are
conserved between yeast and higher eukaryotes. For example, HSF
complexes in S. cerevisiae, Xenopus laevis, and an in vitro reconstituted
system have all been studied [3, 48, 185]. While in S. cerevisiae Cpr7 helps
Hsp90 in the attenuation of the heat shock response under both basal and
induced conditions [48], immunodepletion of its mammalian homolog
CyP40 in the in vitro reconstitution assay fails to elicit HSF1 activation
[185]. Apparently, metazoan and yeast regulation of HSF differs with
regards to the involvement of cyclophilins. On the other hand, regulation of
some substrates exhibits striking similarities. For example, both Ste11 and
its mammalian homolog Raf require Cdc37 for full function and association
with the Hsp90 complex [2, 60]. Along the same lines, the models of Hsp90
regulation of eIF-2α kinases, fungal Gcn2, and mammalian PKR,
demonstrate similar mechanisms with multiple steps of Hsp90 association
and involvement of at least one common co-chaperone, Sba1/p23 [45, 46].
Identical requirements for the components of the Hsp90 complex by a
particular substrate in S. cerevisiae and mammalian cells cannot be assumed,

and will have to be established on an individual basis.
5 THE ROLE OF CO-CHAPERONES IN HSP90

COMPLEXES
It is clear from a large number of studies in model systems, as well as

from in vitro approaches, that co-chaperones are versatile modulators of
Hsp90 action that are involved with virtually every aspect of Hsp90
functioning. Underlying this adaptability is the fact that Hsp90 shares at least
some of its co-chaperones with other molecular chaperones. Hop/Sti1, for
example, binds both Hsp70 and Hsp90. In S. cerevisiae, Sti1, Cpr7, and
Cns1 interact with a molecular chaperone Hsp104, which is responsible for
both thermotolerance and maintenance of prion phenotypes in yeast cells [1].
Therefore, co-chaperones are likely to have very versatile roles both in the
regulation of Hsp90 and other cellular functions in general.
As we have seen, some co-chaperones can regulate the ATPase activity
of Hsp90. Presence of purified Sti1 decreases the ATPase activity of purified
yeast Hsp82 [135]. The ATPase activity of human Hsp90 is inhibited by the
addition of p23 [100]. Given the coupling of the ATPase cycle to the
chaperoning activities of Hsp90, the regulation of ATPase activity by the co-
chaperones is likely to be one of the most important aspects of the co-
chaperone involvement in the Hsp90 function.
Chaperoning of substrates by Hsp70 and Hsp90 has been well established,
but this activity may also be performed by the co-chaperones themselves.
p23, CyP40, and FKBP52 all have chaperone activities in vitro [13, 58].
Some co-chaperones, including Ydj1, p23, and ARA9/XAP2/AIP are
capable of binding client proteins directly [57, 71, 78, 102]. The formation
of such ternary complexes may be important for the adoption of proper
conformations by the substrates, or for the progress of the substrate to the
next step in the refolding cycle.
Hsp90 co-chaperones may also play a role in the proper localization of the
substrates. For example, FKBPs apparently use their interaction with dynein
to facilitate nuclear translocation of the steroid receptors [39]. Since CyP40
localizes to the nucleolus [97, 120], it may serve to localize Hsp90 to this
cellular compartment. In further support for this role of co-chaperones,
ARA9/XAP2/AIP regulates cellular distribution of AhR [82, 90, 125].
In spite of numerous experiments affirming the presence of co-chaperones
in Hsp90 complexes, and mounting evidence that co-chaperones play
important functional roles in these complexes, the question of whether any
of the co-chaperones are absolutely required for a specific Hsp90 function
remains unanswered. In vitro, two chaperones alone, Hsp70 and Hsp90, have
been reported to mediate maturation of steroid receptors [136], arguing
against an unconditional requirement for the co-chaperones. Furthermore,
although the Hsp90 MEEVD sequence is considered to be sufficient for the
binding of TPR domain co-chaperones, its deletion has no effect on the
viability of S. cerevisiae cells [94]. This suggests that interactions of Hsp90
with all proteins that bind to it through their TPR motifs are dispensable. If
this is the case, then TPR domain co-chaperones may simply be modulators
of Hsp90 functions. Alternatively, it may not be the physical association
with Hsp90 per se that is necessary for substrate regulation, but the presence
of co-chaperones in the Hsp90 complex, which can also be achieved through
binding to substrates directly. While some co-chaperones, like p23, are most
likely generally required by Hsp90, others may be specific for certain classes
of substrates, or only in some cell types, and under certain cellular
conditions.
Although it chaperones a large number of substrates, Hsp90 does not
interact indiscriminately with partially unfolded or inactive proteins,
suggesting that triage decisions are made in the cell that determine Hsp90
substrate specificity. One of the models of co-chaperone action, which posits
that co-chaperones determine specificity of the Hsp90 complex for indivi-
dual substrates, has been difficult to prove. Some Hsp90 co- chaperones
show clear preference for one class of substrates. ARA9/XAP2/AIP is the
only co-chaperone identified so far that apparently interacts with only one
Hsp90 substrate, AhR [6, 19, 82, 83, 90, 102, 103, 125]. All other co-
chaperones show a less stringent predilection for Hsp90 substrates. Cdc37,
for example, is present almost exclusively in protein kinase–Hsp90
complexes. However, Cdc37 also interacts with at least one steroid
receptor, AR. Proteins containing TPR domains are the most obvious
candidates for co-chaperones that determine specificity of the Hsp90
complex because they bind Hsp90 directly through the TPR domain, while
the other domain in each co-chaperone (isomerase in the case of
immunophilins, or phosphatase in the case of PP5/Ppt1) can mediate binding
to the appropriate substrate. Especially intriguing is the idea that Hsp90 can
accommodate more than one TPR domain co-chaperone (Cns1 and Cpr7, for
example), thus increasing the number of possible co-chaperone combinations
and, consequently, the number of substrates Hsp90 acts upon. However, it is
still equally likely that co-chaperones determine specificity of Hsp90
function not by bridging the chaperone to the substrate, but by keeping
Hsp90 itself in a conformation that favors some client proteins over others.
Consistent with this idea, defining the substrate recognition site for Hsp90
has proven to be difficult, suggesting that this is a highly versatile chaperone
capable of discriminating between similar proteins. Future work should be
able to distinguish the two hypotheses of Hsp90 substrate determination,

and, given the variety among co-chaperones, it is likely that the co-
chaperones will have widely differing roles in this process.
6 HSP90 CO-CHAPERONES IN CANCER
Hsp90 is of major interest to the area of cancer research as a direct target

of an antitumor agent which is currently in clinical trials, a GA derivative
17-allylaminogeldanamycin (17-AAG) [127]. Although it is known that GA
inhibits the ATPase activity of Hsp90, the mechanistic basis for the
antineoplastic properties of this class of benzoquinone ansamycins is not
clearly understood. Therefore, it is possible that a small number of key
tumorigenic substrates become unregulated when Hsp90 function is partially
lost. It is also possible that, given the involvement of Hsp90 in a number of
processes regulating cellular growth at multiple steps, there is a cumulative
requirement for Hsp90 by a large number of regulatory proteins, which leads
to the observed effect of GA-related drugs.
Several components of the Hsp90 complex are involved in the regulation
of cell cycle proteins. For example, phosphorylation and subsequent
degradation of Cln3, a yeast cyclin, depends on Ydj1 [179]. Wis2, a CyP40
homolog in S. pombe, can suppress a cell cycle defect [173]. As mentioned,
Cdc37 interacts with Cdk4 and Cdk6 (but not Cdk2 and Cdc2) [165]. Hsp90
itself interacts with Wee1 kinase, which phosphorylates cyclin B [4].
Therefore, various components of the Hsp90 complex may be involved at
multiple points of control of cell cycle, and mutations in these proteins could
lead to the loss of regulation and cellular transformation.
Certain substrates of Hsp90 have been implicated in tumorigenesis. Some
are oncogenic kinases, such as v-Src, v-Yes, v-Fps, and v-Fgr [14, 91, 184].
Telomerase, which is involved in cellular immortalization, also requires
Hsp90 complex for function [70]. A particularly important Hsp90 substrate
is p53, a well-known tumor suppressor; differences in the interaction of
Hsp90 with p53 wild type and mutant forms may have significant impact on
our understanding of cancer development [8]. As the list of known Hsp90
client proteins is growing, the number of those involved in tumorigenic
processes is likely to increase as well.
Some co-chaperones, such as Cdc37 and TTC4, are themselves potential
oncogenes or tumor suppressors [128, 163, 166, 167]. It is not clear,
however, whether the role of Cdc37 as an oncogene is directly related to its
involvement in Cdk4–Hsp90 complexes, and maybe also complexes with
other oncogenic kinases, or the oncogenic role is an unrelated property of
this co-chaperone.
Because a large number of proteins depend on Hsp90 for normal levels and
activity, it is a concern that indiscriminate inhibition of Hsp90 function, as is
achieved by inhibiting its function through GA, may have widely pleotropic
effects. If co-chaperones indeed determine the specificity for Hsp90, instead
of inhibiting Hsp90 function directly, it might be more advantageous to
inhibit the function of a specific co-chaperone involved in a complex with a
tumorigenic substrate. Additionally, instead of completely inhibiting the
function of a certain co-chaperone, specific inhibition of the physical
interaction between Hsp90 and the co-chaperone might provide even greater
specificity for the desired antitumor agent. In order to accomplish this, it will
be necessary to understand the details of the physical interaction between the
co-chaperones and Hsp90, as well as the various aspects of regulation by the
co-chaperones of both Hsp90 and the substrates, in the context of an in vivo
cell; for this approach, the yeast system is particularly well suited.
ACKNOWLEDGMENTS
The authors would like to thank Ellen Nollen, Joshua Schnell, and
Sricharan Bandhakavi for their critical reading of the manuscript and helpful
suggestions.
REFERENCES
1. Abbas-Terki, T., O. Donze, P. A. Briand, and D. Picard. 2001. Hsp104 interacts with
Hsp90 cochaperones in respiring yeast. Mol. Cell. Biol. 21:7569–7575.
2. Abbas-Terki, T., O. Donze, and D. Picard. 2000. The molecular chaperone Cdc37 is
required for Ste11 function and pheromone-induced cell cycle arrest. FEBS Lett.
467:111–116.
3. Ali, A., S. Bharadwaj, R. O’Carroll, and N. Ovsenek. 1998. HSP90 interacts with and
regulates the activity of heat shock factor 1 in Xenopus oocytes. Mol. Cell. Biol.
18:4949–4960.
4. Aligue, R., H. Akhavan-Niak, and P. Russell. 1994. A role for Hsp90 in cell cycle
control: Wee1 tyrosine kinase activity requires interaction with Hsp90. EMBO J.
13:6099–6106.
5. Ballinger, C. A., P. Connell, Y. Wu, Z. Hu, L. J. Thompson, L. Y. Yin, and
C. Patterson. 1999. Identification of CHIP, a novel tetratricopeptide repeat-containing
protein that interacts with heat shock proteins and negatively regulates chaperone
functions. Mol. Cell. Biol. 19:4535–4545.
6. Bell, D. R., and A. Poland. 2000. Binding of aryl hydrocarbon receptor (AhR) to AhR-
interacting protein. The role of hsp90. J. Biol. Chem. 275:36407–36414.
7. Berardini, T. Z., K. Bollman, H. Sun, and R. S. Poethig. 2001. Regulation of
vegetative phase change in Arabidopsis thaliana by cyclophilin 40. Science 291:2405–2407.
8. Blagosklonny, M. V., J. Toretsky, S. Bohen, and L. Neckers. 1996. Mutant

conformation of p53 translated in vitro or in vivo requires functional Hsp90. PNAS
93:8379–8383.
9. Blatch, G. L., and M. Lassle. 1999. The tetratricopeptide repeat: a structural motif
mediating protein- protein interactions. Bioessays 21:932–939.
10. Bohen, S. P. 1998. Genetic and biochemical analysis of p23 and ansamycin antibiotics
in the function of Hsp90-dependent signaling proteins. Mol. Cell. Biol. 18:3330–3339.
11. Bohen, S. P., and K. R. Yamamoto. 1993. Isolation of Hsp90 mutants by screening for
decreased steroid receptor function. PNAS 90:11424–11428.
12. Borkovich, K. A., F. W. Farrelly, D. B. Finkelstein, J. Taulien, and S. Lindquist.
1989. Hsp82 is an essential protein that is required in higher concentrations for growth
of cells at higher temperatures. Mol. Cell. Biol. 9:3919–3930.
13. Bose, S., T. Weikl, H. Bugl, and J. Buchner. 1996. Chaperone function of Hsp90-
associated proteins. Science 274:1715–1717.
14. Brugge, J. S. 1986. Interaction of the Rous sarcoma virus protein pp60src with the
cellular proteins pp50 and pp90. Curr. Top. Microbiol. Immunol. 123:1–22.
15. Brugge, J. S., G. Jarosik, J. Andersen, A. Queral-Lustig, M. Fedor-Chaiken, and
J. R. Broach. 1987. Expression of Rous sarcoma virus transforming protein pp60v-src
in Saccharomyces cerevisiae cells. Mol. Cell. Biol. 7:2180–2187.
16. Caplan, A. J., and M. G. Douglas. 1991. Characterization of YDJ1: a yeast homologue
of the bacterial dnaJ protein. J. Cell Biol. 114:609–621.
17. Carver, L. A., and C. A. Bradfield. 1997. Ligand-dependent interaction of the aryl
hydrocarbon receptor with a novel immunophilin homolog in vivo. J. Biol. Chem. 272:
11452–11456.
18. Carver, L. A., V. Jackiw, and C. A. Bradfield. 1994. The 90-kDa heat shock protein is
essential for Ah receptor signaling in a yeast expression system. J. Biol. Chem. 269:
30109–30112.
19. Carver, L. A., J. J. LaPres, S. Jain, E. E. Dunham, and C. A. Barfield. 1998. Charac-
terization of the Ah receptor-associated protein, ARA9. J. Biol. Chem. 273:33580–
33587.
20. Chadli, A., I. Bouhouche, W. Sullivan, B. Stensgard, N. McMahon, M. G. Catelli,
and D. O. Toft. 2000. Dimerization and N-terminal domain proximity underlie the
function of the molecular chaperone heat shock protein 90. Proc. Natl. Acad. Sci. USA
97:12524–12529.
21. Chang, H.-C. J., D. F. Nathan, and S. Lindquist. 1997. In vivo analysis of the
Hsp90 cochaperone Sti1 (p60). Mol. Cell. Biol. 17:318–325.
22. Chang, H. C., and S. Lindquist. 1994. Conservation of Hsp90 macromolecular
complexes in Saccharomyces cerevisiae. J. Biol. Chem. 269:24983–24988.
23. Cheetham, M. E., A. P. Jackson, and B. H. Anderton. 1994. Regulation of 70-kDa
heat-shock-protein ATPase activity and substrate binding by human DnaJ-like proteins,
HSJ1a and HSJ1b. Eur. J. Biochem. 226:99–107.
24. Chen, M.-S., A. m. Silverstein, W. B. Pratt, and M. Chinkers. 1996. The
tetratricopeptide domain of protein phosphatase 5 mediates binding to glucocorticoid
receptor heterocomplexes and acts as a dominant negative mutant. J. Biol. Chem. 271:
32315–32320.
25. Chen, M. X., and P. T. Cohen. 1997. Activation of protein phosphatase 5 by limited
proteolysis or the binding of polyunsaturated fatty acids to the TPR domain. FEBS Lett.
400:136–140.
26. Chen, M. X., A. E. McPartlin, L. Brown, Y. H. Chen, H. M. Barker, and P. T.

Cohen. 1994. A novel human protein serine/threonine phosphatase, which possesses
four tetratricopeptide repeat motifs and localizes to the nucleus. EMBO J. 13:4278–4290.
27. Chen, S., V. Prapapanich, R. A. Rimerman, B. Honore, and D. F. Smith. 1996.
Interactions of p60, a mediator of progesterone receptor assembly, with heat shock
proteins hsp90 and hsp70. Mol. Endocrinol. 10:682–693.
28. Chen, S., and D. F. Smith. 1998. Hop as an adaptor in the heat shock protein 70
(Hsp70) and Hsp90 chaperone machinery. J. Biol. Chem. 273:35194–35200.
29. Chen, S., W. P. Sullivan, D. O. Toft, and D. F. Smith. 1998. Differential interactions
of p23 and the TPR-containing proteins Hop, Cyp40, FKBP52 and FKBP51 with Hsp90
mutants. Cell Stress Chaperones 3:118–129.
30. Cheng, L., K. Hirst, and P. W. Piper. 1992. Authentic temperature-regulation of a heat
shock gene inserted into yeast on a high copy number vector. Influences of
overexpression of HSP90 protein on high temperature growth and thermotolerance.
Biochim. Biophys. Acta. 1132:26–34.
31. Cheung, J., and D. F. Smith. 2000. Molecular chaperone interactions with steroid
receptors: an update. Mol. Endocrinol. 14:939–946.
32. Chinkers, M. 2001. Protein phosphatase 5 in signal transduction. Trends Endocrinol.
Metab. 12:28–32.
33. Connell, P., C. A. Ballinger, J. Jiang, Y. Wu, L. J. Thompson, J. Hohfeld, and
C. Patterson. 2001. The co-chaperone CHIP regulates protein triage decisions mediated
by heat-shock proteins. Nat. Cell Biol. 3:93–96.
34. Costanzo, M. C., M. E. Crawford, J. E. Hirschman, J. E. Kranz, P. Olsen, L. S.
Robertson, M. S. Skrzypek, B. R. Braun, K. L. Hopkins, P. Kondu, C. Lengieza,
J. E. Lew-Smith, M. Tillberg, and J. I. Garrels. 2001. YPD, PombePD and WormPD:
model organism volumes of the BioKnowledge library, an integrated resource for
protein information. Nucleic Acids Res. 29:75–79.
35. Counter, C. M., A. A. Avilion, C. E. LeFeuvre, N. G. Stewart, C. W. Greider, C. B.
Harley, and S. Bacchetti. 1992. Telomere shortening associated with chromosome
instability is arrested in immortal cells which express telomerase activity. EMBO J.
11:1921–1929.
36. Crevel, G., H. Bates, H. Huikeshoven, and S. Cotterill. 2001. The Drosophila Dpit47
protein is a nuclear Hsp90 co-chaperone that interacts with DNA polymerase alpha. J.
Cell Sci. 114:2015–2025.
37. Cutforth, T., and G. M. Rubin. 1994. Mutations in Hsp83 and cdc37 impair signaling
by the sevenless receptor tyrosine kinase in Drosophila. Cell 77:1027–1036.
38. Das, A. K., P. T. W. Cohen, and D. Bradford. 1998. The structure of the
tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated
protein-protein interactions. EMBO J. 17:1192–1199.
39. Davies, T. H., Y. M. Ning, and E. R. Sanchez. 2002. A new first step in activation of
steroid receptors: hormone-induced switching of FKBP51 and FKBP52 immunophilins.
J. Biol. Chem. 277:4597–4600.
40. Dey, B., A. J. Caplan, and F. Boschelli. 1996. The Ydj1 molecular chaperone
facilitates formation of active p60v-src in yeast. Mol. Biol. Cell. 7:91–100.
41. Dey, B., J. J. Lightbody, and F. Boschelli. 1996. CDC37 is required for p60v-src
activity in yeast. Mol. Biol. Cell. 7:1405–1417.
42. Dittmar, K. D., D. R. Demady, L. F. Stancato, P. Krishna, and W. B. Pratt. 1997.
Folding of the glucocorticoid receptor by the heat shock protein (hsp) 90-based
chaperone machinery. The role of p23 is to stabilize receptor.hsp90 heterocomplexes
formed by hsp90.p60.hsp70. J. Biol. Chem. 272:21213–21220.
43. Dittmar, K. D., K. A. Hutchison, J. K. Owens-Grillo, and W. B. Pratt. 1996.

Reconstitution of the steroid receptor.hsp90 heterocomplex assembly system of rabbit
reticulocyte lysate. J. Biol. Chem. 271:12833–12839.
44. Dolinski, K., M. Cardenas, and J. Heitman. 1998. CNS1 encodes an essential
p60/Sti1 homolog in Saccharomyces cerevisiae that suppresses cyclophilin 40 mutations
and interacts with Hsp90. Mol. Cell. Biol. 18:7344–7352.
45. Donze, O., T. Abbas-Terki, and D. Picard. 2001. The Hsp90 chaperone complex is
both a facilitator and a repressor of the dsRNA-dependent kinase PKR. EMBO J.
20:3771–3780.
46. Donze, O., and D. Picard. 1999. Hsp90 binds and regulates Gcn2, the ligand-inducible
kinase of the alpha subunit of eukaryotic translation initiation factor 2 [corrected]. Mol.
Cell. Biol. 19:8422–8432.
47. Duina, A. A., H.-C. J. CHang, J. A. Marsh, S. Lindquist, and R. F. Gaber. 1996. A
Cyclophilin function in Hsp90-dependent signal transduction. Science 274:1713–1715.
48. Duina, A. A., H. M. Kalton, and R. F. Gaber. 1998. Requirement for Hsp90 and a
CyP-40-type cyclophilin in negative regulation of the heat shock response. J. Biol.
Chem. 273:18974–18978.
49. Duina, A. A., J. A. Marsh, and R. F. Gaber. 1996. Identification of two CyP-40-like
cyclophilins in Saccharomyces cerevisiae, one of which is required for normal growth.
yeast 12:943–952.
50. Duina, A. A., J. A. Marsh, R. B. Kurtz, H.-C. J. Chang, S. Lindquist, and R. F.
Gaber. 1998. The peptidyl-prolyl isomerase domain of the CyP-40 cyclophilin homolog
is not required to support growth or glucocorticoid receptor activity in Saccharomyces
cerevisiae. J. Biol. Chem. 273:10819–10822.
51. Fabrizio, P., F. Pozza, S. D. Pletcher, C. M. Gendron, and V. D. Longo. 2001.
Regulation of longevity and stress resistance by Sch9 in yeast. Science 292:288–290.
52. Fang, Y., A. E. Fliss, J. Rao, and A. J. Caplan. 1998. SBA1 encodes a yeast Hsp90
cochaperone that is homologous to vertebrate p23 proteins. Mol. Cell. Biol. 18:3727–
3734.
53. Fang, Y., A. E. Fliss, D. M. Robins, and A. J. Caplan. 1996. Hsp90 regulates
androgen receptor hormone binding affinity in vivo. J. Biol. Chem. 271:28697–28702.
54. Finkelstein, D. B., S. Strausberg, and L. McAlister. 1982. Alterations of transcription
during heat shock of Saccharomyces cerevisiae. J. Biol. Chem. 257:8405–8411.
55. Fliss, A. E., Y. Fang, F. Boschelli, and A. J. Caplan. 1997. Differential in vivo regu-
lation of steroid hormone receptor activation by Cdc37p. Mol. Biol. Cell 8:2501–2509.
56. Fliss, A. E., J. Rao, M. W. Melville, M. E. Cheetham, and A. J. Caplan. 1999.
Domain requirements of DnaJ-like (Hsp40) molecular chaperones in the activation of a
steroid hormone receptor. J. Biol. Chem. 274:34045–34052.
57. Freeman, B. C., S. J. Felts, D. O. Toft, and K. R. Yamamoto. 2000. The p23
molecular chaperones act at a late step in intracellular receptor action to differentially
affect ligand efficacies. Genes Dev. 14:422–434.
58. Freeman, B. C., D. O. Toft, and R. I. Morimoto. 1996. Molecular chaperone
machines: chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-
associated protein p23. Science 274:1718–1720.
59. Frydman, J., and J. Hohfeld. 1997. Chaperones get in touch: the hip-hop connection.
Trends Biol. Sci. 22:87–92.
60. Grammatikakis, N., J. H. Lin, A. Grammatikakis, P. N. Tsichlis, and B. H.
Cochran. 1999. p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function.
Mol. Cell. Biol. 19:1661–1672.
61. Grandin, N., and M. Charbonneau. 2001. Hsp90 levels affect telomere length in
yeast. Mol. Genet. Genomics 265:126–134.
62. Grenert, J. P., B. D. Johnson, and D. O. Toft. 1999. The importance of ATP binding
and hydrolysis by hsp90 in formation and function of protein heterocomplexes. J. Biol.
Chem. 274:17525–17533.
63. Hamilton, G. S., and J. P. Steiner. 1998. Immunophilins: beyond immunosuppression.
J. Med. Chem. 41:5119–5143.
64. Harris, N., M. MacLean, K. Hatzianthis, B. Panaretou, and P. W. Piper. 2001.
Increasing Saccharomyces cerevisiae stress resistance, through the overactivation of the
heat shock response resulting from defects in the Hsp90 chaperone, does not extend
replicative life span but can be associated with slower chronological ageing of
nondividing cells. Mol. Genet. Genomics 265:258–263.
65. Hastie, N. D., M. Dempster, M. G. Dunlop, A. M. Thompson, D. K. Green, and
R. C. Allshire. 1990. Telomere reduction in human colorectal carcinoma and with
ageing. Nature 346:866–868.
66. Hernandez, M. P., A. Chadli, and D. O. Toft. 2002. hsp40 binding is the first step in
the hsp90 chaperoning pathway for the progesterone receptor. J. Biol. Chem. 23:23.
67. Hodges, P. E., A. H. McKee, B. P. Davis, W. E. Payne, and J. I. Garrels. 1999. The
yeast proteome database (YPD): a model for the organization and presentation of
genome-wide functional data. Nucleic Acids Res. 27:69–73.
68. Hoffmann, K., and R. Handschumacher. 1995. Cyclophilin-40: evidence for a dimeric
complex with Hsp90. Biochem. J. 307:5–8.
69. Hoggard, N., Y. Hey, B. Brintnell, L. James, D. Jones, E. Mitchell, J. Weissenbach,
and J. M. Varley. 1995. Identification and cloning in yeast artificial chromosomes of a
region of elevated loss of heterozygosity on chromosome 1p31.1 in human breast
cancer. Genomics 30:233–243.
70. Holt, S. E., D. L. Aisner, J. Baur, V. M. Tesmer, M. Dy, M. Ouellette, J. B. Trager,
G. B. Morin, D. O. Toft, J. W. Shay, W. E. Wright, and M. A. White. 1999.
Functional requirement of p23 and Hsp90 in telomerase complexes. Genes Dev. 13:817–
826.
71. Hon, T., H. C. Lee, A. Hach, J. L. Johnson, E. A. Craig, H. Erdjument-Bromage,
P. Tempst, and L. Zhang. 2001. The Hsp70-Ydj1 molecular chaperone represses the
activity of the heme activator protein Hap1 in the absence of heme. Mol. Cell. Biol.
21:7923–7932.
72. Hu, J., D. O. Toft, and C. Seeger. 1997. Hepadnavirus assembly and reverse
transcription require a multi-component chaperone complex which is incorporated into
nucleocapsids. EMBO J. 16:59–68.
73. Hunter, T., and R. Y. C. Poon. 1997. Cdc37: a protein kinase chaperone? Trends Cell
Biol. 7:157–161.
74. Hutchison, K. A., K. D. Dittmar, and W. B. Pratt. 1994. All of the factors required
for assembly of the glucocorticoid receptor into a functional heterocomplex with heat
shock protein 90 are preassociated in a self-sufficient protein folding structure, a
“foldosome”. J. Biol. Chem. 269:27894–27899.
75. Jakob, U., and J. Buchner. 1994. Assisting spontaneity: the role of Hsp90 and small
Hsps as molecular chaperones. Trends Biochem. Sci. 19:205–211.
76. Johnson, B. D., R. J. Schumacher, E. D. Ross, and D. O. Toft. 1998. Hop modulates
Hsp70/Hsp90 interactions in protein folding. J. Biol. Chem. 273:3679–3686.
77. Johnson, J. L., and E. A. Craig. 2001. An essential role for the substrate-binding
region of Hsp40s in Saccharomyces cerevisiae. J. Cell. Biol. 152:851–856.
78. Johnson, J. L., and E. A. Craig. 2000. A role for the Hsp40 Ydj1 in repression of basal
steroid receptor activity in yeast. Mol. Cell. Biol. 20:3027–3036.
79. Johnson, J. L., and D. O. Toft. 1995. Binding of p23 and hsp90 during assembly with
the progesterone receptor. Mol. Endocrinol. 9:670–678.
80. Kanelakis, K. C., Y. Morishima, K. D. Dittmar, M. D. Galigniana, S. Takayama,
J. C. Reed, and W. B. Pratt. 1999. Differential effects of the hsp70-binding protein
BAG-1 on glucocorticoid receptor folding by the hsp90-based chaperone machinery. J.
Biol. Chem. 274:34134–34140.
81. Kang, H., S. L. Sayner, K. L. Gross, L. C. Russell, and M. Chinkers. 2001.
Identification of amino acids in the tetratricopeptide repeat and C-terminal domains of
protein phosphatase 5 involved in autoinhibition and lipid activation. Biochemistry
40:10485–10490.
82. Kazlauskas, A., L. Poellinger, and I. Pongratz. 2000. The immunophilin-like protein
XAP2 regulates ubiquitination and subcellular localization of the dioxin receptor.
J. Biol. Chem. 275:41317–41324.
83. Kazlauskas, A., S. Sundstrom, L. Poellinger, and I. Pongratz. 2001. The hsp90
chaperone complex regulates intracellular localization of the dioxin receptor. Mol. Cell.
Biol. 21:2594–25607.
84. Kimura, Y., S. Matsumoto, and I. Yahara. 1994. Temperature-sensitive mutants of
hsp82 of the budding yeast Saccharomyces cerevisiae. Mol. Gen. Genet. 242:517–527.
85. Kimura, Y., S. L. Rutherford, Y. Miyata, I. Yahara, B. C. Freeman, L. Yue, R. I.
Morimoto, and S. Lindquist. 1997. Cdc37 is a molecular chaperone with specific
functions in signal transduction. Genes Dev. 11:1775–1785.
86. Kimura, Y., I. Yahara, and S. Lindquist. 1995. Role of the protein chaperone YDJ1 in
establishing Hsp90-mediated signal transduction pathways. Science 268:1362–1365.
87. King, F. W., A. Wawrzynow, J. Hohfeld, and M. Zylicz. 2001. Co-chaperones Bag-1,
Hop and Hsp40 regulate Hsc70 and Hsp90 interactions with wild-type or mutant p53.
EMBO J. 20:6297–6305.
88. Kornbluth, S., R. Jove, and H. Hanafusa. 1987. Characterization of avian and viral
p60src proteins expressed in yeast. Proc. Natl. Acad. Sci. USA 84:4455–4459.
89. Lamb, J. R., S. Tugendreich, and P. Hieter. 1995. Tetratrico peptide repeat
interactions: to TPR or not to TPR? Trends Biochem. Sci. 20:257–259.
90. LaPres, J. J., E. Glover, E. E. Dunham, M. K. Bunger, and C. A. Bradfield. 2000.
ARA9 modifies agonist signaling through an increase in cytosolic aryl hydrocarbon
receptor. J. Biol. Chem. 275:6153–6159.
91. Lipsich, L. A., J. R. Cutt, and J. S. Brugge. 1982. Association of the transforming
proteins of Rous, Fujinami, and Y73 avian sarcoma viruses with the same two cellular
proteins. Mol. Cell. Biol. 2:875–880.
92. Liu, X. D., K. A. Morano, and D. J. Thiele. 1999. The yeast Hsp110 family member,
Sse1, is an Hsp90 cochaperone. J. Biol. Chem. 274:26654–26660.
93. Louvion, J.-F., T. Abbas-Terki, and D. Picard. 1998. Hsp90 is required for
pheromone signalling in yeast. Mol. Biol. Cell 9:3071–3083.
94. Louvion, J.-F., R. Warth, and D. Picard. 1996. Two eukaryote-specific regions of
Hsp82 are dispensable for its viability and signal transduction functions in yeast. PNAS
93:13937–13942.
95. Luke, M. M., A. Sutton, and K. T. Arndt. 1991. Characterization of SIS1, a
Saccharomyces cerevisiae homologue of bacterial dnaJ proteins. J. Cell Biol. 114:623–638.
96. Marcu, M. G., A. Chadli, I. Bouhouche, M. Catelli, and L. M. Neckers. 2000. The
heat shock protein 90 antagonist novobiocin interacts with a previously unrecognized
ATP-binding domain in the carboxyl terminus of the chaperone. J. Biol. Chem. 275:
37181–37186.
97. Mark, P. J., B. K. Ward, P. Kumar, H. Lahooti, R. F. Minchin, and T. Ratajczak.
2001. Human cyclophilin 40 is a heat shock protein that exhibits altered intracellular
localization following heat shock. Cell Stress Chaperones 6:59–70.
98. Marsh, J. A., H. M. Kalton, and R. F. Gaber. 1998. Cns1 is an essential protein
associated with the Hsp90 chaperone complex in Saccharomyces cerevisiae that can
restore cyclophilin 40-dependent functions in cpr7∆ cells. Mol. Cell. Biol. 18:7353–
7359.
99. Mayr, C., K. Richter, H. Lilie, and J. Buchner. 2000. Cpr6 and Cpr7, two closely
related Hsp90-associated immunophilins from Saccharomyces cerevisiae, differ in their
functional properties. J. Biol. Chem. 275:34140–34146.
100. McLaughlin, S. H., H. W. Smith, and S. E. Jackson. 2002. Stimulation of the weak
ATPase activity of human hsp90 by a client protein. J. Mol. Biol. 315:787–798.
101. Meng, X., J. Devin, W. P. Sullivan, D. Toft, E. E. Baulieu, and M. G. Catelli. 1996.
Mutational analysis of Hsp90 alpha dimerization and subcellular localization: dimer
disruption does not impede “in vivo” interaction with estrogen receptor. J. Cell Sci.
109:1677–1687.
102. Meyer, B. K., and G. H. Perdew. 1999. Characterization of the AhR-hsp90-XAP2 core
complex and the role of the immunophilin-related protein XAP2 in AhR stabilization.
Biochemistry 38:8907–8917.
103. Meyer, B. K., J. R. Petrulis, and G. H. Perdew. 2000. Aryl hydrocarbon (Ah) receptor
levels are selectively modulated by hsp90-associated immunophilin homolog XAP2.
Cell Stress Chaperones 5:243–254.
104. Miao, B., J. Davis, and E. A. Craig. 1997. The Hsp70 family – an overview., p. 3–13.
In M. J. Gethring (ed.), Guidebook to Molecular Chaperones and Protein Catalysis.
Oxford University Press, Oxford.
105. Miller, C. A. 2002. Two tetratricopeptide repeat proteins facilitate human aryl
hydrocarbon receptor signalling in yeast. Cell Signal. 14:615–623.
106. Miller, C. A., 3rd. 1997. Expression of the human aryl hydrocarbon receptor complex in
yeast. Activation of transcription by indole compounds. J. Biol. Chem. 272:32824–
32829.
107. Minami, Y., Y. Kimura, H. Kawasaki, K. Suzuki, and I. Yahara. 1994. The carboxy-
terminal region of mammalian hsp90 is required for its dimerization and function
in vivo. Mol. Cell. Biol. 14:1459–1464.
108. Morano, K. A., N. Santoro, K. A. Koch, and D. J. Thiele. 1999. A trans-activation
domain in yeast heat shock transcription factor is essential for cell cycle progression
during stress. Mol. Cell. Biol. 19:402–411.
109. Morishima, Y., P. J. Murphy, D. P. Li, E. R. Sanchez, and W. B. Pratt. 2000.
Stepwise assembly of a glucocorticoid receptor.hsp90 heterocomplex resolves two
sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the
steroid binding pocket. J. Biol. Chem. 275:18054–18060.
110. Mukai, H., T. Kuno, H. Tanaka, D. Hirata, T. Miyakawa, and C. Tanaka. 1993.
Isolation and characterization of SSE1 and SSE2, new members of the yeast HSP70
multigene family. Gene 132:57–66.
111. Munoz, M. J., E. R. Bejarano, R. R. Daga, and J. Jimenez. 1999. The identification
of Wos2, a p23 homologue that interacts with Wee1 and Cdc2 in the mitotic control of
fission yeasts. Genetics 153:1561–1572.
112. Nadeau, K., A. Das, and C. T. Walsh. 1993. Hsp90 chaperonins possess ATPase
activity and bind heat shock transcription factors and peptidyl prolyl isomerases. J. Biol.
Chem. 268:1479–1487.
113. Nathan, D. F., and S. Lindquist. 1995. Mutational analysis of Hsp90 function:
interactions with a steroid receptor and a protein kinase. Mol. Cell. Biol. 15:3917–3925.
114. Nathan, D. F., M. H. Vos, and S. Lindquist. 1999. Identification of SSF1, CNS1, and
HCH1 as multicopy suppressors of a Saccharomyces cerevisiae Hsp90 loss-of-function
mutation. Proc. Natl. Acad. Sci. USA 96:1409–1414.
115. Nemoto, T., Y. Ohara-Nemoto, and M. Ota. 1992. Association of the 90-kDa heat
shock protein does not affect the ligand-binding ability of androgen receptor. J. Steroid
Biochem. Mol. Biol. 42:803–812.
116. Nicolet, C. M., and E. A. Craig. 1989. Isolation and characterization of STI1, a stress-
inducible gene from Saccharomyces cerevisiae. Mol. Cell. Biol. 9:3638–3646.
117. Obermann, W. M., H. Sondermann, A. A. Russo, N. P. Pavletich, and F. U. Hartl.
1998. In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis.
J. Cell Biol. 143:901–910.
118. Ohara-Nemoto, Y., T. Nemoto, and M. Ota. 1991. The Mr 90,000 heat shock protein-
free androgen receptor has a high affinity for steroid, in contrast to the glucocorticoid
receptor. J. Biochem. (Tokyo) 109:113–119.
119. Owen, B. A., W. P. Sullivan, S. J. Felts, and D. O. Toft. 2002. Regulation of heat
shock protein 90 ATPase activity by sequences in the carboxyl terminus. J. Biol. Chem.
277:7086–7091.
120. Owens-Grillo, J. K., M. J. Czar, K. A. Hutchinson, K. Hoffman, G. H. Perdew, and
W. B. Pratt. 1996. A model of protein targeting mediated by immunophilins and other
proteins that bind to Hsp90 via tetratricopeptide repeat domains. J. Biol. Chem. 271:
13468–13475.
121. Owens-Grillo, J. K., K. Hoffmann, K. A. Hutchinson, A. W. Yem, M. R. Deibel,
R. E. Handschumacher, and W. B. Pratt. 1995. The cyclosporin A-binding
immunophilin CyP-40 and the FK506-binding immunophilin hsp56 bind to a common
site on hsp90 and exist in independent cytosolic heterocomplexes with the
untransformed glucocorticoid receptor. J. Biol. Chem. 270:20479–20484.
122. Panaretou, B., C. Prodromou, S. M. Roe, R. O’Brien, J. E. Ladbury, P. W. Piper,
and L. H. Pearl. 1998. ATP binding and hydrolysis are essential to the function of the
Hsp90 molecular chaperone in vivo. EMBO J. 17:4829–4836.
123. Pearl, L. H., and C. Prodromou. 2000. Structure and in vivo function of Hsp90. Curr.
Opin. Struct. Biol. 10:46–51.
124. Perdew, G. H., and M. L. Whitelaw. 1991. Evidence that the 90-kDa heat shock
protein (HSP90) exists in cytosol in heteromeric complexes containing HSP70 and three
other proteins with Mr of 63,000, 56,000, and 50,000. J. Biol. Chem. 266:6708–6713.
125. Petrulis, J. R., N. G. Hord, and G. H. Perdew. 2000. Subcellular localization of the
aryl hydrocarbon receptor is modulated by the immunophilin homolog hepatitis B virus
X-associated protein 2. J. Biol. Chem. 275:37448–37453.
126. Picard, D., B. Khursheed, M. J. Gabaredian, M. G. Fortin, S. Lindquist, and K. R.
Yamamoto. 1990. Reduced levels of hsp90 compromise steroid receptor action in vivo.
Nature 348:166–168.
127. Piper, P. W. 2001. The Hsp90 chaperone as a promising drug target. Curr. Opin.
Investig. Drugs 2:1606–1610.
128. Poetsch, M., T. Dittberner, J. K. Cowell, and C. Woenckhaus. 2000. TTC4, a novel
candidate tumor suppressor gene at 1p31 is often mutated in malignant melanoma of the
skin. Oncogene 19:5817–5820.
129. Prapapanich, V., S. Chen, S. C. Nair, R. A. Rimerman, and D. F. Smith. 1996.

Molecular cloning of human p48, a transient component of progesterone receptor
complexes and an Hsp70-binding protein. Mol. Endocrinol. 10:420–431.
130. Pratt, W. B. 1998. The hsp90-based chaperone system: involvement in signal
transduction from a variety of hormone and growth factor receptors. Proc. Soc. Exp.
Biol. Med. 217:420–434.
131. Pratt, W. B., and K. D. Dittmar. 1998. Studies with purified chaperones advance the
understanding of the mechanism of glucocorticoid receptor-hsp90 complex assembly.
Trends Endocrinol. Metab. 9:244–252.
132. Pratt, W. B., and D. O. Toft. 1997. Steroid receptor interactions with heat shock
protein and immunophilin chaperones. Endocr. Rev. 18:306–360.
133. Prodromou, C., B. Panaretou, S. Chohan, G. Siligardi, R. O’Brien, J. E. Ladbury,
S. M. Roe, P. W. Piper, and L. H. Pearl. 2000. The ATPase cycle of hsp90 drives a
molecular “clamp” via transient dimerization of the N-terminal domains [In Process
Citation]. EMBO J. 19:4383–4392.
134. Prodromou, C., S. M. Roe, R. O’Brien, J. E. Ladbury, P. W. Piper, and L. H. Pearl.
1997. Identification and structural characterization of the ATP/ADP-binding site in the
Hsp90 molecular chaperone. Cell 90:65–75.
135. Prodromou, C., G. Siligardi, R. O’Brien, D. Woolfson, L. Regan, B. Panaretou,
J. E. Ladbury, P. W. Piper, and L. H. Pearl. 1999. Regulation of Hsp90 ATPase
activity by tetratricopeptide repeat (TPR)-domain co-chaperones. EMBO J. 18:754–762.
136. Rajapandi, T., L. E. Greene, and E. Eisenberg. 2000. The molecular chaperones
Hsp90 and Hsc70 are both necessary and sufficient to activate hormone binding by
glucocorticoid receptor. J. Biol. Chem. 275:22597–22604.
137. Rao, J., P. Lee, S. Benzeno, C. Cardozo, J. Albertus, D. M. Robins, and A. J.
Caplan. 2001. Functional interaction of human Cdc37 with the androgen receptor but
not with the glucocorticoid receptor. J. Biol. Chem. 276:5814–5820.
138. Ratajczak, T., A. Carrello, P. J. Mark, B. J. Warner, R. J. Simpson, R. L. Moritz,
and A. K. House. 1993. The cyclophilin component of the unactivated estrogen
receptor contains a tetratricopeptide repeat domain and shares identity with p59
(FKBP59). J. Biol. Chem. 268:13187–13192.
139. Reed, S. I. 1980. The selection of S. cerevisiae mutants defective in the start event of
cell division. Genetics 95:561–577.
140. Richter, K., P. Muschler, O. Hainzl, and J. Buchner. 2001. Coordinated ATP
hydrolysis by the Hsp90 dimer. J. Biol. Chem. 276:33689–33696.
141. Russell, L. C., S. R. Whitt, M. S. Chen, and M. Chinkers. 1999. Identification of
conserved residues required for the binding of a tetratricopeptide repeat domain to heat
shock protein 90. J. Biol. Chem. 274:20060–20063.
142. Rutherford, S. L., and S. Lindquist. 1998. Hsp90 as a capacitor for morphological
evolution. Nature 396:336–342.
143. Sanchez, E. R. 1990. Hsp56: a novel heat shock protein associated with untransformed
steroid receptor complexes. J. Biol. Chem. 265:22067–22070.
144. Sanchez, E. R., L. E. Faber, W. J. Henzel, and W. B. Pratt. 1990. The 56-59-
kilodalton protein identified in untransformed steroid receptor complexes is a unique
protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat
shock proteins. Biochemistry 29:5145–5152.
145. Scheibel, T., T. Weikl, and J. Buchner. 1998. The chaperone sites on Hsp90 differing
in substrate specificity and ATP dependence. PNAS 95:1495–1499.
146. Scheibel, T., T. Weikl, R. Rimerman, D. Smith, S. Lindquist, and J. Buchner. 1999.
Contribution of N- and C-terminal domains to the function of Hsp90 in Saccharomyces
cerevisiae. Mol. Microbiol. 34:701–713.
147. Schena, M., and K. R. Yamamoto. 1988. Mammalian glucocorticoid receptor
derivatives enhance transcription in yeast. Science 1988:965–967.
148. Scheufler, C., A. Brinker, G. Bourenkov, S. Pegoraro, L. Moroder, H. Bartunik,
F. U. Hartl, and I. Moarefi. 2000. Structure of TPR domain-peptide complexes: critical
elements in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101:199–
210.
149. Schulte, T. W., W. G. An, and L. M. Neckers. 1997. Geldanamycin-induced desta-
bilization of Raf-1 involves the proteasome. Biochem. Biophys. Res. Commun. 239:
655–659.
150. Schulte, T. W., M. V. Blagosklonny, C. Ingui, and L. Neckers. 1995. Disruption of
the Raf-1-Hsp90 molecular complex results in destabilization of Raf-1 and loss of Raf-
1-Ras association. J. Biol. Chem. 270:24585–24588.
151. Silverstein, A. M., M. D. Galigniana, M. S. Chen, J. K. Owens-Grillo, M. Chinkers,
and W. B. Pratt. 1997. Protein phosphatase 5 is a major component of glucocorticoid
receptor.hsp90 complexes with properties of an FK506-binding immunophilin. J. Biol.
Chem. 272:16224–16230.
152. Silverstein, A. M., M. D. Galigniana, K. C. Kanelakis, C. Radanyi, J. M. Renoir,
and W. B. Pratt. 1999. Different regions of the immunophilin FKBP52 determine its
association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein. J. Biol.
Chem. 274:36980–36986.
153. Silverstein, A. M., N. Grammatikakis, B. H. Cochran, M. Chinkers, and W. B.
Pratt. 1998. p50cdc37 binds directly to the catalytic domain of raf as well as to the site
on hsp90 that is topologically adjacent to the tetratricopeptide repeat site. J. Biol. Chem.
273:20090–20095.
154. Skinner, J., C. Sinclair, C. Romeo, D. Armstrong, H. Charbonneau, and S. Rossie.
1997. Purification of a fatty acid-stimulated protein-serine/threonine phosphatase from
bovine brain and its identification as a homolog of protein phosphatase 5. J. Biol. Chem.
272:22464–22471.
155. Smith, D. F., B. A. Stensgard, W. J. Welch, and D. O. Toft. 1992. Assembly of
progesterone receptor with heat shock proteins and receptor activation are ATP
mediated events. J. Biol. Chem. 267:1350–1356.
156. Smith, D. F., W. P. Sullivan, T. N. Marion, K. Zaitsu, B. Madden, D. J.
McCormick, and D. O. Toft. 1993. Identification of a 60-kilodalton stress-related
protein, p60, which interacts with hsp90 and hsp70. Mol. Cell. Biol. 13:869–876.
157. Smith, D. F., and D. O. Toft. 1992. Composition, assembly and activation of the avian
progesterone receptor. J. Steroid Biochem. Mol. Biol. 41:201–207.
158. Smith, D. F., and D. O. Toft. 1993. Steroid receptors and their associated proteins.
Mol. Endocrin. 7:4–11.
159. Soti, C., A. Racz, and P. Csermely. 2002. A Nucleotide-dependent molecular switch
controls ATP binding at the C-terminal domain of Hsp90. N-terminal nucleotide binding
unmasks a C-terminal binding pocket. J. Biol. Chem. 277:7066–7075.
160. Stancato, L. F., Y. H. Chow, K. A. Hutchison, G. H. Perdew, R. Jove, and W. B.
Pratt. 1993. Raf exists in a native heterocomplex with hsp90 and p50 that can be
reconstituted in a cell-free system. J. Biol. Chem. 268:21711–21716.
161. Stancato, L. F., A. M. Silverstein, J. K. Owens-Grillo, Y. H. Chow, R. Jove, and
W. B. Pratt. 1997. The hsp90-binding antibiotic geldanamycin decreases Raf levels and
epidermal growth factor signaling without disrupting formation of signaling complexes

or reducing the specific enzymatic activity of Raf kinase. J. Biol. Chem. 272:4013–4020.
162. Stark, M. J. 1996. Yeast protein serine/threonine phosphatases: multiple roles and
diverse regulation. Yeast 12:1647–1675.
163. Stepanova, L., M. Finegold, F. DeMayo, E. V. Schmidt, and J. W. Harper. 2000.
The oncoprotein kinase chaperone CDC37 functions as an oncogene in mice and
collaborates with both c-myc and cyclin D1 in transformation of multiple tissues. Mol.
Cell. Biol. 20:4462–4473.
164. Stepanova, L., X. Leng, and J. W. Harper. 1997. Analysis of mammalian Cdc37, a
protein kinase targeting subunit of heat shock protein 90. Meth. Enzymol. 283:220–229.
165. Stepanova, L., X. Leng, S. B. Parker, and W. J. Parker. 1996. Mammalian p50cdc37
is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4. Genes
Dev. 10:1491–1502.
166. Stepanova, L., G. Yang, F. DeMayo, T. M. Wheeler, M. Finegold, T. C. Thompson,
and J. W. Harper. 2000. Induction of human Cdc37 in prostate cancer correlates with
the ability of targeted Cdc37 expression to promote prostatic hyperplasia. Oncogene
19:2186–2193.
167. Su, G., T. Roberts, and J. K. Cowell. 1999. TTC4, a novel human gene containing the
tetratricopeptide repeat and mapping to the region of chromosome 1p31 that is
frequently deleted in sporadic breast cancer. Genomics 55:157–163.
168. Tai, P. K., M. W. Albers, H. Chang, L. E. Faber, and S. L. Schreiber. 1992.
Association of a 59-kilodalton immunophilin with the glucocorticoid receptor complex.
Science 256:1315–1318.
169. Toda, T., S. Cameron, P. Sass, and M. Wigler. 1988. SCH9, a gene of Saccharomyces
cerevisiae that encodes a protein distinct from, but functionally and structurally related
to, cAMP-dependent protein kinase catalytic subunits. Genes Dev. 2:517–527.
170. Warth, R., P. A. Briand, and D. Picard. 1997. Functional analysis of the yeast 40 kDa
cyclophilin Cyp40 and its role for viability and steroid receptor regulation. Biol. Chem.
378:381–391.
171. Wartmann, M., and R. J. Davis. 1994. The native structure of the activated Raf protein
kinase is a membrane-bound multi-subunit complex. J. Biol. Chem. 269:6695–6701.
172. Weikl, T., P. Muschler, K. Richter, T. Veit, J. Reinstein, and J. Buchner. 2000. C-
terminal regions of Hsp90 are important for trapping the nucleotide during the ATPase
cycle. J. Mol. Biol. 303:583–592.
173. Weisman, R., J. Creanor, and P. Fantes. 1996. A multicopy suppressor of a cell cycle
defect in S. pombe encodes a heat shock-inducible 40 kDa cyclophilin-like protein.
EMBO J. 15:447–456.
174. Whitelaw, M. L., K. Hutchison, and G. H. Perdew. 1991. A 50-kDa cytosolic protein
complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed
with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus. J. Biol. Chem.
266:16436–16440.
175. Whitelaw, M. L., J. McGuire, D. Picard, J. A. Gustafsson, and L. Poellinger. 1995.
Heat shock protein hsp90 regulates dioxin receptor function in vivo. Proc. Natl. Acad.
Sci. USA 92:4437–4441.
176. Whitesell, L., P. D. Sutphin, E. J. Pulcini, J. D. Martinez, and P. H. Cook. 1998. The
physical association of multiple molecular chaperone proteins with mutant p53 is altered
by geldanamycin, an hsp90-binding agent. Mol. Cell. Biol. 18:1517–1524.
177. Xu, Y., and S. Lindquist. 1993. Heat-shock protein hsp90 governs the activity of
pp60v-src kinase. Proc. Natl. Acad. Sci. USA 90:7074–7078.
178. Xu, Y., M. A. Singer, and S. Lindquist. 1999. Maturation of the tyrosine kinase c-src
as a kinase and as a substrate depends on the molecular chaperone Hsp90. PNAS
96:109–114.
179. Yaglom, J. A., A. L. Goldberg, D. Finley, and M. Y. Sherman. 1996. The molecular
chaperone Ydj1 is required for the p34CDC28-dependent phosphorylation of the cyclin
Cln3 that signals its degradation. Mol. Cell. Biol. 16:3679–3684.
180. Young, J. C., and F. U. Hartl. 2000. Polypeptide release by Hsp90 involves ATP
hydrolysis and is enhanced by the co-chaperone p23. EMBO J. 19:5930–5940.
181. Young, J. C., I. Moarefi, and F. U. Hartl. 2001. Hsp90: a specialized but essential
protein-folding tool. J. Cell. Biol. 154:267–273.
182. Young, J. C., C. Schneider, and F. U. Hartl. 1997. In vitro evidence that hsp90
contains two independent chaperone sites. FEBS Lett. 418:139–143.
183. Ziegelhoffer, T., P. Lopez-Buesa, and E. A. Craig. 1995. The dissociation of ATP
from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydj1p and peptide
substrates. J. Biol. Chem. 270:10412–10419.
184. Ziemiecki, A., M. G. Catelli, I. Joab, and B. Moncharmont. 1986. Association of the
heat shock protein hsp90 with steroid hormone receptors and tyrosine kinase oncogene
products. Biochem. Biophys. Res. Commun. 138:1298–1307.
185. Zou, J., Y. Guo, T. Guettouche, D. F. Smith, and R. Voellmy. 1998. Repression of
heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms
a stress-sensitive complex with HSF1. Cell 94:471–480.
186. Zuo, Z., N. M. Dean, and R. E. Honkanen. 1998. Serine/threonine protein phosphatase
type 5 acts upstream of p53 to regulate the induction of p21(WAF1/Cip1) and mediate
growth arrest. J. Biol. Chem. 273:12250–12258.
187. Zuo, Z., G. Urban, J. G. Scammell, N. M. Dean, T. K. McLean, I. Aragon, and
R. E. Honkanen. 1999. Ser/Thr protein phosphatase type 5 (PP5) is a negative regulator
of glucocorticoid receptor-mediated growth arrest. Biochemistry 38:8849–8857.
188. Tesic, M., J. A. March, S. B. Cullinan, and R. F. Gaber. 2003. Functional interactions
between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae.
J. Biol. Chem. 278:32692–32701. (added in proof)
Chapter 7
YEAST AS A MODEL SYSTEM FOR STUDYING
CELL CYCLE CHECKPOINTS
Carmela Palermo and Nancy C. Walworth

Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School and Joint
Graduate Program in Cellular and Molecular Pharmacology, UMDNJ-Graduate School of
Biomedical Sciences and Rutgers, The State University of New Jersey, 675 Hoes Lane,
Piscataway, NJ 08854-5635
The survival of any organism depends on a cell’s ability to accurately

duplicate the genome and equally distribute one copy to each daughter cell.
Successful cell reproduction entails passage through the discrete G1 (pre-
DNA synthesis), S (DNA synthesis), G2 (post-DNA synthesis), and M
(mitotic) phases of the cell cycle. Remarkably, the events of the cell cycle
have been well conserved among eukaryotic organisms ranging from the
unicellular yeasts to humans [23]. As a result, there is a diverse range of
experimental organisms with evolutionary distinctions that contribute to our
understanding of the mechanics that drive the cell cycle, including the yeasts
Saccharomyces cerevisiae and Schizosaccharomyces pombe, the filamentous
fungus Aspergillus nidulans, genetically tractable multicellular eukaryotes
including the fruit fly Drosophila melanogaster and the nematode
Caenorhabditis elegans, systems easily amenable to cell-free analysis
including eggs and embryos of Xenopus laevis and cell culture systems
derived from a variety of mammalian tissues.
The timing and accuracy of cell division are critical for maintaining the
integrity of the genome during normal growth and development. For
example, the commencement of chromosome segregation is dependent on
the completion of sequential events including DNA replication, spindle pole
body or centrosome duplication, chromosome condensation, and spindle
formation. Studies in the fission yeast S. pombe revealed that at the core of
the cell cycle machinery is the cdc2+ gene product, p34cdc2 kinase, which is
179
© 2007 Springer.
180 C. Palermo and N. C. Walworth
required at both the onset of S-phase and M-phase [4, 24]. When activated,
Cdc2 controls mitotic entry via phosphorylation of a number of proteins.
The activation of Cdc2 is itself tightly regulated requiring association with a
cyclin regulatory subunit (cyclin B), appropriate subcellular localization, as
well as critical phosphorylation and dephosphorylation events on an activa-
ting threonine residue and inhibitory tyrosine (Tyr 15) residue [20].
Figure 1. A schematic model of the DNA damage checkpoint pathway. DNA damage
triggers several proteins to localize to presumptive DNA damage sites. Chk1 becomes
phosphorylated by Rad3/Rad26, concomitant with binding to the 14-3-3 protein, Rad24 or
Rad25. Chk1 phosphorylates Cdc25 and Wee1 to control their activity and/or subcellular
localization resulting in a delay to activation of Cdc2 and therefore, a delay to mitotic entry.
To ensure timely entry into mitosis, activation of the cyclin B/cdc2

complex is tightly regulated by cell cycle checkpoints. Checkpoints monitor
the status of a cell’s DNA and prevent inappropriate transitions into
subsequent phases of the cell cycle when DNA damage or other pertur-
bations to the cell cycle are sensed. Specifically, in the event of potentially
catastrophic damage to the genome, checkpoints communicate with the basic
cell cycle machinery to transiently arrest cell cycle progression. Such
delayed mitotic entry prevents cells from initiating M-phase before DNA
damage is repaired or DNA replication is complete. Chemicals, radiation,
and DNA metabolism itself, incessantly threaten genomic stability by
generating DNA damage. Theoretically, the absence of a checkpoint-
induced delay would allow propagation of unrepaired bases, broken
chromosomes, or cells with an abnormal complement of chromosomes [25].
Chapter 7: Yeast as a Model 181
The ramifications of such events could be detrimental to the cell resulting in

cell death or to the organism, if unrepaired damage leads to uncontrolled
proliferation.
Mutations that affect the fidelity of cell cycle progression could
understandably lead to disease states, such as cancer, characterized by
uncontrolled cell divisions. In order to utilize checkpoint pathways as
pharmacological targets it is critical to fully understand how the surveillance
mechanism normally ensures proper cell division. The fission yeast,
S. pombe has been an extraordinarily useful organism for studying the cell
cycle, identifying components of the DNA damage checkpoint pathways for
the development of a checkpoint-signaling model [26]. S. pombe is a rod-
shaped unicellular ascomycete fungus, which grows by apical extension, and
executes many cellular processes similar to multicellular eukaryotes. In fact,
this characteristic apical growth allowed for the initial identification of cell
division cycle (cdc) genes whose function is required for the completion of
cell division. Specifically, cdc mutants become blocked at specific stages
of the cell cycle where they continue to elongate but fail to divide. Thus,
S. pombe cell division cycle mutants could be easily identified
microscopically as elongated cells. The DNA damage checkpoint pathway
has largely been defined through the identification of loss-of-function
mutants that fail to delay mitotic entry following DNA damage in S. pombe.
Remarkably, components of the checkpoint pathway have been well
conserved spanning the evolutionary distance from unicellular yeast to flies,
frogs, mice, and humans. Indeed, many of the human checkpoint proteins
that have been identified to date have been found exclusively based on their
homology to the fission or budding yeast counterparts (Table 1).
A model of checkpoint signaling has emerged from studies in fission and
budding yeast, complemented by studies in Drosophila, mice, Xenopus, and
cultured cells [19]. At its most rudimentary level, the checkpoint pathway
consists of DNA damage sensors, signal transducers to relay the signal, and
effectors that specifically interact with the cell cycle machinery. In yeast,
checkpoints are generally not required for survival, only becoming essential
when DNA damage or perturbations to DNA replication emerge. Thus,
checkpoint components can be identified through genetic screens that select
for mutants that are sensitive to DNA-damaging agents. For example, when
normal cells are damaged by ultraviolet (UV) irradiation, most will
transiently arrest cell cycle progression, repair DNA damage, and upon
completing repair proceed through the cell cycle [35]. Mutations that render
cells sensitive to DNA damage can be classified as either repair defective or
checkpoint defective [13]. Specifically, mutations that render cells repair
defective will result in a permanent cell cycle arrest. On the other hand,
cells harboring mutant checkpoint components bypass the arrest and proceed
through subsequent phases of the cycle with unrepaired DNA damage,
ultimately resulting in cell death.
Table 1. Checkpoint pathway components

Functional class S. pombe H. sapiens S. cerevisiae
ATM family Red3 ATR MEC1
Complex with Rad3 Rad26 ATRIP LCD1/DDC2
PCNA-like Rad1 hRad1 RAD17
Rad9 hRad9 DDC1
Hus1 hHus1 MED3
Protein kinases CHK1 Chk1 CHK1
Cds1 Cds1/Chk2 RAD53
BRCT containing Cut5 53BP1 RAD9
Crb2 BRCA1
RFC Homology Rad17 hRad17 RAD24
Early genetic studies of radiation-sensitive (rad) mutants of fission yeast,

originally isolated due to their sensitivity to UV and ionizing radiation, led to
the identification of some of the first checkpoint pathway components [22].
Further studies revealed that four of the original rad mutants (rad1, rad3,
rad9, rad17) exhibited a complete loss of viability and aberrant attempts at
mitosis following exposure to hydroxyurea (HU), an inhibitor of DNA
synthesis, [2]. This demonstrates that rad1, rad3, rad9, and rad17 are
normally responsible for recognizing incomplete DNA replication and
ensuring that mitosis is dependent on the completion of S-phase. Addi-
tionally, epistatic analysis of the rad mutants with mutant components of the
cell cycle machinery provided evidence that the DNA damage checkpoint
pathway directly impinges on the cell cycle regulatory machinery [11]. The
major cell cycle regulatory kinase controlling mitotic entry is Cdc2, which is
activated by the Cdc25 phosphatase [27] and inhibited by the kinase activity
of Wee1 and Mik1 proteins [17]. A temperature-sensitive loss-of-function
allele of wee1 (wee1-50) renders cells viable at both permissive and
restrictive temperature, though at restrictive temperature causes cells to
divide at a smaller size than wild type. Double mutants of rad1, rad3, rad9,
or rad17 with wee1-50 are inviable at restrictive temperature and display
mitotic abnormalities as a consequence of inappropriately advancing into
mitosis when wee1 activity is decreased [2]. This synthetic lethality between
the checkpoint genes and cell cycle regulatory genes was the first evidence
in S. pombe demonstrating that the checkpoint pathway that responds to
DNA damage must also communicate with the cell cycle machinery.
While the first checkpoint components emerged by testing the ability of
radiation-sensitive mutants to initiate mitosis amidst a block to DNA
replication, additional genetic screens followed to expand the search for

specific components of the pathway. A genetic screen in which nitroso-
guanidine-mutagenized cells were analyzed for sensitivity to HU led to the
identification of the hus mutants (hydroxyurea sensitive) [10]. Interestingly,
in addition to the “new” hus genes, alleles of the aforementioned rad1, rad3,
and rad17 genes emerged in this genetic screen as well. The hus1 mutant,
like the rad mutants, can be characterized as checkpoint deficient, evidenced
by its increased radiation sensitivity and synthetic lethality with a wee
mutant [10]. However, viability assays of synchronous cultures of the hus1,
rad1, rad3, and rad17 mutants revealed that the cells became irreversibly
arrested in S-phase after HU treatment, whereas a dominant mutant allele of
cdc2, cdc-3w which is independent of cdc25 activation, loses viability as
they undergo abortive mitosis [10]. This finding suggests that while the hus
and rad genes play a role in the checkpoint pathway that couples mitosis to
completion of DNA replication, additionally they assume a secondary
function that is required for recovery from DNA damage.
An alternative method of searching for new checkpoint mutants
employed ethyl methanesulfonate (EMS) mutagenesis of cells harboring a
temperature-sensitive allele of DNA ligase (cdc17-K42) and subsequent
screening for UV- or HU-sensitive mutants. At restrictive temperature DNA
ligase activity is decreased in these cells, leading to unligated DNA
fragments that may generate a DNA damage response. The rationale for
employing the DNA ligase mutant background was to distinguish
components of the checkpoint that respond to DNA damage rather than to a
block in S phase, such as that imposed by the HU treatment [3]. Two
additional checkpoint components, rad26 and rad27, were isolated through
this unique genetic screen. Functional characterization of the rad26 gene by
deletion of an internal region of the ORF and subsequent replacement with a
functional gene for the ura4 auxotrophic marker, revealed that it was
inessential for viability. Consistent with the phenotype of a checkpoint
defect, deletion of rad26 conferred increased sensitivity to HU treatment and
ionizing radiation. Furthermore, similar to the hus1 mutant, the rad26-deleted
strain was unable to reverse the S-phase arrest imposed by HU treatment.
Strikingly, a point mutant of this gene, rad26-T12, was found to be radiation
sensitive, yet displayed a normal mitotic arrest in response to ionizing
radiation [3]. The phenotypic discrepancy between the rad26 point mutant
and null allele can be resolved by proposing that the rad/hus gene products
form a complex [3]. Mutations in any particular component of the complex
could lead to loss of the specific function of that gene product, for example
ability to repair DNA damage, but would not affect the integrity of the
complex as a whole. On the other hand, complete loss of the gene product
might structurally disrupt the complex and hence lead to complete failure of
the signal transduction system. This finding began to illustrate the complex
interactions and multiple feedback controls that exist, yet converge on a
single signal transduction pathway, whose ultimate task is to delay mitotic
progression. Interestingly, the predictions made based on genetic obser-
vations are now supported by biochemical evidence that the Rad1, Rad9, and
Hus1 gene products indeed form a heterotrimeric complex that is related in
structure to the PCNA processivity factor for DNA polymerase δ [7].
As sophisticated genetic screens began to establish the checkpoint
pathway in S. pombe, serendipitous findings also contributed candidates for
the central cast of players that couple the checkpoint pathway to the mitotic
apparatus. As one example, a genetic screen aimed to identify elements that
directly interact with Cdc2 utilized a cold-sensitive cdc2-r4 mutant that
grows as wild type at 36°C, yet undergoes cell cycle arrest at the restrictive
temperature of 25°C [17]. The cdc2-r4 mutation itself suppressed the
lethality resulting from simultaneous loss-of-function of the inhibitory
kinases wee1 and mik1, allowing cells to survive in the absence of tyrosine
phosphorylation of Cdc2 and indicating that other mechanisms of Cdc2
regulation are in operation [17]. Introduction of a multicopy gene library into
the cdc2-r4 strain led to isolation of plasmids that suppressed the effects of
the cold-sensitive mutation at 18°C [33]. Consequently, this screen revealed
one of the core components of the checkpoint pathway known as chk1 [33].
Deletion of the chk1 gene does not affect cell viability; however, a
characteristic checkpoint defect is evident when cells are exposed to UV
irradiation. Notably the UV sensitivity of a chk1 disrupted strain can be
largely rescued by imposing an “artificial” G2 delay, discriminating this
checkpoint component from gene products that are involved in DNA repair
processes [33].
Chk1 is a member of the serine/threonine family of protein kinases and
itself was found to undergo phosphorylation in response to DNA damage
[34]. The phosphorylated form, observable by SDS-PAGE as a decreased
mobility form of the protein, has proven to be a useful tool to dissect how
previously isolated and novel checkpoint components interact with Chk1 to
arrest cell cycle progression in response to DNA damage. In fact, epistatic
analysis with the rad checkpoint genes indicated that damage induced
phosphorylation of Chk1 depends on the function of rad1, rad3, rad9,
rad17, and rad26, [34], rad24 [8], and crb2 [28].
To further define the role of Chk1 in the checkpoint pathway, the full-length
gene was utilized as bait in a yeast two-hybrid screen of an S. pombe cDNA
library for proteins that interact with Chk1. Two fission yeast 14-3-3
proteins, Rad 24 and Rad25, emerged from this screen [8]. Structural data
supports a role for the 14-3-3 family of proteins in signal-transduction

pathways, perhaps as scaffolding or adaptor proteins that bring together
signaling molecules [1]. Intriguingly, rad24 and rad 25 also emerged in the
genetic screen that identified rad26 as a pathway element [3, 12].
Accordingly, the Chk1-Rad24/Rad25 interaction was further pursued and
molecular analysis demonstrated that the interaction indeed occurs in vivo.
Furthermore, the association is stimulated by DNA damage, with the
phosphorylated form of Chk1 preferentially interacting with the 14-3-3
proteins [8]. This data suggests that association of 14-3-3 proteins with Chk1
following DNA damage plays a significant, yet to date undetermined, role in
the checkpoint pathway.
In an independent genetic screen, the cds1 gene (Checking DNA
Synthesis) was isolated as a multicopy suppressor of a temperature-sensitive
DNA polymerase-α mutant [21]. Phenotypic characterization of cds1
revealed that the gene was dispensable for normal growth or survival in
response to UV light, yet essential for survival when DNA replication was
inhibited by treatment with HU. Further assessment of genetic interactions
demonstrated that the wild-type gene almost completely rescued the HU
sensitivity, but not the UV sensitivity, of the rad1, rad3, and rad9 mutants
[21]. The ability of Cds1 to suppress HU sensitivity of the checkpoint
mutants implied that its primary role was to instigate an S-phase specific
checkpoint arrest. However, cells in which the cds1 gene has been
eliminated by a knockout mutation, do arrest in the presence of HU, but fail
to recover from that arrest. Thus, as cells resume cell cycle progression
upon resuming DNA synthesis, they enter a catastrophic mitosis [21]. It has
been proposed, therefore, that Cds1 plays an important role in stabilizing
replication forks that have stalled as a result of depleted pools of
ribonucleotides [15]. Consistent with this suggestion, cells lacking Cds1 that
have been exposed to HU exhibit activation of the DNA damage checkpoint
as manifested by phosphorylation of Chk1 [15]. Thus, while Cds1 and Chk1
are both critical effectors of the checkpoint response to inhibition of DNA
replication, they seem to perform distinct roles in the process. In addition,
the results of assays that measure the response of Cds1 and Chk1 to DNA
damage suggest that while both proteins require the function of Rad3 to be
activated, there is a temporal distinction as to when one or the other kinase
can respond to DNA damage [18].
Loss of mitotic checkpoint control can be plausibly associated with the
development and progression of cancer. Interestingly two additional fission
yeast checkpoint proteins, Cut 5 and Crb2, contain BRCT domains (BRCA1
C Terminus) that are also found in the human breast and ovarian cancer
susceptibility gene, BRCA1 [28, 32]. BRCT domains are found in a
growing number of proteins involved in DNA metabolism and can promote
protein–protein interactions [5]. The cut5 gene was initially identified as a

temperature-sensitive mutation that disrupts the normal coordination
between nuclear division and cytokinesis leading to what is known as the
“cut” phenotype (cell untimely torn) [29, 30]. Subsequent genetic analysis of
this gene revealed that cut5 is required for both the onset of DNA replication
and to establish a DNA damage-dependent mitotic arrest [29]. Interestingly,
cut5 is the only gene that has been assigned two mutually exclusive roles in
DNA replication and checkpoint control.
The second BRCT-containing protein was identified in a yeast two-hybrid
screen for Cut5 interacting proteins [28]. Deletion of the crb2 gene
compromised the DNA damage checkpoint, as in the rad mutants, and
therefore warranted investigation into Crb2’s interaction with the other
checkpoint proteins. Consequently it was found that wild-type copies of the
rad1, rad3, rad9, rad17, or rad26 genes could not suppress the UV
sensitivity of a crb2 null strain, while overexpression of chk1 did improve
the survival of the UV-damaged strain [28]. Thus, the interactions hint that
Crb2 may act downstream or in parallel to the Rad checkpoint proteins,
while functioning upstream of Chk1 in the signaling pathway. Furthermore,
molecular characterization of Crb2 indicated that a Rad-dependent modified
form of the protein appears in response to UV irradiation at the same time
that phosphorylated Chk1 appears. One interpretation of these results assigns
Crb2 a role in the checkpoint pathway in which it is modified in response to
DNA damage and subsequently transmits a signal through Chk1 to mediate
cell cycle arrest.
It seems that from the moment that the DNA damage checkpoint was
discovered in yeast it was anticipated that a correlation would be made
between loss of checkpoint control and onset of cancer. Indeed, one might
assume that loss of checkpoint control is a favorable condition for promoting
unregulated cell division and, therefore, that oncogenic processes might
target key checkpoint regulators to achieve this hallmark trait. Hence the
discovery of checkpoint components, cut5 and crb2, that contain structural
features in common with domains characterized in two human cancer
susceptibility genes increase suspicion that a cancer connection is likely.
Evidence is mounting that other checkpoint genes may play critical roles
in the development of cancer as well. For example, the human ATM protein,
which is a member of a family of proteins homologous to the S. pombe Rad3
protein, also plays a role in signaling the presence of DNA double-strand
breaks to delay cell cycle progression [14]. Mutations in ATM lead to the
recessive human disorder ataxia telangiectasia (AT), in which patients suffer
from progressive neurodegeneration and the tendency to develop cancers,
particularly lymphomas [14]. Experimental analysis of cells isolated from
AT patients demonstrated that they were extremely sensitive to ionizing
radiation as a result of failure to arrest the cell cycle in response to DNA-

double-strand breaks. On the other hand, analysis of Chk1 null mice
embryos revealed that some checkpoint proteins are essential during
embryogenesis, as cells succumb early on in development and exhibit
mitotic abnormalities [16, 31]. Similar phenotypes are observed with mice
null for the Rad3 family member ATR [6, 9]. Undoubtedly in the near
future, further genetic analysis will help to demonstrate how disruption of
checkpoint surveillance may lead to changes in genomic stability that are
key to tumor evolution.
ACKNOWLEDGMENT
Work in the Walworth laboratory is supported by the NIH (GM53194).
REFERENCES
1. Aitken, A. 1996. 14-3-3 and its possible role in co-ordinating multiple signalling
pathways. Trends Cell Biol. 6:341–347.
2. al-Khodairy, F., and A. M. Carr. 1992. DNA repair mutants defining G2 checkpoint
pathways in Schizosaccharomyces pombe. EMBO J. 11:1343–1350.
3. al-Khodairy, F., E. Fotou, K. S. Sheldrick, D. J. F. Griffiths, A. R. Lehmann, and
A. M. Carr. 1994. Identification and characterization of new elements involved in
checkpoints and feedback controls in fission yeast. Mol. Biol. Cell 5:147–160.
5. Bork, P., K. Hofmann, P. Bucher, A. F. Neuwald, S. F. Altschul, and E. V. Koonin.
1997. A superfamily of conserved domains in DNA damage-responsive cell cycle
checkpoint proteins. FASEB J. 11:68–76.
6. Brown, E. J., and D. Baltimore. 2000. ATR disruption leads to chromosomal
fragmentation and early embryonic lethality. Genes Dev. 14:397–402.
7. Caspari, T., M. Dahlen, G. Kanter-Smoler, H. D. Lindsay, K. Hofmann,
K. Papadimitriou, P. Sunnerhagen, and A. M. Carr. 2000. Characterization of
Schizosaccharomyces pombe Hus1: a PCNA-related protein that associates with Rad1
and Rad9. Mol. Cell. Biol. 20:1254–1262.
8. Chen, L., T.-H. Liu, and N. C. Walworth. 1999. Association of Chk1 with 14-3-3
proteins is stimulated by DNA damage. Genes Dev. 13:675–685.
9. de Klein, A., M. Muijtjens, R. van Os, Y. Verhoeven, B. Smit, A. M. Carr, A. R.
Lehmann, and J. H. J. Hoeijmakers. 2000. Targeted disruption of the cell-cycle
checkpoint gene ATR leads to early embryonic lethality in mice. Curr. Biol.
10:479–482.
10. Enoch, T., A. M. Carr, and P. Nurse. 1992. Fission yeast genes involved in coupling
mitosis to the completion of DNA replication. Genes Dev. 6:2035–2046.
11. Enoch, T., and P. Nurse. 1990. Mutation of fission yeast cell cycle control genes
abolishes dependence of mitosis on DNA replication. Cell 60:665–673.
12. Ford, J. C., F. al-Khodairy, E. Fotou, K. S. Sheldrick, D. J. Griffiths, and A. M.

Carr. 1994. 14-3-3 protein homologs required for the DNA damage checkpoint in
fission yeast. Science 265:533–535.
13. Hartwell, L. H., and T. A. Weinert. 1989. Checkpoints: controls that ensure the order
of cell cycle events. Science 246:629–634.
14. Lavin, M. F., and Y. Shiloh. 1997. The genetic defect in Ataxia-Telangiectasia. Annu.
Rev. Immunol. 15:177–202.
15. Lindsay, H. D., D. J. F. Griiffithes, R. J. Edwards, P. U. Christensen, J. M. Murray,
F. Osman, N. Walworth, and A. M. Carr. 1998. S-phase-specific activation of Cds1
kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe.
Genes Dev. 12:382–395.
16. Liu, Q., S. Guntuku, X.-S. Cui, S. Matsuoka, D. Cortez, K. Tamai, G. Luo,
S. Carattini-Rivera, F. DeMayo, A. Bradley, L. A. Donehower, and S. J. Elledge.
2000. Chk1 is an essential kinase that is regulated by Atr and required for the G2/M
DNA damage checkpoint. Genes Dev. 14:1448–1459.
17. Lundgren, K., N. Walworth, R. Booher, M. Dembski, M. Kirschner, and D. Beach.
1991. mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell
64:1111–1122.
18. Martinho, R. G., H. D. Lindsay, G. Flaggs, A. J. DeMaggio, M. F. Hoekstra, A. M.
Carr, and N. J. Bentley. 1998. Analysis of Rad3 and Chk1 protein kinases defines
different checkpoint responses. EMBO J. 17:7239–7249.
19. Melo, J., and D. Toczyski. 2002. A unified view of the DNA-damage checkpoint. Curr.
Opin. Cell Biol. 14:237–245.
20. Morgan, D. O. 1997. Cyclin-dependent kinases: engines, clocks, and microprocessors.
Annu. Rev. Cell Dev. Biol. 13:261–291.
21. Murakami, H., and H. Okayama. 1995. A kinase from fission yeast responsible for
blocking mitosis in S phase. Nature 374:817–819.
22. Nasim, A., and B. P. Smith. 1975. Genetic control of radiation sensitivity in
Schizosaccharomyces pombe. Genetics 79:573–582.
23. Nurse, P. 1990. Universal control mechanism regulating the onset of M-phase. Nature
344:503–508.
24. Nurse, P., and Y. Bissett. 1981. Gene required in G1 for commitment to cell cycle and
in G2 for control of mitosis in fission yeast. Nature 292:558–560.
25. Nyberg, K. A., R. J. Michelson, C. W. Putnam, and T. A. Weinert. 2002. Toward
maintaining the genome: DNA damage and replication checkpoints. Annu. Rev. Genet.
36:617–656.
26. O’Connell, M. J., N. C. Walworth, and A. M. Carr. 2000. The G2-phase DNA-
damage checkpoint. Trends Cell Biol. 10:296–303.
27. Russell, P., and P. Nurse. 1986. cdc25+ functions as an inducer in the mitotic control
of fission yeast. Cell 45:145.
28. Saka, Y., F. Esashi, T. Matsusaka, S. Mochida, and M. Yanagida. 1997. Damage
and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a
protein with BRCT motif, with Cut5 and Chk1. Genes Dev. 11:3387–3400.
29. Saka, Y., and M. Yanagida. 1993. Fission yeast cut5+, required for S phase onset
and M phase restraint, is identical to the radiation-damage repair gene rad4+. Cell
74:383–393.
30. Samejima, I., T. Matsumoto, Y. Nakaseko, D. Beach, and M. Yanagida. 1993.

Identification of seven new cut genes involved in Schizosaccharomyces pombe mitosis.
J. Cell Sci. 105 ( Pt 1):135–143.
31. Takai, H., K. Tominaga, N. Motoyama, Y. A. Minamishima, H. Nagahama,
T. Tsukiyama, K. Ikeda, K. Nakayama, and M. Nakanishi. 2000. Aberrant cell
cycle checkpoint function and early embryonic death in Chk1-/- mice. Genes Dev.
14:1439–1447.
32. Verkade, H. M., and M. J. O’Connell. 1998. Cut5 is a component of the UV-
responsive DNA damage checkpoint in fission yeast. Mol. Gen. Genet. 260:426–433.
33. Walworth, N., S. Davey, and D. Beach. 1993. Fission yeast chk1 protein kinase links
the rad checkpoint pathway to cdc2. Nature 363:368–371.
34. Walworth, N. C., and R. Bernards. 1996. rad-dependent response of the chk1-encoded
protein kinase at the DNA damage checkpoint. Science 271:353–356.
35. Weinert, T. A., and L. H. Hartwell. 1988. The RAD9 gene controls the cell cycle
response to DNA damage in Saccharomyces cerevisiae. Science 241:317–322.
Chapter 8
METABOLISM AND FUNCTION OF
SPHINGOLIPIDS IN SACCHAROMYCES
CEREVISIAE: RELEVANCE TO CANCER
RESEARCH
L. Ashely Cowart*, Yusuf A. Hannun* and Lina M. Obeid#

#Ralph H. Johnson Veterans Administration and the Departments of Medicine and
Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston,
South Carolina
Over the last two decades, sphingolipids have emerged as important cell
regulators. Included in this lipid class are the lipid mediator sphingosine, its
phosphorylated derivative sphingosine-1-phosphate, and ceramide. In
humans, the major known functions of sphingolipid-mediated cell regulation
is the modulation of signaling pathways which control fundamental cellular
processes including cell division, senescence, apoptosis, angiogenesis, and
differentiation [40, 41, 74], all of direct relevance to cancer pathogenesis and
progression.
Because of these myriad activities, the enzymes that generate sphingo-
lipid mediators are potential targets for cancer treatment. Thus, characteri-
zation of these enzymes with respect to activity, regulation, localization, and
cellular function is fundamental to developing strategies for modulating
sphingolipid levels in vivo. The yeast system has emerged as an invaluable
tool for the identification, cloning, and characterization of enzymes of
sphingolipid metabolism. Furthermore, insights gained from yeast studies
of sphingolipid metabolism and function continue to push the forefront of
sphingolipid research, especially since the roles of sphingolipids in stress
and other cell responses appear to be conserved from yeast to human.
191
© 2007 Springer.
192 L. A. Cowart, Y. A. Hannun and L. M. Obeid
1 OVERVIEW OF SPHINGOLIPID METABOLISM
As with many fundamental cellular pathways, sphingolipid metabolic

pathways are highly conserved between yeast and mammalian cells. This
high degree of evolutionary conservation underscores the fundamental
importance of this lipid class and, importantly, allows the use of yeast as a
model system for sphingolipid metabolism.
1.1 Long chain base synthesis
In both yeast and mammalian cells, sphingolipid biosynthesis begins with

the production of 3-keto-dihydrosphingosine by condensation of palmitoyl-
CoA with serine (Figure 1). This enzymatic activity is referred to as serine
palmitoyltranferase (SPT), and the reaction is thought to be the rate-limiting
step in sphingolipid biosynthesis and in yeast is catalyzed by the proteins
lcb1p and lcb2p [86], which form a heterodimer [32]. Molecular modeling
has identified a putative catalytic site in the cleft of the interface between the
two peptides [32]. This structural model is supported by experiments
demonstrating that both components are required for activity [75], and, in
yeast, deletion of either of these subunits is lethal, rendering the cells
auxotrophic for sphingoid bases (the products of the reaction) [85]. Thus,
this pathway is vital to Saccharomyces cerevisiae. SPT has been identified
as the target of the immunosupressant myriocin, which has structural
similarity to sphingosine [14]. Inhibition of SPT by myriocin is conserved
from yeast to mammalian SPT isoforms. Complementation screens in
S. cerevisiae were used to clone both subunits in this organism [11, 75], and
the subsequent characterization and cloning of mammalian isoforms was
aided by information gained from this initial work in yeast [37, 76].
Interestingly, a novel subunit of the lcb1p/lcb2p complex was recently
identified in yeast, tsc3p [33]. This gene is required for maximal SPT
activity in yeast; however, deletion of this gene is not lethal under normal
laboratory conditions as it is not required for basal SPT activity. TSC3
belongs to the group of genes identified as temperature suppressors of
calcium sensitivity, many of which are involved in sphingolipid bio-
synthesis. Experimental data demonstrate that accumulation of the
downstream sphingolipid product inositolphosphoceramide (IPC) leads to
increased cell sensitivity to extracellular calcium; thus, deletion of any gene
upstream which would decrease production of this metabolite, suppresses the
calcium-sensitive phenotype. Importantly, the calcium-sensitive phenotype
in yeast has led to the cloning of several enzymes in sphingolipid
biosynthetic pathways (see Figure 1). Though a mammalian homologue of
TSC3 remains to be identified, yeast model systems that enabled isolation of
Chapter 8: Metabolism and Function of Sphingolipids 193
the TSC genes were and continue to be vital to cloning mammalian

homologues of other enzymes of sphingolipid biosynthesis.
Figure 1
Figure 1. Pathways of sphingolipid metabolism. Steps represented in bold text are shared by
yeast and mammalian cells. Yeast-specific enzymes and compounds in italics and
mammalian-specific enzymes and compounds in plain font.
The product of SPT is reduced to dihydrosphingosine by another gene in

the TSC group, 3-keto-dihydrosphingosine reductase (TSC10). Long-chain
bases have cell regulatory activities of their own; however, a variety of other
lipid mediators are produced by their further metabolism. At this point in
sphingolipid biosynthesis, yeast and mammalian pathways slightly diverge.
In yeast, dihydrosphingosine is hydroxylated to form phytosphingosine by
the SUR2/SYR2 gene product. This gene was identified originally as
suppressing a defect in starvation survival [20] and by conferring sensitivity
to the toxin syringomycin E [17]. Its biochemical activity was identified
based on homology to another yeast sphingolipid hydroxylase [35]. Thus, in
yeast, there are two major sphingoid base species formed by de novo
pathways. In mammalian systems, however, dihydrosphingosine is the only

major sphingoid base formed from de novo biosynthesis.
1.2 Phosphorylation and dephosphorylation

of sphingoid bases
Sphingoid bases are subject to two known downstream metabolic

pathways, one of which is their phosphorylation. Dihydrosphingosine and,
in yeast, phytosphingosine can each be phosphorylated at C1 yielding
dihydrosphingosine-1-phosphate (DHS-1-P) and phytosphingosine-1-phos-
phate, respectively. Additionally, sphingosine, which results from ceramide
catabolism in mammalian cells (see below), can also be phosphorylated at
C1 yielding sphingosine-1-phosphate (S-1-P). Cloning of genes responsible
for sphingoid base kinase activity in yeast, LCB4 and LCB5 [78], laid
important groundwork for the later cloning of mammalian isoforms [54, 62].
DHS-1-P can be cleaved by the DHS-1-P lyase into hexadecanal
(palmitaldehyde) and ethanolamine phosphate in yeast, and in mammals,
S-1-P is cleaved into hexadecenal and ethanolamine phosphate. The yeast
gene responsible for this activity, DPL1, was cloned, and its deletion confers
an increased sensitivity to growth inhibition by sphingoid bases [88]. This
phenotype was integral to the cloning of the sphingoid base kinases LCB4
and LCB5 (above), as it was speculated that the reason for DPL1-dependent
sphingoid base sensitivity was actually due to accumulation of sphingoid
base phosphates [92]. Thus, deletion of the sphingoid base kinases reversed
the phenotype of the DPL1 deletion [52]. This is but one example of the
simple and elegant experimental strategies possible in yeast which have
allowed the cloning of a multitude of genes.
In addition to DPL1-mediated cleavage, sphingoid base phosphates can be
dephosphorylated by sphingoid base phosphate phosphatases. The yeast
enzymes YSR2/LCB3/LBP1 and YSR3/LBP2 were cloned and characterized
in yeast [68, 70], and this facilitated the subsequent cloning of a murine
sphingoid base phosphate phosphatase by sequence homology, especially in
the highly conserved active site regions [67]. Both yeast sequences and the
murine sequence facilitated the cloning of two human sphingosine phosphate
phosphatase isoforms which may eventually become targets for cancer
therapeutics [55, 80].
1.3 Ceramide biosynthesis
In addition to their metabolism by phosphorylation, both dihydro-

sphingosine and phytosphingosine can be N-acylated to form dihydro- or
phytoceramide, respectively, by yeast ceramide synthases. There is little

production of phytoceramide in mammalian cells, rather, dihydrosphingosine
is converted to dihydroceramide by (dihydro)ceramide synthase before
reduction to ceramide by dihydroceramide desaturase. This divergence is
potentially very important, as dihydroceramide has few, if any, known cellular
effects [4]. Thus, dihydroceramide desaturase may be a key regulatory point in
mammalian sphingolipid metabolism, regulating the relative levels of the
active lipid ceramide and the inactive lipid dihydroceramide.
Sphingolipid biosynthesis in yeast continues to diverge slightly from
mammalian pathways at the point of ceramide biosynthesis. Acyl groups
detected in yeast ceramides typically have very long carbon chains (C26);
however, in mammalian ceramides, N-acyl groups range in length from
16 to 24 carbons with varied degrees of saturation [21].
Despite these differences, overall the biosynthesis is highly conserved at a
biochemical as well as a functional level (see below, Figure 1, Table 1).
Table 1. Comparison of sphingolipid metabolism and function in Saccharomyces cerevisiae and

mammals. (Fungal toxin inhibitors are listed in parentheses.)
Activity Yeast Mammalian
Enzyme Function Enzyme Function
Production LCB1 LCB2 Heat stress [49], LBC1 LBC2 Heat stress [48],
of 3-keto- heterodimer cell cycle arrest [49], heterodimer hereditary sensory
dihydro- (myriocin, endocytosis [104], neuropathy type I
sphingosine syringomycin) intracellular protein [32]
transport [101]
N-acylation LAG1, LAC1 Influence life span [18] UOG1 Regulator of growth
of sphingoid (fumonisin) and differentiation
bases [98]
Sphingoid LCB4, LCB5 Clear sphingoid base hSPP1, hSPP2, mSPP1 Clear sphingoid
base kinase uptake [70], clear base phosphates;
sphingoid base change balance
phosphates, cell between ceramide
proliferation [69], and S-1-P
heat stress [69]
Ceramidase YPC1, YDC1 Clear ceramide/ Neutral ceramidase, Clear ceramide/
produce sphingoid Alkaline ceramidase produce sphingoid
bases bases
Addition of AUR1 Calcium sensitivity Sphingomyelin Correlate with
headgroups (aureobasidin) [23] synthase, Glucosyl- transformed pheno-
to ceramide ceramide synthase type [65]; drug
resistance [63, 64]
Breakdown ISC1 Cell wall in Neutral sphingomye- Central link between
of complex Schizosaccharomyes linase 1, neutral apoptosis-inducing
sphingolipids pombe [27] sphingomyelinase 2, factors and ceramide
acid sphingomyelinase formation [38]; lipid-
storage disease [29]
Yeast enzymes required for sphingoid base N-acylation are LAG1 and LAC1
[91]. It is not yet established whether the enzymes encoded by these genes
produce ceramide directly or act as necessary activators of an as yet
unidentified ceramide synthases; however, the dependence of ceramide
formation on these proteins as well as their ability to control intracellular
ceramide levels have been well established biochemically [91]. (Longevity
assurance gene LAG1) was initially identified as a gene whose deletion
conferred increased lifespan in yeast [91]. This is particularly interesting as
studies in mammalian cells indicate that increased ceramide is correlated
with cell senescence (see below). The functions of lag1p and lac1p are
partially redundant, thus, deletion strains of LAG1 or LAC1 are viable;
however, deletion of both genes causes a severe slow growth phenotype,
indicating the importance of ceramide biosynthetic pathways in yeast [91].
Very interestingly, LAG1 homologues have been identified throughout
eukaryotic organisms, including tomato [9], Caenorhabditis elegans [51],
Drosophila [97], and human [51] based on sequence similarity to the yeast
gene. In particular, a mouse homologue (UOG) was recently identified as
necessary for N-stearoyl-sphinganine biosynthesis, and of particular interest
is that this gene had a suspected role in the regulation of cell differentiation
prior to discovery of its role in ceramide synthesis, suggesting not only
biochemical conservation but possibly some degree of functional conser-
vation as well [98].
Ceramide synthase was found to be a target of the group of fungal toxins
known as fumonisins. Fumonisin inhibition of ceramide synthase was first
reported in rat liver microsomes, but provided insight into mechanisms of
the observed correlation between fumonisin consumption and development
of cancer [99]. It appears that not all LAG1 homologues are fumonisin
targets; however, the bulk of ceramide synthase activity in mammalian cells
is inhibited by fumonisins.
Similarly to sphingoid bases, ceramide can be phosphorylated at C1. A
mammalian ceramide kinase was cloned based on sequence similarity to
mouse and human isoforms of sphingosine kinase [93]. Interestingly, no
yeast homologue for ceramide kinase has been identified. Presently,
however, at least 50% of the yeast genome (i.e., protein-encoding open
reading frames) are of unknown function. Thus, further characterization of
ceramide kinase may well lead to identification of a yeast isoform, and, if so,
yeast systems may be useful in functional characterization of ceramide-
1-phosphate.
1.4 Formation of complex sphingolipids
A variety of complex sphingolipids are formed in both yeast and

mammalian cells by the addition of various headgroups to the ceramide
molecule. Again, though there is significant divergence between yeast and
mammalian pathways, several aspects of these pathways are conserved
between the two. In yeast, a glycerophospholipid-derived phosphoinositol
headgroup is added to ceramide forming IPC. IPC can be mannosylated to
form mannosyl-IPC (MIPC), and yet another phosphoinositol headgroup is
added to MIPC, forming M(IP)2C. Together, these comprise the set of
complex sphingolipids in yeast. Biosynthesis of these lipids is carried out by
the IPC synthase AUR1, the MIPC synthases CSG1 and CSG2, and the
M(IP)2C synthase IPT1 [23]. AUR1 was originally identified as the target of
the antifungal agent aureobasidin A, as mutations in this gene conferred
resistance to aureobasidin A [43]. Deletion of this gene is lethal, indicating a
strict requirement for complex sphingolipids in yeast (and/or toxicity of the
accumulated ceramide). Later biochemical studies identified the function of
AUR1 as the IPC synthase and confirmed the inhibition of this enzyme by
aureobasidin A [77]. Of particular interest is that the homologue of this gene
in Cryptococcus neoformans, IPC1, has been shown to be involved in the
pathogenesis of this virulent fungus [66].
As with the TSC group of genes, CSG1 and CSG2 also play roles in cell
sensitivity to extracellular calcium, as a previous study demonstrated that
mutants in either gene were hypersensitive to extracellular calcium [2, 3].
Thus, it appears that mannosylated IPC derivatives are important for
resistance to high extracellular calcium concentrations.
The mammalian set of complex sphingolipids is much larger and more
diverse. Whereas in yeast the inositol headgroup derives from phosphati-
dylinositol, the analogous mammalian headgroup, phosphocholine, derives
from phosphatidylcholine through an analogous transferase reaction which
also generates diacylglycerol from the glycerophospholipid (reviewed in
[39]). Unlike ceramide, which is associated with attenuation of cell growth
through cell cycle arrest, apoptosis, or senescence (reviewed in [38, 42]),
diacylglycerol is recognized as the physiological agonist of protein kinase C
and is often associated with mitogenesis [7]. Importantly, in producing
sphingomyelin, sphingomyelin synthase catalyzes both the consumption of
ceramide and the formation of diacylglycerol, perhaps shifting the cellular
balance of growth-attenuating mediators to growth-promoting mediators
[39]. Therefore, this activity may be a key point of cell growth regulation,
making it an excellent target for chemotherapeutic agents. Unfortunately,
mammalian sphingomyelin synthase has evaded purificationand cloning.
Hopefully further knowledge of the yeast orthologue will assist in this goal
as well.
Though mannose is the only sugar in S. cerevisiae sphingolipids,
mammalian complex sphingolipids incorporate a variety of carbohydrate
groups including lactose, glucose, galactose, and sialic acid. These activities
in mammalian cells give rise to glycosphingolipids, gangliosides, and
cerebrosides. It should be noted that in some other fungal species including
Candida albicans, glucosylceramide synthase has been detected [60],
though the significance of this activity is not known [59].
1.5 Sphingolipid catabolism
Though complex sphingolipids in yeast are structurally different from

complex sphingolipids in mammalian cells, there is surprising functional
conservation of sphingolipid catabolic pathways. These pathways are
important not only because of their ability to regulate sphingolipid levels,
but also with respect to their participation in formation of ceramide and
sphingoid bases as they break down more complex sphingolipids. Thus, two
major routes contribute to formation of bioactive sphingolipids: de novo
biosynthesis and catabolic pathways.
In yeast, IPCs and their mannosylated derivatives are hydrolyzed to
produce phytoceramide by the enzyme isc1p. The analogous enzyme in
mammalian cells, neutral sphingomyelinase, hydrolyzes sphingomyelin to
produce ceramide. The extended family of sphingomyelinases consists of
several isoforms based on expression, intracellular localization, and pH
optima [61].
Just as hydrolysis of inositolphosphoceramide and sphingomyelin
produces phytoceramide and ceramide, respectively, hydrolysis of ceramide
by ceramidases produces sphingoid bases. In yeast, the phytoceramidase
YPC1 was first identified by yeast genetic screens, and the dihydro-
ceramidase YDC1 was cloned based on sequence homology [71, 72]. The
sequences of these two genes greatly assisted the cloning of several
mammalian ceramidase isoforms [73].
Importantly, the same pathways that metabolize de novo sphingolipids
also may metabolize ceramide and sphingoid bases derived from catabolic
reactions.
2 SPHINGOLIPID FUNCTIONS
2.1 Modulation of signaling pathways
Studies in yeast have demonstrated the modulation of signaling pathways

by sphingolipids, including the modulation of the protein kinases pkh1p and
pkh2p by sphingoid bases [31]. These kinases act upstream of pkc1p and the
YPK kinases 1 and 2, which control endocytosis and other cell processes
[90]. Ongoing research is focused on discovering new pathways of
sphingolipid action in yeast since these pathways may be conserved between
yeast and mammalian systems.
One of the original signaling activities attributed to sphingolipids was the
modulation of protein kinases [5]. Indeed, modulation of protein kinase C
activity by sphingosine has been implicated in a variety of downstream
effects of sphingolipid production. Other studies led to the identification of
the ceramide-activated protein phosphatase (CAPP) [24]. This phosphatase
activity was demonstrated to be directly activated by exogenous ceramide,
and to have characteristics of protein phosphatase 2A (PP2A) including
cation independence and inhibition by okadaic acid [24]. Other studies in
mammalian systems also identified protein phosphatase 1 (PP1)-dependent
CAPP activity, suggesting that other phosphatases may also act as CAPPs
[12]. Work in yeast demonstrated the conservation of this activity in
S. cerevisiae, where phosphatase activity was shown to mediate ceramide
induced growth inhibition [30]. Importantly, genetic studies in cerevisiae
demonstrated that the activity is comprised of the regulatory subunits TPD3
and CDC55 and the catalytic subunit SIT4 [79]. Ceramide interactions with
the mammalian CAPP have been linked to numerous cellular activities
including downregulation of c-myc in response to TNF [103], dephospho-
rylation of the oncogene c-jun [87], induction of cell cycle arrest through
dephosphorylation of the retinoblastoma gene product Rb [56], regulation of
alternative mRNA splicing via dephosphorylation of SR proteins [13], and
the inactivation of PKCα and Bcl2 by dephosphorylation [57]. Other studies
have demonstrated direct effects of ceramide on the kinase KSR (kinase
suppressor of ras) which may be involved in mediating effects of ceramide
on MAP kinases [105]. The protease cathepsin D was also identified as a
direct target for ceramide, and, given its lysosomal localization, cathepsin D
may define a compartment-specific pathway for ceramide action [45]. The
discovery of these pathways regulated by ceramide underscores the
significant influence of sphingolipid metabolism in the regulation of cell
division, apoptosis, and differentiation. Thus, elucidation of the pathways of
ceramide action in yeast and mammals is important, as these pathways may

become excellent targets for development of chemotherapeutics.
2.2 Stress responses
Perhaps the most studied activities of sphingolipids in yeast involve yeast

stress responses. In yeast, heat stress induces an acute upregulation of
de novo sphingolipid biosynthesis. As early as 5 min, increases of several
fold are seen in dihydrosphingosine and phytosphingosine, immediately
followed by increases in phosphorylated sphingoid bases [92]. Subsequently
the ceramides are observed to rise, around 20–30 min, when sphingoid bases
decrease [22, 50].
Several components of the yeast heat stress response, including cell cycle
arrest and proteolysis are dependent on de novo sphingolipid biosynthesis
(Figure 2), as revealed in studies using a temperature-sensitive mutant in
LCB1, lcb1-100, which cannot generate sphingolipids upon heat stress. This
mutant does not undergo transient cell cycle arrest [49], and addition of
exogenous phytosphingosine induces a transient cell cycle arrest [15, 16,
50]; thus, demonstrating an essential role for the acute formation of
sphingolipids in the heat stress response.
Figure 2. Heat stress induces many activities in yeast, some of which are mediated by stress-
induced de novo sphingolipid synthesis. Direct targets for sphingolipids in yeast include
ceramide-activated protein phosphatase, comprised of sit4p, cdc55p, and tpd3p. Additionally,
heat stress induces sphingolipid synthesis in some mammalian cell types. Interestingly,
several chemotherapeutic agents induce de novo sphingolipid production and thus, their
effects are likely mediated in part by sphingolipid biosynthesis. (See text for further
discussion.)
Another key feature of the yeast heat stress response is the internalization
and ubiquitin-mediated proteolysis of nutrient permeases. Previous studies
in yeast have demonstrated that de novo sphingolipid biosynthesis is
required for these activities, and that exogenously added phytosphingosine
initiates this degradation in the absence of heat stress [15, 16]. This
sphingolipid activity is at least somewhat conserved in some mammalian
cells, as ceramide has been demonstrated to modulate the ubiquitin-mediated
proteolysis of c-myc [82]. More investigation is needed in both yeast and
mammalian systems to determine the specific mechanisms of sphingolipid
modulation of this pathway. Interestingly, a recent study implicates lipid
rafts in the transport and degradation of the nutrient permease Fur4p [44],
and thus, the propensity of sphingolipids to form lipid microdomains [10, 34,
36, 47] may facilitate these activities.
These findings in yeast led to the hypothesis that mammalian heat stress
responses may depend at least partially on de novo generated sphingolipids.
Indeed, subsequent studies in MOLT-4 human leukemia cells [48] confirmed
this hypothesis. Heat stress in these cells induced a flux through sphingolipid
biosynthetic pathways similar to those observed in yeast. Heat-induced
de novo sphingolipid biosynthesis in MOLT-4 cells was shown to influence
mRNA splicing by the modulation of SR protein phosphorylation state [48].
One difference between heat-induced de novo sphingolipid biosynthesis in
yeast and in the MOLT-4 cells is that yeast accumulate sphingoid bases prior
to ceramide formation, whereas the MOLT-4 cells showed no accumulation
in sphingoid bases after heat stress, but accumulated ceramide, which is
thought to be the active intermediate in modulation of many mammalian
signaling pathways. Whether this situation is typical of mammalian systems
and whether the sphingoid bases may play specific roles remains to be
determined.
Downstream regulatory roles of sphingolipids generated during stress
responses is of interest to cancer research, as several chemotherapeutic
agents including etoposide, daunorubicin, and gemciytabine have been
demonstrated to cause increased de novo sphingolipid synthesis, and
furthermore, many effects of these drugs are blocked by inhibitors of
ceramide synthesis ([8], reviewed in [81, 89]). Thus, these agents act at least
partially through the de novo pathway of sphingolipid generation.
Therefore, understanding the mechanisms of formation and action of
sphingoid bases and ceramides may lead to direct insight into the
mechanisms of action of several chemotherapeutic agents and, especially,
the mechanisms by which they activate stress response pathways.
2.3 Ceramide and cell senescence
Many lines of evidence in a spectrum of systems indicate that ceramide

levels are correlated with regulation of life span and cell senescence. These
studies stemmed from early work indicating elevated ceramide levels in
senescent human diploid fibroblasts (HDF) [96]. Furthermore, addition of
ceramide to proliferating HDF induced several markers of senescence
including cessation of DNA replication and dephosphorylation of Rb [19],
suggesting a role for ceramide in mediating the conversion of cell phenotype
from proliferating to senescent.
Several clues exist as to the specific mechanisms by which ceramide may
mediate the regulation of lifespan. A key phenotype of senescent cells is the
lack of response to mitogens. Investigations into this particular facet of cell
senescence led to the discovery that lack of mitogenic response is mediated
through inhibition of phospholipase D (PLD). Senescent HDF showed
ceramide-dependent inability to activate PLD, resulting in lack of PKC
activation which in turn mediates the transcriptional response to mitogens.
Treatment of quiescent young HDF with exogenous ceramide mimicked the
inactivation of PLD and its sequelae observed in senescent HDF [82, 83, 96].
Other investigation has demonstrated that ceramide inhibits telomerase
activity in a lung cancer cell line via inactivation of c-Myc [82]. This
inactivation partially involves increased ubiquitylation and subsequent
proteolytic degradation, as do some stress responsive events, as discussed
above [82]. Current investigation focuses on the roles of ceramide in
coordinating the development of these senescent phenotypes.
Importantly, links between ceramide generation and cell senescence
appear to be conserved in yeast. In S. cerevisiae, deletion of the longevity
assurance genes LAG1 and LAC1 confers an increased life span in yeast by
30% [91]. As is the case with mammalian cells, each yeast cell has a defined
number of cell division cycles it can undergo before reaching a nondividing
state, or senescence. It may follow that LAG1 and LAC1 regulate yeast life
span via the regulation of ceramide levels. Intriguingly, some of the
Lac1/Lag1 homologues in other eukaryotes appear to contain not only
LAG-1 homologous domains, but transcriptional regulatory HOX-motifs,
suggesting the possibility of coupling between ceramide binding and
transcriptional regulation [97]. The structural and functional conservation of
these proteins, as well as their potential roles in the regulation of ceramide
formation, is of intense interest. Indeed, ongoing studies in yeast and
mammalian system will likely shed light on the relationship of LAG1 and its
homologues to ceramide levels and cell growth control.
In sum, studies in both yeast and mammalian systems have enabled the
compilation of a general picture of ceramide influences on cell senescence
and aging; this apparent functional conservation attests to the evolutionary
importance of these pathways. Eventually these studies may provide novel

targets for cancer therapeutics.
2.4 Sphingosine kinase-mitogenesis and vascularization
Tumor progression depends on successful vascularization to provide

factors which enable tumor growth. These ideas have led to the hypothesis
that reducing tumor vascularization is an effective way to block tumor
progression. Novel insights came from work demonstrating that S-1-P is a
high affinity ligand for the endothelium development and growth receptor-1
(EDG-1) [58] and other EDG receptors, recently renamed S-1-P receptors
[46]. These are G-protein coupled receptors (GPCRs) which are involved
in many cell processes including vascularization and mitogenesis [25].
Subsequent studies have further supported roles for S-1-P and sphingosine
kinase in activation of these receptors [1, 53, 84, 95, 100, 102]. Of particular
interest is that a member of this receptor family, the mammalian EDG-2
lysophosphatidic acid receptor, when heterologously expressed in yeast, was
shown to couple to the pheromone signaling pathway [26], indicating
evolutionary conservation between these two receptors. It will be interesting
to see if sphingoid base phosphate activation of GPCRs is conserved in all
eukaryotic systems.
3 CONCLUSIONS
Yeast studies have contributed to our understanding of sphingolipid

formation, metabolism, and function in mammalian cells and yeast sphingo-
lipid studies have become intertwined with mammalian studies, complement-
ting, supporting, and indeed, often spurring research into mammalian
sphingolipid biosynthesis and function. Key areas for further investigation
include mechanisms of ceramide biosynthesis induction by chemotherapeutic
agents and factors such as TNF, and determining functions of individual
sphingolipids such as sphingoid bases and sphingoid base phosphates, and
their mechanisms of action. In general, more research is needed into how
sphingolipid metabolic pathways are coordinated to regulate relative cellular
levels of sphingolipid and other metabolites including diacylglycerols.
Undoubtedly, yeast systems will continue to be exploited to maximum
advantage for increasing our understanding of sphingolipid formation and
function in all eukaryotic cells.
ACKNOWLEDGMENTS
This work was supported by a VA merit award (LMO), and NIH grants
AG16583 (LMO), GM62887 (LMO), GM63465 (YAH), and GM4825
(YAH).
REFERENCES
1. An, S., T. Bleu, and Y. Zheng. 1999. Transduction of intracellular calcium signals
through G protein-mediated activation of phospholipase C by recombinant sphingosine
1-phosphate receptors. Mol. Pharmacol. 55:787–794.
2. Beeler, T., K. Gable, C. Zhao, and T. Dunn. 1994. A novel protein, CSG2p, is
required for Ca2+ regulation in Saccharomyces cerevisiae. J. Biol. Chem. 269:7279–
7284.
3. Beeler, T. J., D. Fu, J. Rivera, E. Monaghan, K. Gable, and T. M. Dunn. 1997.
SUR1 (CSG1/BCL21), a gene necessary for growth of Saccharomyces cerevisiae in the
presence of high Ca2+ concentrations at 37 degrees C, is required for mannosylation of
inositolphosphorylceramide. Mol. Gen. Genet. 255:570–579.
4. Bielawska, A., H. M. Crane, D. Liotta, L. M. Obeid, and Y. A. Hannun. 1993.
Selectivity of ceramide-mediated biology. Lack of activity of erythro-dihydroceramide.
J. Biol. Chem. 268:26226–26232.
5. Bielawska, A., C. M. Linardic, and Y. A. Hannun. 1992. Modulation of cell growth
and differentiation by ceramide. FEBS Lett. 307:211–214.
6. Birchwood, C. J., J. D. Saba, R. C. Dickson, and K. W. Cunningham. 2001. Calcium
influx and signaling in yeast stimulated by intracellular sphingosine 1-phosphate
accumulation. J. Biol. Chem. 276:11712–11718.
7. Blobe, G. C., S. Stribling, L. M. Obeid, and Y. A. Hannun. 1996. Protein kinase C
isoenzymes: regulation and function. Cancer Surv. 27:213–248.
8. Bose, R., M. Verheij, A. Haimovitz-Friedman, K. Scotto, Z. Fuks, and
R. Kolesnick. 1995. Ceramide synthase mediates daunorubicin-induced apoptosis: an
alternative mechanism for generating death signals. Cell 82:405–414.
9. Brandwagt, B. F., L. A. Mesbah, F. L. Takken, P. L. Laurent, T. J. Kneppers,
J. Hille, and H. J. Nijkamp. 2000. A longevity assurance gene homolog of tomato
mediates resistance to Alternaria alternata f. sp. lycopersici toxins and fumonisin B1.
10. Brown, D. 2002. Structure and function of membrane rafts. Int. J. Med. Microbiol.
291:433–437.
11. Buede, R., C. Rinker-Schaffer, W. J. Pinto, R. L. Lester, and R. C. Dickson. 1991.
Cloning and characterization of LCB1, a Saccharomyces gene required for biosynthesis
of the long-chain base component of sphingolipids. J. Bacteriol. 173:4325–4332.
12. Chalfant, C. E., K. Kishikawa, M. C. Mumby, C. Kamibayashi, A. Bielawska, and
Y. A. Hannun. 1999. Long chain ceramides activate protein phosphatase-1 and protein
phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid. J. Biol.
Chem. 274:20313–20317.
13. Chalfant, C. E., B. Ogretmen, S. Galadari, B. J. Kroesen, B. J. Pettus, and Y. A.
Hannun. 2001. FAS activation induces dephosphorylation of SR proteins; dependence
on the de novo generation of ceramide and activation of protein phosphatase 1. J. Biol.

Chem. 276:44848–44855.
14. Chen, J. K., W. S. Lane, and S. L. Schreiber. 1999. The identification of myriocin-
binding proteins. Chem. Biol. 6:221–235.
15. Chung, N., G. Jenkins, Y. A. Hannun, J. Heitman, and L. M. Obeid. 2000.
Sphingolipids signal heat stress-induced ubiquitin-dependent proteolysis. J. Biol. Chem.
275:17229–17232.
16. Chung, N., C. Mao, J. Heitman, Y. A. Hannun, and L. M. Obeid. 2001.
Phytosphingosine as a specific inhibitor of growth and nutrient import in Saccharomyces
17. Cliften, P., Y. Wang, D. Mochizuki, T. Miyakawa, R. Wangspa, J. Hughes, and
J. Y. Takemoto. 1996. SYR2, a gene necessary for syringomycin growth inhibition of
Saccharomyces cerevisiae. Microbiology 142 (Pt 3):477–484.
18. D’Mello N. P., A. M. Childress, D. S. Franklin, S. P. Kale, C. Pinswasdi, and S. M.
Jazwinski. 1994. Cloning and characterization of LAG1, a longevity-assurance gene in
yeast. J. Biol. Chem. 269:15451–15459.
19. Dbaibo, G. S., M. Y. Pushkareva, S. Jayadev, J. K. Schwarz, J. M. Horowitz, L. M.
Obeid, and Y. A. Hannun. 1995. Retinoblastoma gene product as a downstream target
for a ceramide-dependent pathway of growth arrest. Proc. Natl. Acad. Sci. USA
92:1347–1351.
20. Desfarges, L., P. Durrens, H. Juguelin, C. Cassagne, M. Bonneu, and M. Aigle.
1993. Yeast mutants affected in viability upon starvation have a modified phospholipid
composition. Yeast 9:267–277.
21. Dickson, R. C. 1998. Sphingolipid functions in Saccharomyces cerevisiae: comparison
to mammals. Annu. Rev. Biochem. 67:27–48.
22. Dickson, R. C., E. E. Nagiec, M. Skrzypek, P. Tillman, G. B. Wells, and R. L.
Lester. 1997. Sphingolipids are potential heat stress signals in Saccharomyces. J. Biol.
Chem. 272:30196–30200.
23. Dickson, R. C., E. E. Nagiec, G. B. Wells, M. M. Nagiec, and R. L. Lester. 1997.
Synthesis of mannose-(inositol-P)2-ceramide, the major sphingolipid in Saccharomyces
cerevisiae, requires the IPT1 (YDR072c) gene. J. Biol. Chem. 272:29620–29625.
24. Dobrowsky, R. T., and Y. A. Hannun. 1992. Ceramide stimulates a cytosolic protein
phosphatase. J. Biol. Chem. 267:5048–5051.
25. English, D., D. N. Brindley, S. Spiegel, and J. G. Garcia. 2002. Lipid mediators of
angiogenesis and the signalling pathways they initiate. Biochim. Biophys. Acta
1582:228–239.
26. Erickson, J. R., J. J. Wu, J. G. Goddard, G. Tigyi, K. Kawanishi, L. D. Tomei, and
M. C. Kiefer. 1998. Edg-2/Vzg-1 couples to the yeast pheromone response pathway
selectively in response to lysophosphatidic acid. J. Biol. Chem. 273:1506–1510.
27. Feoktistova, A., P. Magnelli, C. Abeijon, P. Perez, R. L. Lester, R. C. Dickson, and
K. L. Gould. 2001. Coordination between fission yeast glucan formation and growth
requires a sphingolipase activity. Genetics 158:1397–1411.
28. Ferguson-Yankey, S. R., M. S. Skrzypek, R. L. Lester, and R. C. Dickson. 2002.
Mutant analysis reveals complex regulation of sphingolipid long chain base phosphates
and long chain bases during heat stress in yeast. Yeast 19:573–586.
29. Ferlinz, K., R. Hurwitz, and K. Sandhoff. 1991. Molecular basis of acid
sphingomyelinase deficiency in a patient with Niemann-Pick disease type A. Biochem.
Biophys. Res. Commun. 179:1187–1191.
30. Fishbein, J. D., R. T. Dobrowsky, A. Bielawska, S. Garrett, and Y. A. Hannun.

1993. Ceramide-mediated growth inhibition and CAPP are conserved in Saccharomyces
31. Friant, S., R. Lombardi, T. Schmelzle, M. N. Hall, and H. Riezman. 2001. Sphingoid
base signaling via Pkh kinases is required for endocytosis in yeast. EMBO J.
20:6783–6792.
32. Gable, K., G. Han, E. Monaghan, D. Bacikova, M. Natarajan, R. Williams, and
T. M. Dunn. 2002. Mutations in the yeast LCB1 and LCB2 genes, including those
corresponding to the hereditary sensory neuropathy type I mutations, dominantly
inactivate serine palmitoyltransferase. J. Biol. Chem. 277:10194–10200.
33. Gable, K., H. Slife, D. Bacikova, E. Monaghan, and T. M. Dunn. 2000. Tsc3p is an
80-amino acid protein associated with serine palmitoyltransferase and required for
optimal enzyme activity. J. Biol. Chem. 275:7597–7603.
34. Gkantiragas, I., B. Brugger, E. Stuven, D. Kaloyanova, X. Y. Li, K. Lohr,
F. Lottspeich, F. T. Wieland, and J. B. Helms. 2001. Sphingomyelin-enriched
microdomains at the Golgi complex. Mol. Biol. Cell 12:1819–1833.
35. Grilley, M. M., S. D. Stock, R. C. Dickson, R. L. Lester, and J. Y. Takemoto. 1998.
Syringomycin action gene SYR2 is essential for sphingolipid 4-hydroxylation in
Saccharomyces cerevisiae. J. Biol. Chem. 273:11062–11068.
36. Hakomori, S., S. Yamamura, and A. K. Handa. 1998. Signal transduction through
glyco(sphingo)lipids. Introduction and recent studies on glyco(sphingo)lipid-enriched
microdomains. Ann. N. Y. Acad. Sci. 845:1–10.
37. Hanada, K., T. Hara, M. Nishijima, O. Kuge, R. C. Dickson, and M. M. Nagiec.
1997. A mammalian homolog of the yeast LCB1 encodes a component of serine
palmitoyltransferase, the enzyme catalyzing the first step in sphingolipid synthesis.
J. Biol. Chem. 272:32108–32114.
38. Hannun, Y. A. 1994. The sphingomyelin cycle and the second messenger function of
ceramide. J. Biol. Chem. 269:3125–3128.
39. Hannun, Y. A., and R. M. Bell. 1993. The sphingomyelin cycle: a prototypic
sphingolipid signaling pathway. Adv. Lipid Res. 25:27–41.
40. Hannun, Y. A., and L. M. Obeid. 1997. Ceramide and the eukaryotic stress response.
Biochem. Soc. Trans. 25:1171–1175.
41. Hannun, Y. A., and L. M. Obeid. 2002. The ceramide-centric universe of lipid-mediated
cell regulation: stress encounters of the lipid kind. J. Biol. Chem. 277:25847–25850.
42. Hannun, Y. A., L. M. Obeid, and R. A. Wolff. 1993. The novel second messenger
ceramide: identification, mechanism of action, and cellular activity. Adv. Lipid Res.
25:43–64.
43. Hashida-Okado, T., A. Ogawa, M. Endo, R. Yasumoto, K. Takesako, and I. Kato.
1996. AUR1, a novel gene conferring aureobasidin resistance on Saccharomyces
cerevisiae: a study of defective morphologies in Aur1p-depleted cells. Mol. Gen. Genet.
251:236–244.
44. Hearn, J. D., R. L. Lester, and R. C. Dickson. 2002. The uracil transporter Fur4p
associates with lipid rafts. J. Biol. Chem. 278:3679–3686.
45. Heinrich, M., M. Wickel, W. Schneider-Brachert, C. Sandberg, J. Gahr,
R. Schwandner, T. Weber, P. Saftig, C. Peters, J. Brunner, M. Kronke, and
S. Schutze. 1999. Cathepsin D targeted by acid sphingomyelinase-derived ceramide.
EMBO J. 18:5252–5263.
46. Hla, T., M. J. Lee, N. Ancellin, S. Thangada, C. H. Liu, M. Kluk, S. S. Chae, and
M. T. Wu. 2000. Sphingosine-1-phosphate signaling via the EDG-1 family of
G-protein-coupled receptors. Ann. N. Y. Acad. Sci. 905:16–24.
47. Holopainen, J. M., M. Subramanian, and P. K. Kinnunen. 1998. Sphingomyelinase

induces lipid microdomain formation in a fluid phosphatidylcholine/sphingomyelin
membrane. Biochemistry 37:17562–17570.
48. Jenkins, G. M., L. A. Cowart, P. Signorelli, B. J. Pettus, C. E. Chalfant, and Y. A.
Hannun. 2002. Acute activation of de novo sphingolipid biosynthesis upon heat shock
causes an accumulation of ceramide and subsequent dephosphorylation of SR proteins.
J. Biol. Chem. 277:42572–42578.
49. Jenkins, G. M., and Y. A. Hannun. 2001. Role for de novo sphingoid base
biosynthesis in the heat-induced transient cell cycle arrest of Saccharomyces cerevisiae.
J. Biol. Chem. 276:8574–8581.
50. Jenkins, G. M., A. Richards, T. Wahl, C. Mao, L. Obeid, and Y. Hannun. 1997.
Involvement of yeast sphingolipids in the heat stress response of Saccharomyces
51. Jiang, J. C., P. A. Kirchman, M. Zagulski, J. Hunt, and S. M. Jazwinski. 1998.
Homologs of the yeast longevity gene LAG1 in Caenorhabditis elegans and human.
Genome Res. 8:1259–1272.
52. Kim, S., H. Fyrst, and J. Saba. 2000. Accumulation of phosphorylated sphingoid long
chain bases results in cell growth inhibition in Saccharomyces cerevisiae. Genetics
156:1519–1529.
53. Kimura, T., T. Watanabe, K. Sato, J. Kon, H. Tomura, K. Tamama, A. Kuwabara,
T. Kanda, I. Kobayashi, H. Ohta, M. Ui, and F. Okajima. 2000. Sphingosine
1-phosphate stimulates proliferation and migration of human endothelial cells possibly
through the lipid receptors, Edg-1 and Edg-3. Biochem. J. 348 Pt 1:71–76.
54. Kohama, T., A. Olivera, L. Edsall, M. M. Nagiec, R. Dickson, and S. Spiegel. 1998.
Molecular cloning and functional characterization of murine sphingosine kinase. J. Biol.
Chem. 273:23722–23728.
55. Le Stunff, H., C. Peterson, R. Thornton, S. Milstien, S. M. Mandala, and S. Spiegel.
2002. Characterization of murine sphingosine-1-phosphate phosphohydrolase. J. Biol.
Chem. 277:8920–8927.
56. Lee, J. Y., A. E. Bielawska, and L. M. Obeid. 2000. Regulation of cyclin-dependent
kinase 2 activity by ceramide. Exp. Cell Res. 261:303–311.
57. Lee, J. Y., Y. A. Hannun, and L. M. Obeid. 1996. Ceramide inactivates cellular
protein kinase Calpha. J. Biol. Chem. 271:13169–13174.
58. Lee, M. J., J. R. Van Brocklyn, S. Thangada, C. H. Liu, A. R. Hand, R. Menzeleev,
S. Spiegel, and T. Hla. 1998. Sphingosine-1-phosphate as a ligand for the G protein-
coupled receptor EDG-1. Science 279:1552–1555.
59. Leipelt, M., D. Warnecke, U. Zahringer, C. Ott, F. Muller, B. Hube, and E. Heinz.
2001. Glucosylceramide synthases, a gene family responsible for the biosynthesis of
glucosphingolipids in animals, plants, and fungi. J. Biol. Chem. 276:33621–33629.
60. Leipelt, M., D. C. Warnecke, B. Hube, U. Zahringer, and E. Heinz. 2000.
Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms.
Biochem. Soc. Trans. 28:751–752.
61. Levade, T., and J. P. Jaffrezou. 1999. Signalling sphingomyelinases: which, where,
how and why? Biochim. Biophys. Acta 1438:1–17.
62. Liu, H., M. Sugiura, V. E. Nava, L. C. Edsall, K. Kono, S. Poulton, S. Milstien,
T. Kohama, and S. Spiegel. 2000. Molecular cloning and functional characterization of
a novel mammalian sphingosine kinase type 2 isoform. J. Biol. Chem. 275:19513–
19520.
63. Liu, Y. Y., T. Y. Han, A. E. Giuliano, and M. C. Cabot. 2001. Ceramide
glycosylation potentiates cellular multidrug resistance. FASEB J. 15:719–730.
64. Liu, Y. Y., T. Y. Han, A. E. Giuliano, and M. C. Cabot. 1999. Expression of

glucosylceramide synthase, converting ceramide to glucosylceramide, confers
adriamycin resistance in human breast cancer cells. J. Biol. Chem. 274:1140–1146.
65. Luberto, C., and Y. A. Hannun. 1998. Sphingomyelin synthase, a potential regulator
of intracellular levels of ceramide and diacylglycerol during SV40 transformation. Does
sphingomyelin synthase account for the putative phosphatidylcholine-specific
phospholipase C? J. Biol. Chem. 273:14550–14559.
66. Luberto, C., D. L. Toffaletti, E. A. Wills, S. C. Tucker, A. Casadevall, J. R. Perfect,
Y. A. Hannun, and M. M. Del Poeta. 2001. Roles for inositol-phosphoryl ceramide
synthase 1 (IPC1) in pathogenesis of C. neoformans. Genes Dev. 15:201–212.
67. Mandala, S. M., R. Thornton, I. Galve-Roperh, S. Poulton, C. Peterson, A. Olivera,
J. Bergstrom, M. B. Kurtz, and S. Spiegel. 2000. Molecular cloning and
characterization of a lipid phosphohydrolase that degrades sphingosine-1- phosphate and
induces cell death. Proc. Natl. Acad. Sci. USA 97:7859–7864.
68. Mandala, S. M., R. Thornton, Z. Tu, M. B. Kurtz, J. Nickels, J. Broach,
R. Menzeleev, and S. Spiegel. 1998. Sphingoid base 1-phosphate phosphatase: a key
regulator of sphingolipid metabolism and stress response. Proc. Natl. Acad. Sci. USA
95:150–155.
69. Mao, C., J. D. Saba, and L. M. Obeid. 1999. The dihydrosphingosine-1-phosphate
phosphatases of Saccharomyces cerevisiae are important regulators of cell proliferation
and heat stress responses. Biochem. J. 342 Pt 3:667–675.
70. Mao, C., M. Wadleigh, G. M. Jenkins, Y. A. Hannun, and L. M. Obeid. 1997.
Identification and characterization of Saccharomyces cerevisiae dihydrosphingosine-1-
phosphate phosphatase. J. Biol. Chem. 272:28690–28694.
71. Mao, C., R. Xu, A. Bielawska, and L. M. Obeid. 2000. Cloning of an alkaline
ceramidase from Saccharomyces cerevisiae. An enzyme with reverse (CoA-independent)
ceramide synthase activity. J. Biol. Chem. 275:6876–6884.
72. Mao, C., R. Xu, A. Bielawska, Z. M. Szulc, and L. M. Obeid. 2000. Cloning and
characterization of a Saccharomyces cerevisiae alkaline ceramidase with specificity for
dihydroceramide. J. Biol. Chem. 275:31369–31378.
73. Mao, C., R. Xu, Z. M. Szulc, A. Bielawska, S. H. Galadari, and L. M. Obeid. 2001.
Cloning and characterization of a novel human alkaline ceramidase. A mammalian
enzyme that hydrolyzes phytoceramide. J. Biol. Chem. 276:26577–26588.
74. Merrill, A. H., Jr., Y. A. Hannun, and R. M. Bell. 1993. Introduction: sphingolipids
and their metabolites in cell regulation. Adv. Lipid. Res. 25:1–24.
75. Nagiec, M. M., J. A. Baltisberger, G. B. Wells, R. L. Lester, and R. C. Dickson.
1994. The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of
serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis. Proc. Natl.
Acad. Sci. USA 91:7899–7902.
76. Nagiec, M. M., R. L. Lester, and R. C. Dickson. 1996. Sphingolipid synthesis:
identification and characterization of mammalian cDNAs encoding the Lcb2 subunit of
serine palmitoyltransferase. Gene 177:237–241.
77. Nagiec, M. M., E. E. Nagiec, J. A. Baltisberger, G. B. Wells, R. L. Lester, and R. C.
Dickson. 1997. Sphingolipid synthesis as a target for antifungal drugs. Comple-
mentation of the inositol phosphorylceramide synthase defect in a mutant strain of
Saccharomyces cerevisiae by the AUR1 gene. J. Biol. Chem. 272:9809–9817.
78. Nagiec, M. M., M. Skrzypek, E. E. Nagiec, R. L. Lester, and R. C. Dickson. 1998.
The LCB4 (YOR171c) and LCB5 (YLR260w) genes of Saccharomyces encode
sphingoid long chain base kinases. J. Biol. Chem. 273:19437–19442.
79. Nickels, J. T., and J. R. Broach. 1996. A ceramide-activated protein phosphatase mediates
ceramide-induced G1 arrest of Saccharomyces cerevisiae. Genes Dev. 10:382–394.
80. Ogawa, C., A. Kihara, M. Gokoh, and Y. Igarashi. 2003. Identification and
characterization of a novel human sphingosine-1-phosphate phosphohydrolase, hSPP2.
J. Biol. Chem. 278:1268–1272.
81. Ogretmen, B., and Y. A. Hannun. 2001. Updates on functions of ceramide in
chemotherapy-induced cell death and in multidrug resistance. Drug Resist Updat.
4:368–377.
82. Ogretmen, B., J. M. Kraveka, D. Schady, J. Usta, Y. A. Hannun, and L. M. Obeid.
2001. Molecular mechanisms of ceramide-mediated telomerase inhibition in the A549
human lung adenocarcinoma cell line. J. Biol. Chem. 276:32506–32514.
83. Ogretmen, B., D. Schady, J. Usta, R. Wood, J. M. Kraveka, C. Luberto, H. Birbes,
Y. A. Hannun, and L. M. Obeid. 2001. Role of ceramide in mediating the inhibition of
telomerase activity in A549 human lung adenocarcinoma cells. J. Biol. Chem.
276:24901–24910.
84. Okamoto, H., N. Takuwa, Y. Yatomi, K. Gonda, H. Shigematsu, and Y. Takuwa.
1999. EDG3 is a functional receptor specific for sphingosine 1-phosphate and
sphingosylphosphorylcholine with signaling characteristics distinct from EDG1 and
AGR16. Biochem. Biophys. Res. Commun. 260:203–208.
85. Patton, J. L., B. Srinivasan, R. C. Dickson, and R. L. Lester. 1992. Phenotypes of
sphingolipid-dependent strains of Saccharomyces cerevisiae. J. Bacteriol. 174:7180–7184.
86. Pinto, W. J., G. W. Wells, and R. L. Lester. 1992. Characterization of enzymatic
synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains
exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase
activity. J. Bacteriol. 174:2575–2581.
87. Reyes, J. G., I. G. Robayna, P. S. Delgado, I. H. Gonzalez, J. Q. Aguiar, F. E.
Rosas, L. F. Fanjul, and C. M. Galarreta. 1996. c-Jun is a downstream target for
ceramide-activated protein phosphatase in A431 cells. J. Biol. Chem. 271:21375–21380.
88. Saba, J. D., F. Nara, A. Bielawska, S. Garrett, and Y. A. Hannun. 1997. The BST1
gene of Saccharomyces cerevisiae is the sphingosine-1-phosphate lyase. J. Biol. Chem.
272:26087–26090.
89. Saba, J. D., L. M. Obeid, and Y. A. Hannun. 1996. Ceramide: an intracellular
mediator of apoptosis and growth suppression. Philos. Trans. R. Soc. Lond. B. Biol. Sci.
351:233–240; discussion 240–241.
90. Schmelzle, T., S. B. Helliwell, and M. N. Hall. 2002. Yeast protein kinases and the
RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in
yeast. Mol. Cell Biol. 22:1329–1339.
91. Schorling, S., B. Vallee, W. P. Barz, H. Riezman, and D. Oesterhelt. 2001. Lag1p
and Lac1p are essential for the acyl-CoA-dependent ceramide synthase reaction in
Saccharomyces cerevisae. Mol. Biol. Cell 12:3417–3427.
92. Skrzypek, M. S., M. M. Nagiec, R. L. Lester, and R. C. Dickson. 1999. Analysis of
phosphorylated sphingolipid long-chain bases reveals potential roles in heat stress and
growth control in Saccharomyces. J. Bacteriol. 181:1134–1140.
93. Sugiura, M., K. Kono, H. Liu, T. Shimizugawa, H. Minekura, S. Spiegel, and
T. Kohama. 2002. Ceramide kinase, a novel lipid kinase. Molecular cloning and
functional characterization. J. Biol Chem. 277:23294–23300.
94. Van Brocklyn, J. R., M. J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier,
D. M. Thomas, P. J. Coopman, S. Thangada, C. H. Liu, T. Hla, and S. Spiegel.
1998. Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled
receptor Edg-1 and intracellular to regulate proliferation and survival. J. Cell Biol.
142:229–240.
95. Van Brocklyn, J. R., Z. Tu, L. C. Edsall, R. R. Schmidt, and S. Spiegel. 1999.
Sphingosine 1-phosphate-induced cell rounding and neurite retraction are mediated by
the G protein-coupled receptor H218. J. Biol. Chem. 274:4626–4632.
96. Venable, M. E., G. C. Blobe, and L. M. Obeid. 1994. Identification of a defect in
the phospholipase D/diacylglycerol pathway in cellular senescence. J. Biol. Chem.
269:26040–2604.
97. Venkataraman, K., and A. H. Futerman. 2002. Do longevity assurance genes
containing Hox domains regulate cell development via ceramide synthesis? FEBS Lett.
528:3–4.
98. Venkataraman, K., C. Riebeling, J. Bodennec, H. Riezman, J. C. Allegood, M. C.
Sullards, A. H. Merrill, Jr., and A. H. Futerman. 2002. Upstream of growth and
differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity
assurance gene 1 (LAG1), regulates N-stearoyl-sphinganine (C18-(dihydro)ceramide)
synthesis in a fumonisin B1-independent manner in mammalian cells. J. Biol. Chem.
277:35642–35649.
99. Wang, E., W. P. Norred, C. W. Bacon, R. T. Riley, and A. H. Merrill, Jr. 1991.
Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases
associated with Fusarium moniliforme. J. Biol. Chem. 266:14486–14490.
100. Wang, F., J. R. Van Brocklyn, J. P. Hobson, S. Movafagh, Z. Zukowska-Grojec,
S. Milstien, and S. Spiegel. 1999. Sphingosine 1-phosphate stimulates cell migration
through a G(i)-coupled cell surface receptor. Potential involvement in angiogenesis.
J. Biol. Chem. 274:35343–35350.
101. Watanabe, R., K. Funato, K. Venkataraman, A. H. Futerman, and H. Riezman.
2002. Sphingolipids are required for the stable membrane association of glycosyl-
phosphatidylinositol-anchored proteins in yeast. J. Biol. Chem. 277:49538–49544.
102. Windh, R. T., M. J. Lee, T. Hla, S. An, A. J. Barr, and D. R. Manning. 1999.
Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and
H218/Edg-5 to the G(i), G(q), and G(12) families of heterotrimeric G proteins. J. Biol.
Chem. 274:27351–27358.
103. Wolff, R. A., R. T. Dobrowsky, A. Bielawska, L. M. Obeid, and Y. A. Hannun.
1994. Role of ceramide-activated protein phosphatase in ceramide-mediated signal
transduction. J. Biol. Chem. 269:19605–19609.
104. Zanolari, B., S. Friant, K. Funato, C. Sutterlin, B. J. Stevenson, and H. Riezman.
2000. Sphingoid base synthesis requirement for endocytosis in Saccharomyces
cerevisiae. EMBO J. 19:2824–2833.
105. Zhang, Y., B. Yao, S. Delikat, S. Bayoumy, X. H. Lin, S. Basu, M. McGinley, P. Y.
Chan-Hui, H. Lichenstein, and R. Kolesnick. 1997. Kinase suppressor of Ras is
ceramide-activated protein kinase. Cell 89:63–72.
Chapter 9
EXPLORING AND RESTORING THE p53
PATHWAY USING THE p53 DISSOCIATOR
ASSAY IN YEAST
Rainer K. Brachmann
Department of Medicine, University of California at Irvine, Irvine, California 92697, USA
1 BRIEF INTRODUCTION TO p53 BIOLOGY
For more than a decade, the tumor suppressor protein p53 has been
recognized as a, and maybe the, central protein that protects humans from
cancer. Its role as “guardian of the genome” [51] and its frequent
inactivation in human cancers has drawn numerous researchers into the p53
field, guaranteeing a continuous flow of new research data that is regularly
summarized in excellent reviews (see for example, 20, 49, 56, 79, 97, 98; see
also the chapter by Inga and colleagues for an in-depth review of p53
biology). Therefore, this introduction will be short, but nevertheless
sufficient to appreciate the p53-related questions that are being pursued in
the p53 dissociator assay.
The p53 protein was initially isolated in 1979 as a 53 kD protein that was
associated with SV40 large T antigen [53, 57]. It was a decade before p53
was recognized as an important tumor suppressor because of its frequent
mutation in human cancers [38, 52, 73]. This key finding raised two central
questions: Why was p53 so important and what was its mode of action?
It was quickly established that wild-type p53 exerts its effects, at least in
part, as a transcription factor that recognizes its direct target genes through
binding of its core domain (amino acids 96–292) to specific DNA
sequences. In unstressed cells, p53 is relatively inactive and has a short half-
life of 15 min because of negative feedback loops, such as with MDM2.
211
© 2007 Springer.
212 R. K. Brachmann
p53 drives expression of MDM2, and the Mdm2 protein then binds to the
p53 amino-terminus and induces ubiquitin-mediated degradation [55].
Activation of the p53 protein occurs through upstream signals, such as DNA
damage, hypoxia, overexpression of oncogenes and others. These signals
typically lead to posttranslational modifications of p53, in particular amino-
terminal phosphorylation and carboxy-terminal acetylation. These modify-
cations stabilize and activate p53. p53 is then able to induce its numerous
target genes and causes cell cycle arrest, DNA repair, and/or apoptosis [20,
49, 56, 79, 97, 98].
The central role of p53 in protecting humans from cancer is reflected in
the enormous number of human cancers with p53 mutations. It is estimated
that 50% of all human cancers have p53 mutations, and such mutations have
been catalogued in large international databases [5, 18, 32, 36, 37, 62, 77].
The latest update of the IARC TP53 Mutation Database contains 19,809
somatic p53 mutations. 14,123 or 71% of these are missense mutations
affecting the p53 core domain, and close to 1,000 different amino acid
changes have been described for this p53 region (R9, http://www.
iarc.fr/p53/).
It has been, and continues to be, a significant challenge to understand the
complexity of wild-type p53, p53 cancer mutants and downstream target
genes. The chapter by Inga and colleagues summarizes how powerful
genetic approaches in yeast assays have made important contributions to
characterizing the p53 network. This chapter will focus on two aspects of
p53 research that are addressed in the p53 dissociator assay: restoring
function to p53 cancer mutants and identifying proteins that play important
roles in p53 biology, be it as a p53 inhibitor, a p53 coactivator, or a protein
that is regulated by p53.
2 THE p53 DISSOCIATOR ASSAY IN YEAST
2.1 The URA3 gene and counterselection in the yeast

Saccharomyces cerevisiae
The p53 dissociator assay is based on the principles of yeast experimental

systems developed by Fields and others that allow for the study of
macromolecular interactions by simple phenotypic readouts [22, 23, 30,
100]. Like these systems, the p53 dissociator assay uses a reporter gene,
URA3, activation of which enables yeast cells to grow on media lacking
uracil, thus allowing for the selection of a macromolecular interaction. But
Chapter 9: p53 Dissociator Assay 213
the dissociator assay has a significant difference to the other systems in that
it also allows for selection against a particular interaction. This is possible
because the URA3 gene product is involved in converting 5-fluoro-orotic
acid (Foa) into a toxic substance, thereby arresting cell growth [7]. This twist
has been exploited as the “reverse one- or two-hybrid system”, a powerful
tool that allows for the selection of novel factors, “dissociators”, able to
interfere with a macromolecular interaction [10, 95, 103].
The p53 dissociator assay consists of a tightly repressed reporter
construct, 1cUAS53::URA3, typically integrated into the yeast genome, in
which a p53 consensus DNA-binding site in the context of the artificially
introduced SPO13 promoter is placed upstream of the URA3 gene [11, 95].
p53 binds to its DNA-binding site and activates transcription of URA3. The
activity of wild-type p53 can be phenotypically scored in two ways: yeast
growth on plates without Uracil (Ura+) and lack of growth on Foa-containing
plates, i.e., Foa-sensitive (FoaS).
Figure 1. Principles of the p53 dissociator assay in yeast. The p53-dependent reporter gene
URA3 allows for selection against active p53, because URA3 expression results in conversion
of 5-fluoro-orotic acid (Foa) into a toxic substance, thus preventing yeast growth. The concept
of counterselection can be exploited for the identification of human proteins that reduce p53
activity in the yeast assay. Not only p53 inhibitors score as “dissociators”, but also p53
coactivators and proteins that are regulated by p53. This is likely the case because the human
proteins are overexpressed, often amino-terminally truncated (due to the process of the library
construction) and evaluated in an artificial yeast assay. Since the p53 dissociator assay relies
on native p53 and a consensus p53 DNA-binding site (contrary to, for example, two-hybrid
assays for p53), a variety of p53-inhibitory mechanisms can be envisioned: for example, a
“dissociator” (“?” in the figure) may conceal the p53 transactivation domain (A), but could
also achieve its effect by binding to the p53 DNA-binding site (B).
214 R. K. Brachmann
The FoaS phenotype of the p53 dissociator assay provides the essential
tool to perform cDNA expression library screens (“dissociator screens”).
Proteins that interfere with wild-type p53 activity will reduce URA3
expression and allow growth on Foa plates, i.e., Foa-resistant (FoaR). p53
dissociators might utilize a variety of mechanisms, such as binding to and
masking the transactivation domain of p53 (Figure 1A) or the actual p53
DNA-binding site (Figure 1B), p53 degradation, or posttranslational
modifications of p53. The p53 dissociator assay should clearly identify
proteins that negatively regulate p53. But proteins with quite different
functions in human cells might be isolated as well, since the yeast assay is
different from human cells in many ways. The library proteins are expressed
at higher levels than p53 and outside their usual context, thus potentially
lacking important regulators of their activity. For example, a p53 enhancer
may require a protein complex for its activity in human cells. Missing these
important cofactors in yeast, the p53 enhancer may now have the opposite
effect by binding to and sequestering p53. Additionally, due to the mode of
library construction, most of the cDNA expression plasmids encode for
amino-terminally truncated proteins that may behave in a dominant-negative
fashion because they lack, for example, a regulatory domain.
2.2 Established p53 inhibitors in the p53 dissociator

assay
A useful p53 dissociator assay requires that already known regulators of

p53 are able to change the yeast phenotype from FoaS to FoaR. The first
validation of the assay came from the protein that led to the identification of
p53, SV40 large T antigen. As predicted, this viral protein was able to
interfere with p53 activity in the p53 dissociator assay and resulted in Foa-
resistance [101].
The p53 dissociator assay can also recapitulate more complex situations,
such as the interplay of p53, E6-associated protein (E6-AP) and E6 of high-
risk human papilloma viruses (HPV). Exposure to high-risk HPV is linked to
cervical cancers, and the E6 protein of high-risk HPV has been shown to
induce p53 degradation. To this purpose, E6 recruits the human host factor
E6-AP, an E3 in the ubiquitin cascade that forms a direct thioester bond with
ubiquitin. The trimeric complex of p53, high-risk HPV E6, and E6-AP
results in the degradation of p53 via the ubiquitin cascade (Figure 2A) [20,
28, 70, 84, 96].
Expression of E6-AP and high-risk HPV E6 in the p53 dissociator assay
conferred the FoaR phenotype. Controls demonstrated that both high-risk
HPV E6 and E6-AP were required for this phenotype. The E6 protein from a
low-risk HPV, unable to cause cervical cancers or to induce p53 degradation,

was inactive in the p53 dissociator assay (Figure 2B). Western blot analysis
for p53 in the presence of high-risk HPV E6 and E6-AP showed decreased
p53 protein levels as compared to the controls suggesting that the two
proteins induced degradation of p53 also in yeast (Figure 2C). Experiments
with two E6-AP mutants with mutations in cysteine 833 (provided by
J. Huibregtse, Rutgers University, Piscataway, NJ) confirmed that the
thioester bond between E6-AP and ubiquitin was essential for the FoaR
phenotype in yeast (Figure 2D; Brachmann and Boeke, unpublished data).
Similar results were obtained with an assay in Schizosaccharomyces pombe
that utilized the principle of p53 counterselection through the p53-dependent
ura4+ reporter gene [99].
2.3 p53 dissociator screens to identify proteins

important for p53 biology
In order to perform large-scale screens, further optimization of the assay

to reduce the number of false-positive clones was required. False-positives
were mainly due to recessive mutations in the URA3 reporter gene and to a
lesser extent in the p53 cDNA. The use of a diploid reporter strain with two
copies each of the URA3 reporter gene and the p53 cDNA eliminated most
of this undesired background [11, 101].
Thus far, three dissociator screens have been performed. The discussed
modifications of the assay resulted in a powerful and very stringent selection
system: approximately 1 × 107 yeast transformants resulted in 600 FoaR
clones, and one third of those passed all confirmatory tests. Thus, only one
in 50,000 plasmids was able to confer an FoaR phenotype (Figure 3) [101].
Besides a large group of proteins without connection to p53 biology in
the literature, several established p53-interacting proteins were isolated. This
included the known inhibitor of p53, MDM2 [101]. High-risk HPV E6 was
also isolated when a screen was performed in the presence of E6-AP with a
cDNA expression library from HeLa cells, a cervical cancer cell line known
to express E6 and other HPV proteins (see Figure 2; Brachmann, and Boeke
unpublished data). Besides p53 inhibitors, the screens also isolated 53BP1, a
protein that physically interacts with the p53 core domain and that, in
mammalian assays, can enhance p53 activity [43, 44].
216 R. K. Brachmann
Figure 2. Recapitulating the biology of p53, E6-associated protein and high-risk human
papilloma virus (HPV) E6 in the p53 dissociator assay. Infection with high-risk HPV is
associated with more than 80% of human cervical cancers. (A) The E6 protein induces
ubiquitin-mediated degradation of p53 through recruitment of the host factor E6-associated
protein (E6-AP), an E3 in the ubiquitin cascade whose cysteine 833 forms a direct thioester
with ubiquitin (“~S-” in the figure). (B) The complex interplay between two human and one
viral protein(s) can be recreated in the p53 dissociator assay, since the combination of E6-AP
and high-risk HPV E6 results in an Foa resistant (FoaR) phenotype on plates containing
Foa (“+Foa0.1%”). The interference with p53 is highly specific, since low-risk E6 from
an HPV unable to cause cervical cancer does not confer Foa resistance. (C) The FoaR
phenotype corresponds to reduced p53 protein levels that are seen not only in galactose
media (high expression of E6-AP), but also in glucose media (minimal expression of E6-AP).
(D) E6-AP mutants in which cysteine 833 is changed to alanine or serine suggest that
thioester formation between E6-AP and ubiquitin is essential for inhibition of p53. In all
experiments (B–D), E6-AP proteins were expressed under the control of the GAL1 promoter
that is induced on galactose, “Gal”, but not induced on glucose-containing media. E6 was
expressed under the control of the constitutive ADH1 promoter. The two plasmids containing
the marker genes LEU2 or TRP1 were selected on media lacking leucine and tryptophane
(Leu–Trp). The ADH1-p53 expression cassette was either integrated into the genome at the
LYS2 locus (B and D) or maintained on a plasmid with the HIS3 marker gene (C).
Figure 3. Summary of three p53 dissociator screens. The use of a diploid yeast reporter strain
with two copies each of the assay components (p53 expression cassette and p53-dependent
URA3 reporter gene) results in very stringent screens that allow only 1 in 50,000 plasmids to
score with an FoaR phenotype in the p53 dissociator assay.
2.4 The example of hADA3 as a p53 dissociator
hADA3 was one of the first proteins to be isolated in the HeLa p53
dissociator screen. At the time, this human homologue of the yeast protein
Ada3p had just been described as a part of histone acetyltransferase (HAT)
complexes containing the human HATs hGCN5 or PCAF [76]. The yeast
gene ADA3 had previously been identified because its inactivation protected
Saccharomyces cerevisiae from the toxic effects of an overexpressed
artificial transcription factor, the GAL4–VP16 fusion protein [78]. Sub-
sequent work established that yeast Ada3p physically interacts with Ada2p
and the yeast HAT Gcn5p, forming HAT complexes termed Ada or SAGA
(if additional proteins are present) [29]. These HAT complexes acetylate
histones thereby relaxing tightly packed chromatin and giving transcription
factors access to promoters. They also appear to regulate other proteins by
acetylating them. Both Ada2p and Ada3p interact with the transactivation
domains of transcription factors, and the whole Ada complex is required for
transcriptional activity of many transcription factors [29, 60, 89, 90].
218 R. K. Brachmann
Interestingly, prior to any studies of hADA3, work by Candau and

colleagues had already suggested that Ada proteins are important for trans-
criptional activity of p53 in yeast [17]. They took advantage of the yeast Ada
complex to further dissect the transactivation domain of p53. In this context,
they showed that gene knockouts of ada2, ada3, and gcn5 resulted in loss of
transcriptional activity of p53 in yeast. However, it was not further explored
why the Ada complex was required for transcriptional activity of p53.
The characterization of full-length hADA3 in human cells by us
indicated that hADA3, very much like its yeast counterpart, serves as an
adaptor or docking protein that allows transcription factors to communicate
with HAT complexes. In the case of p53, the interaction with hADA3
appears to be very tightly regulated. Although a basal interaction between
p53 and hADA3 can be detected, p53 that is activated by upstream stress
signals shows a dramatically enhanced interaction with p53. This enhanced
interaction depends on amino-terminal phosphorylation of p53. Carboxy-
terminally truncated hADA3 acts as a dominant-negative protein and
abrogates p53 transcriptional activity and p53-mediated apoptosis. Not only
does hADA3 interact with activated p53, but it is also required for p53
activity following stress stimuli, since absence of hADA3 (through antisense
approaches) results in significantly reduced p53 transcriptional activity
following DNA damage [101].
Thus, the p53 dissociator assay identified a new coactivator of p53 whose
further characterization suggested novel mechanisms of blunting the p53
pathway. For instance, mutations that truncate hADA3 may result in
dominant-negative proteins that prevent access of p53 to HAT complexes
and, thus, full activation of p53. This hypothesis is readily testable by
screening human cancer specimens for hADA3 mutations and aberrant
hADA3 protein sizes. A knockout mouse model for ADA3 will be another
important strategy to assess the significance of ADA3 for the p53 pathway.
It was recently shown that high-risk HPV E6 not only targets p53 for
degradation, but also hADA3 [50], suggesting that removal of hADA3 is an
important step in HPV-mediated carcinogenesis.
2.5 p53 dissociator screens to characterize specific p53

inactivations in cancers
Considering the central role of p53 and its frequent inactivation through
p53 gene mutations, it is very likely that the remaining human cancers with
wild-type p53 must significantly reduce the activity of the p53 pathway. But
compared to the detailed knowledge regarding p53 mutations, much less is
known about the p53 status of the remaining 50% of human cancers with no
p53 mutations.
One obvious way of eliminating the p53 pathway would be to abrogate
p53 gene expression. This is indeed the case for a subset of breast cancers.
Raman and colleagues determined that the transcription factor HOXA5 is
important for expression of the p53 gene [81]. In breast cancers with loss of
p53 expression, the promoter of the HOXA5 gene was frequently methylated
resulting in lack of HoxA5 expression and HOXA5 protein. It is currently
unknown whether this mechanism is found in other cancers as well.
A second efficient mode to eliminate the p53 pathway is direct
inactivation of the p53 protein. There are two well-documented examples: a
large fraction of sarcomas, as well as a small percentage of other human
cancers show overexpression of MDM2, thus turning the physiological
negative feedback loop for p53 into a tool to rid the cell of p53 activity [20,
28, 56, 59, 68, 96]. Also, high-risk HPV cause more than 80% of cervical
cancers, and the HPV E6 protein is responsible for inducing the degradation
of p53 [20, 28, 70, 84, 96]. Firm numbers are not available, but it is unlikely
that the majority of human cancers with wild-type p53 utilize inactivation of
p53 by high-risk HPV E6 or MDM2 overexpression.
There are two additional ways of inactivating wild-type p53 protein
whose underlying molecular mechanisms have not yet been fully elucidated.
A variety of human cancers show complete or partial mislocalization of p53
to the cytoplasm. This has been described for primary tissues and/or cell
lines of breast cancers, colon cancers, neuroblastomas, malignant, melanomas
and retinoblastomas [8, 9, 65, 67, 85, 91, 102], and impairs p53’s ability to
induce G1 arrest and cause growth inhibition [48, 66]. Several underlying
genetic alterations can be envisioned, including loss of factors required for
active import of p53 into the nucleus (Figure 4A, I) and overexpression or
constitutive activation of factors that actively hold p53 in the cytoplasm
(Figure 4A, II). The latter model is supported by indirect evidence from
several studies that suggest that an unidentified short-lived anchor protein
might tether p53 to the vimentin scaffold [26, 47, 48] and the identification
of a cytoplasmic anchor protein, Parc, for p53 [45, 74]. Stommel and col-
leagues provided strong support for a third possibility, inappropriate nuclear
export of p53 [88]. They showed that disruption of p53 homo-tetramers
results in exposure of a nuclear export signal in the carboxy-terminus.
Inappropriate activity of the nuclear factor(s) involved in this mechanism
could thus result in excessive nuclear export of p53 (Figure 4A, III).
220 R. K. Brachmann
Figure 4. p53 dissociator screens to elucidate specific modes of wild-type p53 inactivation in
human cancers. (A) A significant percentage of human cancers carry wild-type p53 that is
mislocalized to the cytoplasm, thus rendering it inactive. Several mechanisms may underlie
this finding. I. p53 may be imported into the nucleus inefficiently. II. p53 may be actively
held in the cytoplasm. III. p53 may be excessively exported from the nucleus because of
inappropriate exposure of a normally hidden nuclear export signal (NES) in the region of the
tetramerization domain. (B) Testicular cancers carry wild-type p53 that is stabilized and
transcriptionally inactive, suggesting the presence of a p53-inhibitory protein in the nuclei of
these cancers. Targeted p53 dissociator screens with cDNA expression libraries representative
of these p53 inactivations should be able to decipher the underlying molecular mechanisms.
Testicular cancers have a distinctly different phenotype of p53 inacti-

vation. They typically have elevated p53 protein levels, yet the p53 genes do
not show mutations [69]. Based on work with murine teratocarcinoma cell
lines [61], wild-type p53 in this type of cancer is not transcriptionally active.
But the inactivation can be overcome through DNA damage or induction of
differentiation with retinoic acid. These findings are highly suggestive of the
presence of a protein that sequesters and functionally inactivates p53 in the
nucleus (Figure 4B).
The p53 dissociator assay provides an excellent tool to perform targeted

dissociator screens with cDNA expression libraries of cell lines that reflect
the two specific inactivations of p53.
3 INTRAGENIC SUPPRESSOR MUTATIONS

AND THE QUEST TO ENDOW FUNCTION
TO p53 CANCER MUTANTS
3.1 Strategies to restore p53 function to human cancers
Because of its central role in tumor suppression, p53 and its pathways
have been recognized as a prime target for developing new cancer therapies
[13, 24, 27, 35, 40, 46, 58, 64], and various strategies have been pursued.
Wild-type p53 may be delivered through gene therapy approaches regardless
of the p53 status of the cancer [3, 19, 71, 83, 92, 105]. Cancer cells lacking
p53 function can be specifically targeted with mutant adenovirus [6, 63] or
adeno-associated virus [80]. Inactive wild-type p53 and some p53 cancer
mutants may be reactivated through targeting of the very carboxy-terminal
putative autoregulatory domain of p53 with antibodies [1, 33, 41, 72] or
peptides spanning part of this region [2, 42, 86]. If MDM2 overexpression is
responsible for p53 inactivation, the inappropriate interaction between
MDM2 and p53 may be disrupted by peptides and compounds [21, 40, 87].
The pursuit of this strategy has resulted in very potent small-molecule
inhibitors of MDM2 [54, 94]. Yet, all these strategies have their limitations,
such as lack of efficient local and/or systemic delivery, undesirable res-
ponses of the host immune system to the therapeutic agent, lack of
specificity for the cancer cell and/or targeting of a small subset of human
cancers.
Besides these strategies, one approach appears particularly appealing:
small chemical compounds that endow p53 cancer mutants with function by
restoring the integrity of the p53 core domain. Such compounds are highly
desirable, because of the high frequency of p53 mutations and the large
number of patients who could potentially benefit. It has been estimated that
every year approximately 360,000 patients in the USA and 2.6 million
patients worldwide are diagnosed with cancers that contain p53 mutations
[34, 35]. This subset of human cancers (including lung, prostate, colorectal,
breast, head and neck, pancreatic, and gastric cancers) is often resistant to
conventional therapies and difficult to treat at advanced stages [5, 18, 24, 31,
35, 38, 58].
222 R. K. Brachmann
The strategy is, at least in theory, possible because of the unique pattern
of p53 missense mutations in human cancers. Typically, p53 cancer mutants
are full-length proteins with an intact transactivation domain and carboxy-
terminal tetramerization domain. The problem is the altered core domain
(amino acids 96–292) whose structural integrity needs to be restored in order
to have functional p53. Compounds able to achieve this goal are predicted to
be specific for the cancer cells, since the structurally intact wild-type p53
core domain of surrounding normal cells should not be affected. Further-
more, p53 cancer mutants are typically present at very high levels (thus
providing a large drug target), since their lack of transcriptional activity
abrogates negative feedback loops, such as with MDM2 [55]. The approach
also has the advantage of combining systemic delivery with lack of a host
immune response.
The attractive features of this strategy are easily rivaled by the enormous
challenges it poses: such compounds may simply not exist, or, if they do,
they may rescue only a very small fraction of the close to 1,000 different
known p53 cancer mutants that have one amino acid change in the core
domain. On a more optimistic note, looking at missense mutations of the p53
core domain (71% of the database entries; R9, http://www.iarc.fr/p53/), the
50 most frequent amino acid changes account for 55% of the total number,
and of these 50, the 8 most common amino acid changes comprise the
majority of them (30% of the total). Thus, chemical compounds that rescue a
subset of these p53 mutants are likely to correspond to a large number of
cancer patients.
Also, as a proof of principle, two classes of compounds have already
been identified in large drug screens that appear to have the desired
characteristics. The first screen utilized an antibody-based screening strategy
that relied on partially unfolded wild-type p53 due to elevated temperatures.
The isolated compounds appeared to partially restore function to a few p53
cancer mutants [25]. However, further characterization of the lead
compound CP-31398 has thus far not established the exact mechanism of
rescue. In addition, CP-31398 appears to have strong p53-independent
activities [13, 16, 82, 93].
The second class of compound was isolated using a mammalian growth
suppression assay [16]. Very impressively, this compound rescued the most
common p53 cancer mutant R175H whose core domain is very likely
entirely denatured [13–16, 106]. It still remains to be determined how many
others of the most common p53 cancer mutants will be rescued by this
compound and what the exact mechanism of rescue is.
3.2 Intragenic suppressor mutations for p53 cancer

mutants
The p53 yeast dissociator assay has allowed a broader look at the
challenge of rescuing p53 cancer mutants; it has been used for identifying
intragenic suppressor mutations that overcome the deleterious effects of
cancer mutations. Such suppressor mutations are highly informative, as they
identify key regions within the p53 core domain that, upon alteration,
provide increased stability to the core domain. Such suppressor mutations
have also been pursued by computer modeling, but this strategy generated
only one successful suppressor mutation and several false predictions [104].
Additionally, “suppressor mutation design” was not as wide in scope as
genetic screens in yeast.
Figure 5. Search for intragenic suppressor mutations that endow function to common p53
cancer mutants. (A) Starting with a common p53 cancer mutant (with mutation “C”) that is
transcriptionally inactive (Ura– phenotype), (B) the up- or downstream regions of the cDNA
are mutagenized using PCR mutagenesis. (C) In a separate reaction, the cDNA expression
plasmid is gapped in the same region. (D) Both products are transformed into yeast, and the
overlap between PCR product and gapped plasmid allows the yeast to repair the plasmid by
homologous recombination. After confirmatory experiments, the plasmid is eventually
sequenced to identify the suppressor mutation(s) (“S”).
Screens for suppressor mutations in the p53 dissociator assay take

advantage of PCR mutagenesis and gap repair in yeast through homologous
recombination. Starting with a p53 cancer mutant that is Ura– (Figure 5A),
the regions up- and downstream of the cDNA are PCR-amplified under
mutagenic conditions (Figure 5B). The expression plasmid for the p53
224 R. K. Brachmann
cancer mutant is gapped, so as to create an overlap with the PCR products

(Figure 5C). PCR products and gapped plasmids are then transformed into a
yeast reporter strain that repairs the plasmid by homologous recombination
utilizing a PCR product (Figure 5D).
A pilot study identified several suppressor mutations for three cancer
mutations: N268D for V143A, T123P, N239Y and S240N for G245S and
T123A in combination with H168R for R249S [12]. The suppressor
mechanism for H168R appeared to be very specific to R249S [12, 75], but
two other isolated suppressor mutations, N268D and N239Y, increased the
overall stability of two cancer mutants, G245S and V143A. This suggested
that, in principle, small molecules should be able to bind to and stabilize the
core domain of such p53 cancer mutants in a similar manner [75].
3.3 Identification of a global suppressor motif for p53

cancer mutants
The initial findings with the p53 dissociator assay were very promising,
yet several technical limitations needed to be overcome to allow for a
systematic analysis of the most common p53 cancer mutants. To this end, an
engineered open reading frame for p53 was constructed [39] that contained
numerous silent restriction sites [4].
Analysis for the eight most common p53 cancer mutants (30% of core
domain missense mutants) identified suppressor mutations for four of them
(G245S, R249S, R273C, and R273H; 12% of core domain missense
mutants). Several combinations of suppressor mutations were identified,
and, intriguingly, N239Y or S240R were involved in the rescue of all four
p53 mutants [4]. This suggested that amino acids 239 and 240 may be part of
a global suppressor motif, a hypothesis that was further pursued by
oligonucleotide-mediated mutagenesis of a small region of the core domain,
codons 225–241. The mutagenesis included directed changes of codons 239
and 240 to all other 19 possible amino acids plus a background mutagenesis
resulting in approximately 1 in 100 nucleotide misincorporations. This stra-
tegy resulted in the rescue of 16 out of 30 of the most common p53 mutants
tested, representing 18% of all reported p53 core domain missense mutants.
As expected, the suppressor mutations included changes of codons 239
and/or 240, but, in addition, changes of amino acid 235 to lysine were also
frequently found. The resulting p53 mutants were not only transcriptionally
active in yeast, but had also activity in mammalian reporter gene and
apoptosis assays [4].
These findings strongly argue that manipulation of a small region of the
p53 core domain is sufficient to endow p53 cancer mutants with function.
This region therefore comprises a global suppressor motif. Structural studies
of this motif will determine the exact rescue mechanism and provide the
basis to exploit this motif further by designing small compounds able to
recreate the suppressor mechanism (Figure 6). By restoring function to p53
cancer mutants, these small molecules would, in effect, reactivate a major
apoptotic pathway and “relicense” the cancer cell to kill itself.
Figure 6. Strategy for the development of a new anticancer therapy that aims to endow p53
cancer mutants with wild-type function. (A) A pilot study established that the concept of and
search for intragenic suppressor mutations was a viable strategy to achieve a better
understanding of how mutant p53 core domains can be stabilized. (B) The subsequent
comprehensive screen using a new engineered p53 open reading frame identified amino acids
235, 239, and 240 a part of an important global suppressor motif that rescued 16 of the 30
most common p53 cancer mutants. (C) Crystal structures for p53 cancer mutants and p53
suppressor/cancer mutants will establish the underlying mechanism that allows the 235–239–
240 suppressor motif to rescue p53 cancer mutants. (D) This information will lay the
foundation to pursue the most challenging aspect of the project: design, identify, and
characterize small compounds able to recreate the rescue mechanism of the suppressor motif.
This step requires a multidisciplinary approach that combines the expertise of computer
scientists, chemists, structural biologists, and molecular biologists.
4 CONCLUSIONS
Baker’s yeast or S. cerevisiae is a highly valuable research tool for

studies of the human tumor suppressor protein and transcription factor p53.
Reporter gene assays for p53 in yeast have made important contributions to
our understanding of this central tumor suppressor protein, and the chapter
by Inga and colleagues provides an excellent overview on these efforts. This
chapter highlighted the specific contributions of the p53 dissociator assay.
The hunt for intragenic suppressor mutations illustrates that, similar to other
p53 yeast assays, it is able to efficiently analyze and select for p53 activity.
But the p53 dissociator assay also provides additional genetic strategies in
226 R. K. Brachmann
yeast, as it exploits the concept of selection against p53 activity, a twist that
allows for powerful screens for proteins important to p53 biology.
As the biology of p53 is bound to become more and more complex, p53
yeast assays will continue to contribute to our understanding of this crucial
tumor suppressor protein. The focus of this review was p53, but many of the
discussed concepts can be readily applied to the studies of other fundamental
cellular mechanisms. Undoubtedly, many human proteins, particularly
transcription factors, are just waiting to be plugged into a yeast assay of their
own.
ACKNOWLEDGMENTS
R.K.B. thanks Carrie Baker Brachmann for critically reading the

manuscript and Jef D. Boeke whose vision and support were instrumental for
the development of the p53 dissociator assay.
REFERENCES
1. Abarzua, P., J. E. LoSardo, M. L. Gubler, and A. Neri. 1995. Microinjection of
monoclonal antibody PAb421 into human SW480 colorectal carcinoma cells restores the
transcription activation function to mutant p53. Cancer Res. 55:3490–3494.
2. Abarzua, P., J. E. LoSardo, M. L. Gubler, R. Spathis, Y. A. Lu, A. Felix, and
A. Neri. 1996. Restoration of the transcription activation function to mutant p53 in
human cancer cells. Oncogene 13:2477–2482.
3. Barinaga, M. 1997. From bench top to bedside. Science 278:1036–1039.
4. Baroni, T. E., T. Wang, H. Qian, L. R. Dearth, L. N. Truong, J. Zeng, A. E. Denes,
S. W. Chen, and R. K. Brachmann. 2004. A global suppressor motif for p53 cancer
mutants. Proc. Natl. Acad. Sci. USA in press.
5. Beroud, C., and T. Soussi. 1998. p53 gene mutation: software and database. Nucleic
Acids Res. 26:200–204.
6. Bischoff, J. R., D. H. Kirn, A. Williams, C. Heise, S. Horn, M. Muna, L. Ng, J. A.
Nye, A. Sampson-Johannes, A. Fattaey, and F. McCormick. 1996. An adenovirus
mutant that replicates selectively in p53-deficient human tumor cells. Science 274:373–
376.
7. Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants
lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid
resistance. Mol. Gen. Genet. 197:345–346.
8. Bosari, S., G. Viale, P. Bossi, M. Maggioni, G. Coggi, J. J. Murray, and A. K. Lee.
1994. Cytoplasmic accumulation of p53 protein: an independent prognostic indicator in
colorectal adenocarcinomas. J. Natl. Cancer Inst. 86:681–687.
9. Bosari, S., G. Viale, M. Roncalli, D. Graziani, G. Borsani, A. K. Lee, and G. Coggi.
1995. p53 gene mutations, p53 protein accumulation and compartmentalization in
colorectal adenocarcinoma. Am. J. Pathol. 147:790–798.
10. Brachmann, R. K., and J. D. Boeke. 1997. Tag games in yeast: the two-hybrid system
and beyond. Curr. Opin. Biotechnol. 8:561–568.
11. Brachmann, R. K., M. Vidal, and J. D. Boeke. 1996. Dominant-negative p53 muta-
tions selected in yeast hit cancer hot spots. Proc. Natl. Acad. Sci. USA 93:4091–4095.
12. Brachmann, R. K., K. Yu, Y. Eby, N. P. Pavletich, and J. D. Boeke. 1998. Genetic
selection of intragenic suppressor mutations that reverse the effect of common p53
cancer mutations. EMBO J. 17:1847–1859.
13. Bullock, A. N., and A. R. Fersht. 2001. Rescuing the function of mutant p53. Nat. Rev.
Cancer 1:68–76.
14. Bullock, A. N., J. Henckel, B. S. DeDecker, C. M. Johnson, P. V. Nikolova, M. R.
Proctor, D. P. Lane, and A. R. Fersht. 1997. Thermodynamic stability of wild-type
and mutant p53 core domain. Proc. Natl. Acad. Sci. USA 94:14338–4342.
15. Bullock, A. N., J. Henckel, and A. R. Fersht. 2000. Quantitative analysis of residual
folding and DNA binding in mutant p53 core domain: definition of mutant states for
rescue in cancer therapy. Oncogene 19:1245–1256.
16. Bykov, V. J. N., N. Issaeva, A. Shilov, M. Hultcrantz, E. Pugacheva, P. Chumakov,
J. Bergman, K. G. Wiman, and G. Selivanova. 2002. Restoration of the tumor
suppressor function to mutant p53 by a low-molecular-weight compound. Nat. Med.
8:282–288.
17. Candau, R., D. M. Scolnick, P. Darpino, C. Y. Ying, T. D. Halazonetis, and S. L.
Berger. 1997. Two tandem and independent sub-activation domains in the amino
terminus of p53 require the adaptor complex for activity. Oncogene 15:807–816.
18. Cariello, N. F., G. R. Douglas, N. J. Gorelick, D. W. Hart, J. D. Wilson, and
T. Soussi. 1998. Databases and software for the analysis of mutations in the human p53
gene, human hprt gene and both the lacI and lacZ gene in transgenic rodents. Nucleic
Acids Res. 26:198–199.
19. Clayman, G. L., A. K. el-Naggar, S. M. Lippman, Y. C. Henderson, M. Frederick,
J. A. Merritt, L. A. Zumstein, T. M. Timmons, T. J. Liu, L. Ginsberg, J. A. Roth,
W. K. Hong, P. Bruso, and H. Goepfert. 1998. Adenovirus-mediated p53 gene
transfer in patients with advanced recurrent head and neck squamous cell carcinoma. J.
Clin. Oncol. 16:2221–2232.
20. Donehower, L. A., and A. Bradley. 1993. The tumor suppressor p53. Biochim.
Biophys. Acta 1155:181–205.
21. Duncan, S. J., S. Gruschow, D. H. Williams, C. McNicholas, R. Purewal, M. Hajek,
M. Gerlitz, S. Martin, S. K. Wrigley, and M. Moore. 2001. Isolation and structure
elucidation of chlorofusin, a novel p53-MDM2 antagonist from a Fusarium sp. J. Am.
Chem. Soc. 123:554–560.
22. Durfee, T., K. Becherer, P. L. Chen, S. H. Yeh, Y. Yang, A. E. Kilburn, W. H. Lee,
and S. J. Elledge. 1993. The retinoblastoma protein associates with the protein
phosphatase type 1 catalytic subunit. Genes Dev. 7:555–569.
23. Fields, S., and O. Song. 1989. A novel genetic system to detect protein-protein
interactions. Nature 340:245–246.
24. Fisher, D. E. 1994. Apoptosis in cancer therapy: crossing the threshold. Cell 78:539–
542.
25. Foster, B. A., H. A. Coffey, M. J. Morin, and F. Rastinejad. 1999. Pharmacological
rescue of mutant p53 conformation and function. Science 286:2507–2510.
26. Gannon, J. V., and D. P. Lane. 1991. Protein synthesis required to anchor a mutant
p53 protein which is temperature-sensitive for nuclear transport. Nature 349:802–806.
228 R. K. Brachmann
27. Gibbs, J. B., and A. Oliff. 1994. Pharmaceutical research in molecular oncology. Cell
79:193–198.
28. Gottlieb, T. M., and M. Oren. 1996. p53 in growth control and neoplasia. Biochim.
29. Grant, P. A., D. E. Sterner, L. J. Duggan, J. L. Workman, and S. L. Berger. 1998.
The SAGA unfolds: convergence of transcription regulators in chromatin-modifying
complexes. Trends Cell Biol. 8:193–197.
30. Gyuris, J., E. Golemis, H. Chertkov, and R. Brent. 1993. Cdi1, a human G1 and S
phase protein phosphatase that associates with Cdk2. Cell 75:791–803.
31. Hainaut, P., T. Hernandez, A. Robinson, P. Rodriguez-Tome, T. Flores,
M. Hollstein, C. C. Harris, and R. Montesano. 1998. IARC database of p53 gene
mutations in human tumors and cell lines: updated compilation, revised formats and new
visualisation tools. Nucleic Acids Res. 26:205–213.
32. Hainaut, P., and M. Hollstein. 2000. p53 and human cancer: the first ten thousand
mutations. Adv. Cancer Res. 77:81–137.
33. Halazonetis, T. D., and A. N. Kandil. 1993. Conformational shifts propagate from the
oligomerization domain of p53 to its tetrameric DNA binding domain and restore DNA
binding to select p53 mutants. EMBO J. 12:5057–5064.
34. Harris, C. C. 1996. p53 tumor suppressor gene: from the basic research laboratory to
the clinic – an abridged historical perspective. Carcinogenesis 17:1187–1198.
35. Harris, C. C. 1996. Structure and function of the p53 tumor suppressor gene: clues for
rational cancer therapeutic strategies. J. Natl. Cancer Inst. 88:1442–1454.
36. Hernandez-Boussard, T., R. Montesano, and P. Hainaut. 1999. Sources of bias in the
detection and reporting of p53 mutations in human cancer: analysis of the IARC p53
mutation database. Genet. Anal. 14:229–233.
37. Hernandez-Boussard, T., P. Rodriguez-Tome, R. Montesano, and P. Hainaut. 1999.
IARC p53 mutation database: a relational database to compile and analyze p53
mutations in human tumors and cell lines. International Agency for Research on Cancer.
Hum. Mutat. 14:1–8.
38. Hollstein, M., D. Sidransky, B. Vogelstein, and C. C. Harris. 1991. p53 mutations in
human cancers. Science 253:49–53.
39. Holowachuk, E. W., and M. S. Ruhoff. 1995. Efficient gene synthesis by Klenow
assembly/extension-Pfu polymerase amplification (KAPPA) of overlapping oligonu-
cleotides. PCR Methods Appl. 4:299–302.
40. Hupp, T. R., D. P. Lane, and K. L. Ball. 2000. Strategies for manipulating the p53
pathway in the treatment of human cancer. Biochem. J. 352 Pt 1:1–17.
41. Hupp, T. R., D. W. Meek, C. A. Midgley, and D. P. Lane. 1993. Activation of the
cryptic DNA binding function of mutant forms of p53. Nucleic Acids Res. 21:3167–3174.
42. Hupp, T. R., A. Sparks, and D. P. Lane. 1995. Small peptides activate the latent
sequence-specific DNA binding function of p53. Cell 83:237–245.
43. Iwabuchi, K., P. L. Bartel, B. Li, R. Marraccino, and S. Fields. 1994. Two cellular
proteins that bind to wild-type but not mutant p53. Proc. Natl. Acad. Sci. USA 91:6098–
6102.
44. Iwabuchi, K., B. Li, H. F. Massa, B. J. Trask, T. Date, and S. Fields. 1998.
Stimulation of p53-mediated transcriptional activation by the p53- binding proteins,
53BP1 and 53BP2. J. Biol. Chem. 273:26061–26068.
45. Kastan, M. B., and G. P. Zambetti. 2003. Parc-ing p53 in the cytoplasm. Cell 112:
1–2.
46. Kinzler, K. W., and B. Vogelstein. 1994. Cancer therapy meets p53. N. Engl. J. Med.
331:49–50.
47. Klotzsche, O., D. Etzrodt, H. Hohenberg, W. Bohn, and W. Deppert. 1998.
Cytoplasmic retention of mutant tsp53 is dependent on an intermediate filament protein
(vimentin) scaffold. Oncogene 16:3423–3434.
48. Knippschild, U., M. Oren, and W. Deppert. 1996. Abrogation of wild-type p53
mediated growth-inhibition by nuclear exclusion. Oncogene 12:1755–1765.
49. Ko, L. J., and C. Prives. 1996. p53: puzzle and paradigm. Genes Dev. 10:1054–1072.
50. Kumar, A., Y. Zhao, G. Meng, M. Zeng, S. Srinivasan, L. M. Delmolino, Q. Gao,
G. Dimri, G. F. Weber, D. E. Wazer, H. Band, and V. Band. 2002. Human
papillomavirus oncoprotein E6 inactivates the transcriptional coactivator human ADA3.
Mol. Cell. Biol. 22:5801–5812.
51. Lane, D. P. 1992. p53, guardian of the genome. Nature 358:15–16.
52. Lane, D. P., and S. Benchimol. 1990. p53: oncogene or anti-oncogene? Genes Dev.
4:1–8.
53. Lane, D. P., and L. V. Crawford. 1979. T antigen is bound to a host protein in SV40-
transformed cells. Nature 278:261–263.
54. Lane, D. P., and P. M. Fischer. 2004. Turning the key on p53. Nature 427:789–790.
55. Lane, D. P., and P. A. Hall. 1997. MDM2 – arbiter of p53’s destruction. Trends
Biochem. Sci. 22:372–4.
56. Levine, A. J. 1997. p53, the cellular gatekeeper for growth and division. Cell 88:323–
331.
57. Linzer, D. I., and A. J. Levine. 1979. Characterization of a 54K dalton cellular SV40
tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma
cells. Cell 17:43–52.
58. Lowe, S. W. 1995. Cancer therapy and p53. Curr. Opin. Oncol. 7:547–553.
59. Lozano, G., and R. Montes de Oca Luna. 1998. MDM2 function. Biochim. Biophys.
Acta 1377:M55–59.
60. Luo, R. X., and D. C. Dean. 1999. Chromatin remodeling and transcriptional
regulation. J. Natl. Cancer Inst. 91:1288–1294.
61. Lutzker, S. G., and A. J. Levine. 1996. A functionally inactive p53 protein in
teratocarcinoma cells is activated by either DNA damage or cellular differentiation. Nat.
Med. 2:804–810.
62. Martin, A. C., A. M. Facchiano, A. L. Cuff, T. Hernandez-Boussard, M. Olivier,
P. Hainaut, and J. M. Thornton. 2002. Integrating mutation data and structural
analysis of the TP53 tumor-suppressor protein. Hum. Mutat. 19:149–164.
63. McCormick, F. 1999. Cancer therapy based on p53. Cancer J. Sci. Am. 5:139–144.
64. Milner, J. 1995. DNA damage, p53 and anticancer therapies. Nat. Med. 1:879–880.
65. Moll, U. M., M. LaQuaglia, J. Benard, and G. Riou. 1995. Wild-type p53 protein
undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in
differentiated tumors. Proc. Natl. Acad. Sci. USA 92:4407–4411.
66. Moll, U. M., A. G. Ostermeyer, R. Haladay, B. Winkfield, M. Frazier, and
G. Zambetti. 1996. Cytoplasmic sequestration of wild-type p53 protein impairs the G1
checkpoint after DNA damage. Mol. Cell Biol. 16:1126–1137.
67. Moll, U. M., G. Riou, and A. J. Levine. 1992. Two distinct mechanisms alter p53
in breast cancer: mutation and nuclear exclusion. Proc. Natl. Acad. Sci. USA 89:
7262–7266.
68. Momand, J., and G. P. Zambetti. 1997. Mdm-2: “big brother” of p53. J. Cell
Biochem. 64:343–352.
230 R. K. Brachmann
69. Murty, V. V., and R. S. Chaganti. 1998. A genetic perspective of male germ cell
tumors. Semin. Oncol. 25:133–144.
70. Neil, J. C., E. R. Cameron, and E. W. Baxter. 1997. p53 and tumour viruses: catching
the guardian off-guard. Trends Microbiol. 5:115–120.
71. Nielsen, L. L., and D. C. Maneval. 1998. P53 tumor suppressor gene therapy for
cancer. Cancer Gene. Ther. 5:52–63.
72. Niewolik, D., B. Vojtesek, and J. Kovarik. 1995. p53 derived from human tumour cell
lines and containing distinct point mutations can be activated to bind its consensus target
sequence. Oncogene 10:881–890.
73. Nigro, J. M., S. J. Baker, A. C. Preisinger, J. M. Jessup, R. Hostetter, K. Cleary,
S. H. Bigner, N. Davidson, S. Baylin, P. Devilee, T. Glover, F. S. Collins, A. Weslon,
R. Modali, C. C. Harris, and B. Vogelstein. 1989. Mutations in the p53 gene occur in
diverse human tumour types. Nature 342:705–708.
74. Nikolaev, A. Y., M. Li, N. Puskas, J. Qin, and W. Gu. 2003. Parc: a cytoplasmic
anchor for p53. Cell 112:29–40.
75. Nikolova, P. V., K. B. Wong, B. DeDecker, J. Henckel, and A. R. Fersht. 2000.
Mechanism of rescue of common p53 cancer mutations by second-site suppressor
mutations. EMBO J. 19:370–378.
76. Ogryzko, V. V., T. Kotani, X. Zhang, R. L. Schlitz, T. Howard, X. J. Yang, B. H.
Howard, J. Qin, and Y. Nakatani. 1998. Histone-like TAFs within the PCAF histone
acetylase complex. Cell 94:35–44.
77. Olivier, M., R. Eeles, M. Hollstein, M. A. Khan, C. C. Harris, and P. Hainaut. 2002.
The IARC TP53 database: new online mutation analysis and recommendations to users.
Hum. Mutat. 19:607–614.
78. Pina, B., S. Berger, G. A. Marcus, N. Silverman, J. Agapite, and L. Guarente. 1993.
ADA3: a gene, identified by resistance to GAL4-VP16, with properties similar to and
different from those of ADA2. Mol. Cell. Biol. 13:5981–5989.
79. Prives, C., and P. A. Hall. 1999. The p53 pathway. J. Pathol. 187:112–126.
80. Raj, K., P. Ogston, and P. Beard. 2001. Virus-mediated killing of cells that lack p53
activity. Nature 412:914–917.
81. Raman, V., S. A. Martensen, D. Reisman, E. Evron, W. F. Odenwald, E. Jaffee,
J. Marks, and S. Sukumar. 2000. Compromised HOXA5 function can limit p53
expression in human breast tumours. Nature 405:974–978.
82. Rippin, T. M., V. J. Bykov, S. M. Freund, G. Selivanova, K. G. Wiman, and A. R.
Fersht. 2002. Characterization of the p53-rescue drug CP-31398 in vitro and in living
cells. Oncogene 21:2119–2129.
83. Roth, J. A., D. Nguyen, D. D. Lawrence, B. L. Kemp, C. H. Carrasco, D. Z. Ferson,
W. K. Hong, R. Komaki, J. J. Lee, J. C. Nesbitt, K. M. Pisters, J. B. Putnam,
R. Schea, D. M. Shin, G. L. Walsh, M. M. Dolormente, C. I. Han, F. D. Martin,
N. Yen, K. Xu, L. C. Stephens, T. J. McDonnell, T. Mukhopadhyay, and D. Cai.
1996. Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung
cancer. Nat. Med. 2:985–991.
84. Scheffner, M., U. Nuber, and J. M. Huibregtse. 1995. Protein ubiquitination
involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373:81–83.
85. Schlamp, C. L., G. L. Poulsen, T. M. Nork, and R. W. Nickells. 1997. Nuclear
exclusion of wild-type p53 in immortalized human retinoblastoma cells. J. Natl. Cancer
Inst. 89:1530–1536.
86. Selivanova, G., V. Iotsova, I. Okan, M. Fritsche, M. Strom, B. Groner, R. C.
Grafstrom, and K. G. Wiman. 1997. Restoration of the growth suppression function
of mutant p53 by a synthetic peptide derived from the p53 C-terminal domain. Nat.
Med. 3:632–638.
87. Stoll, R., C. Renner, S. Hansen, S. Palme, C. Klein, A. Belling, W. Zeslawski,
M. Kamionka, T. Rehm, P. Muhlhahn, R. Schumacher, F. Hesse, B. Kaluza,
W. Voelter, R. A. Engh, and T. A. Holak. 2001. Chalcone derivatives antagonize inter-
actions between the human oncoprotein MDM2 and p53. Biochemistry 40:336–344.
88. Stommel, J. M., N. D. Marchenko, G. S. Jimenez, U. M. Moll, T. J. Hope, and
G. M. Wahl. 1999. A leucine-rich nuclear export signal in the p53 tetramerization
domain: regulation of subcellular localization and p53 activity by NES masking. EMBO
J. 18:1660–1672.
89. Struhl, K. 1998. Histone acetylation and transcriptional regulatory mechanisms. Genes
Dev. 12:599–606.
90. Struhl, K., and Z. Moqtaderi. 1998. The TAFs in the HAT. Cell 94:1–4.
91. Sun, X. F., J. M. Carstensen, H. Zhang, O. Stal, S. Wingren, T. Hatschek, and
B. Nordenskjold. 1992. Prognostic significance of cytoplasmic p53 oncoprotein in
colorectal adenocarcinoma. Lancet 340:1369–1373.
92. Swisher, S. G., J. A. Roth, J. Nemunaitis, D. D. Lawrence, B. L. Kemp, C. H.
Carrasco, D. G. Connors, A. K. El-Naggar, F. Fossella, B. S. Glisson, W. K. Hong,
F. R. Khuri, J. M. Kurie, J. J. Lee, J. S. Lee, M. Mack, J. A. Merritt, D. M.
Nguyen, J. C. Nesbitt, R. Perez-Soler, K. M. Pisters, J. B. Putnam, Jr., W. R.
Richli, M. Savin, D. S. Schrump, D. M. Shin, A. Shylkin, G. L. Walsh, J. Wait,
D. Weill, and M. K. A. Waugh. 1999. Adenovirus-mediated p53 gene transfer in
advanced non-small-cell lung cancer. J. Natl. Cancer Inst. 91:763–771.
93. Takimoto, R., W. Wang, D. T. Dicker, F. Rastinejad, J. Lyssikatos, and W. S.
El-Deiry. 2002. The mutant p53-conformation modifying drug, CP-31398, can induce
apoptosis of human cancer cells and can stabilize wild-type p53 protein. Cancer Biol.
Therapy 1:in press.
94. Vassilev, L. T., B. T. Vu, B. Graves, D. Carvajal, F. Podlaski, Z. Filipovic, N. Kong,
U. Kammlott, C. Lukacs, C. Klein, N. Fotouhi, and E. A. Liu. 2004. In vivo
activation of the p53 pathway by small-molecule antagonists of MDM2. Science
303:844–848.
95. Vidal, M., R. K. Brachmann, A. Fattaey, E. Harlow, and J. D. Boeke. 1996. Reverse
two-hybrid and one-hybrid systems to detect dissociation of protein–protein and DNA–
protein interactions. Proc. Natl. Acad. Sci. USA 93:10315–10320.
96. Vogelstein, B., and K. W. Kinzler. 1992. p53 function and dysfunction. Cell 70:523–526.
97. Vogelstein, B., D. Lane, and A. J. Levine. 2000. Surfing the p53 network. Nature
408:307–310.
98. Vousden, K. H. 2000. p53: death star. Cell 103:691–694.
99. Waddell, S., and J. R. Jenkins. 1998. Defining the minimal requirements for papilloma
viral E6-mediated inhibition of human p53 activity in fission yeast. Oncogene 16:
1759–1765.
100. Wang, M. M., and R. R. Reed. 1993. Molecular cloning of the olfactory neuronal
transcription factor Olf-1 by genetic selection in yeast. Nature 364:121–126.
101. Wang, T., T. Kobayashi, R. Takimoto, A. E. Denes, E. L. Snyder, W. S. El-Deiry,
and R. K. Brachmann. 2001. hADA3 is required for p53 activity. EMBO J. 20:6404–
6413.
102. Weiss, J., M. Heine, B. Korner, H. Pilch, and E. G. Jung. 1995. Expression of p53
protein in malignant melanoma: clinicopathological and prognostic implications. Br.
J. Dermatol. 133:23–31.
232 R. K. Brachmann
103. White, M. A. 1996. The yeast two-hybrid system: forward and reverse. Proc. Natl.
Acad. Sci. USA 93:10001–10003.
104. Wieczorek, A. M., J. L. Waterman, M. J. Waterman, and T. D. Halazonetis. 1996.
Structure-based rescue of common tumor-derived p53 mutants. Nat. Med. 2:1143–1146.
105. Wolf, J. K., G. B. Mills, L. Bazzet, R. C. Bast, Jr., J. A. Roth, and D. M.
Gershenson. 1999. Adenovirus-mediated p53 growth inhibition of ovarian cancer cells
is independent of endogenous p53 status. Gynecol. Oncol. 75:261–266.
106. Wong, K. B., B. S. DeDecker, S. M. Freund, M. R. Proctor, M. Bycroft, and A. R.
Fersht. 1999. Hot-spot mutants of p53 core domain evince characteristic local structural
changes. Proc. Natl. Acad. Sci. USA 96:8438–8442.
Chapter 10
FUNCTIONAL ANALYSIS OF THE HUMAN p53
TUMOR SUPPRESSOR AND ITS MUTANTS
USING YEAST
Alberto Inga, Francesca Storici and Michael A. Resnick

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH,
P.O. Box 12233, Research Triangle Park, NC 27709
1 FUNCTIONAL ALTERATION OF p53:

A HALLMARK OF CANCER
Multiple perturbations of the complex network of signaling pathways that

define the precise role of a cell in its tissue microenvironment and that
regulate stress responses are accumulated through genetic and epigenetic
changes during processes of transformation that lead to cancer [129, 201]. In
particular, the acquisition of self-sufficiency in growth signal and the
resistance to growth inhibitory and apoptotic signals are likely to be required
for initiation and early progression [77, 174]. Limitless replicative potential,
associated with the selection of canonical or alternative ways of maintaining
the stability of chromosome ends, the ability to induce angiogenesis and to
acquire invasive potential are additional steps in the transformation process
that could lead to a tumor [77].
Stepwise and orderly accumulation of functional changes at proto-
oncogenes and tumor suppressors has been associated with different stages
of cancer in specific types of tissue [70, 129]. However, accumulating
evidence of the multiple levels of complex interactions between survival,
proliferation, and growth restraint pathways suggest that the underlying
genetic alterations may be codependent and specific combinations of
changes may be selected according to the microenvironment and the genetic/
epigenetic makeup of the transforming cell [212].
233
© 2007 Springer.
234 A. Inga, F. Storici and M. A. Resnick
The tumor suppressor gene p53 plays a major role in cellular response to
various environmental stresses [107, 114]. The p53 protein is a sequence-
specific transcription factor that can lead to transactivation of over 50 genes,
many of which are involved in apoptotic or cell cycle arrest responses
(Figure 1). Its role in biological responses may also relate to the ability to
interact with a vast number of proteins [164]. Functional alterations in the
p53 pathway likely occur in nearly all human cancers. In almost half the
human malignancies, there is a mutation in the p53 gene itself [71, 79].
Interestingly, ~80% of p53 mutations are missense changes that lead to
single amino acid substitutions, a feature that distinguishes p53 from other
tumor suppressor genes (e.g., APC, NF1, BRCA1) [22, 67, 155]. The
incidence of p53 mutations and the types of mutations can vary among
tumors in different tissues or populations [71]. Yeast provides unique
advantages for finding and characterizing human p53 mutations.
Figure 1. The tumor suppressor p53 is a sequence-specific transcription factor. Many types of
stress induce signal transduction pathways that lead to p53 stabilization and activation, mainly
by posttranslational modifications. The p53 protein is shown as a tetrameric (dimer of dimers)
sequence-specific transcription factor that recognizes promoter response elements (RE). The
solid arrows indicate the organization of the RE sequence as a closely spaced pair of
Chapter 10: Analysis of p53 and its Mutants Using Yeast 235
dimer-binding sites each consisting of two monomer-binding sites in inverted orientation (see
text for details). A list of the principal p53 target genes and their functions is shown.
In this chapter we first describe the role of p53 in mammalian cells and
the approaches that are commonly used to identify and evaluate the
consequences of p53 mutations. This is followed by a broad discussion of
the many yeast-based assays that have been developed for investigating p53.
1.1 Anatomy of the p53 protein and p53 tumor

mutations
The human p53 protein consists of 393 amino acids. Starting from
the amino terminus (Nter), the following functional domains have been
identified: 2 transactivation domains, a proline-rich sequence likely involved
in protein–protein interactions, a DNA-binding domain, a tetramerization
domain and a basic carboxy-terminal (Cter) region with regulatory functions
[189] (see Figure 2). The protein contains three nuclear localization signals
toward the Cter and one or possibly two nuclear export signals [107, 219].
Figure 2. p53 structural organization, topological domains, functional regulation by

posttranslational modifications and location of tumor mutations.
a
Functional domains. TA I and II = acidic transactivation domains; amino acids 22 and 23
are required for TA I while 53 and 54 are crucial for TA II. One nuclear export signal NES
whose activity is influenced by the phosphorylation status of p53 overlaps with TA I (220).
PxxP = proline-rich domain with possible role in protein–protein interactions [175]. TD =
tetramerization domain; NLS = nuclear localization signal; RR = basic regulatory region.
b
Conserved domains. The positions of the 5 evolutionary-conserved regions along the 393
p53 amino acid sequence are indicated. Four of them correspond to sequences within the
sequence-specific DNA-binding domain. With the exception of domains I and II, the major
p53 hotspots are located in the conserved domains. Presented is the distribution of the tumor
p53 mutations along the protein (hotspots are indicated by the number in the graph). Data
from the R5 release of the IARC p53 mutation database (containing more than 15,000
mutations; http://www.iarc.fr/P53/Somatic.html) were used to generate the graph.
Posttranslational modifications. The numbers indicate the amino acids at which
posttranslational modifications have been described [5]. The p53 sequence-specific DNA-
binding domain does not appear to be modified. P = phosphorylation; Ac = acetylation; Ubi =
ubiquitination (sumolation of lysine 386 is also shown [173]). Phosphorylation of p53
residues at the Nter might affect interaction with acetylases and acetylation of p53 (dotted
arrows).
Acetylation of lysines in the p53 Cter inhibits Mdm2-mediated ubiquitination. The
serine/threonine protein kinases that can phosphorylate p53 are also indicated above the
posttranslational modification sites.
A database of p53 mutations reported in tumor samples and cell lines has
been developed and is updated yearly at the International Agency for Cancer
Research (IARC), Lyon, France [75, 79]. Most (~80%) of the p53 mutations
associated with cancer occur as missense changes in the sequence-specific
DNA-binding domain that comprises about half of the entire protein
sequence (amino acid 100–300). This suggests that most tumor-associated
changes are likely to affect the ability to transcribe p53 responsive genes.
The unusually high frequency of missense changes in the p53 mutation
spectrum has provided opportunities in the field of molecular epidemiology
to investigate the role of carcinogens as etiologic agents of cancer [83]. The
similarity between p53 mutational fingerprints in tumors among exposed
populations and mutational fingerprints obtained following in vitro treatment
with an agent has been used to indicate its role in the etiology of some
cancers [15, 78] (discussed in section 2).
Germline p53 mutations lead to the dominant tumor-susceptibility Li–
Fraumeni syndrome (LFS) characterized by a high incidence of various
cancers with clear tissue-specific effects that in some cases relate to the
nature of the p53 mutations in terms of impact on protein function [74]. The
germline p53 mutation spectrum is similar to the somatic tumor spectrum.
An LFS-like phenotype has also been identified in families that do not
harbor germline p53 defects. Mutations in the checkpoint kinase Chk2 gene,
an upstream regulator of p53 activity, have been observed in these families
suggesting that the p53 pathway can be altered in multiple steps [112].
Alterations in the upstream regulators or downstream effectors of p53
may lead to a hypomorphic functional state of the protein that could impact
on the selective pressure for additional p53 gene mutations during cancer
development. An issue that has received relatively little attention is that
partial inactivation or changes in the spectrum of p53 functions may be
sufficient for, or even favor, tumor progression depending on the cell type
and physiological state (see sections 1.4, 2.1.3, and 2.5). This is relevant to
the prognostic use of tumor p53 status to estimate tumor aggressiveness and
therapeutic responses. Although reports differ possibly also because of lack
of standardization, p53 status can be a prognostic factor based on
multivariate analyses, with loss-of-function p53 mutations correlating with
poor prognosis [34, 51, 120, 124, 153, 181, 188].
The analysis of p53 mutation spectra from tumors suggests that much of
the p53 gene is a target for missense mutations that can inactivate its
function. In fact, more than 1,300 different amino acid changes have been
identified in the protein domain that provides for sequence-specific DNA
binding [126]. Although there are several strong hotspots, the majority of
tumor-associated p53 alleles do not occur at hot spot codons [79] (Figure 2).
Information on the specific functional defects associated with p53 mutations,
particularly at non-hot spot codons, is limited. These functional defects
could range from total loss of transactivation potential to partial loss that
affects only a subset of p53 target genes, to subtle differences in activity
compared with the wild-type (WT) protein, and also to enhanced and altered
DNA binding specificity leading to gain of functions (see sections 2.1.3, 2.5,
and 2.8).
The crystal structure of the human p53 DNA-binding domain and the
comparison of p53 sequences among many species show that the most
frequent mutations affect amino acids directly involved in establishing DNA
contacts and that hot spots tend to occur at evolutionary conserved residues
[36, 209]. The p53 DNA-binding domain has an immunoglobulin-like fold.
Three loops are involved in DNA contacts, and hydrophobic interactions of
two beta-sheets plus the coordination of a zinc atom provide for correct
positioning of the loops. This conformation may explain why many single
amino acid changes would affect the activity of the protein, even if distant
from the DNA contact sites. However, structural information for the
complete protein is not available, which limits structure/function analysis.
Several tumor-associated mutations affect amino acids that belong to the
DNA-binding domain but do not face the DNA [72]. Since the active form
of p53 is a tetramer (a dimer of dimers) (Figure 1), these mutations might
affect protein–protein interactions that stabilize the tetramer and indirectly
lead to inefficient DNA contacts [130].
The observation that p53 is active as a tetramer has led to the hypothesis
that the selection for missense changes in the DNA-binding domain can be
explained by the capacity of the mutant proteins to form hetero-tetramers
with WT p53, leading to dominant-negative effects [24]. The extent, the

mechanisms and the physiological relevance of this dominant-negative
potential are still under debate, especially since the WT allele is generally
lost [8, 19, 59]. It is possible that the requirement for WT p53 activity, a
specific partial function, or complete loss-of-function may change during the
evolution of a tumor. Notably, many tumor p53 mutant proteins are present
at high levels in tumor cells and some p53 mutants acquire oncogenic
attributes [73, 162].
1.2 Biochemical functions of the p53 protein
Different activities and many protein–protein interactions have been

ascribed to the WT p53 protein [164]. Some of these suggest a direct role for
p53 in preserving genome stability, particularly in the regulation of
homologous recombination and modulation of DNA repair capacity [3, 47,
111, 176]. However, mounting evidence indicates that the main p53 function
in tumor suppression results from its activity as a rapidly inducible,
sequence-specific transcription factor [206, 208] (see Figure 1).
1.2.1 Regulation of the p53 transcription factor
In normal unstressed cells, p53 is expressed constitutively but the protein

has a short half-life. Various types of stress signals, including proliferative
stimuli, viral infection, nucleotide pool unbalance, hypoxia, with DNA
damage being the best characterized, lead to p53 stabilization and activation.
These are achieved mainly through posttranslational modifications, com-
prising phosphorylation, acetylation, and sumolation in the Nter and the Cter
regions [7, 69, 154]. Interestingly, the large sequence-specific DNA-binding
domain appears to be largely excluded from posttranslational modifications
(see Figure 2), with the exception of proline isomerization [227]. The precise
effect of these changes is not completely understood. Multiple Nter
serine/threonine phosphorylations (~10 sites) can lead to a change in the
relative binding affinity of p53 toward negative (e.g., MDM2, an E3-
ubiquitin ligase that targets p53 for proteasome-mediated degradation) and
positive (e.g., p300/CBP, a histone acetylase) regulatory factors, or basal
elements of the transcription machinery (e.g., TAFII40, 60, 31) [154]. This
results in increased availability of nuclear p53 protein and stimulation of
transcription [5, 165].
p53 activity is tightly regulated [7, 68] (see Figure 2). Different types of
stress including DNA damage may lead to specific phosphorylations of p53
via different upstream protein kinases. This provides flexibility in p53
abundance, localization, and DNA-binding activity. Notably, the regulation
of p53 activity also involves one major, and possibly more, negative
feedback loop(s). The MDM2 gene is directly activated by p53 [163]. The
importance of this negative regulation is exemplified by the oncogenic
potential of Mdm2 gene amplification and by the observation that Mdm2
knockout (KO) mice are embryonic lethal and exhibit rapid and massive
apoptosis, while the double knockout with p53 is viable [135].
1.2.2 Transactivation of p53 responsive genes
The induction of p53 responses can lead to different biological effects

according to the cell type or the activating stimuli. For example, temporary
G1 or G2 cell cycle arrest, premature senescence, and programmed cell
death can all be induced (or maintained) by p53 [208]. The mechanisms of
how the choice between these pathways is brought about are still elusive,
although several possibilities have been proposed. Differential regulation of
downstream genes, both in terms of the extent and the kinetics of
transactivation and repression, is likely to be an important factor for
dictating the specificity of p53 biological responses. Locus-specific features
of chromatin assembly and remodeling are proposed to affect the
transcriptional status of specific genes [110]. Possibly, the combinatorial
action of different transcription factors, coregulators and corepressors
provides for specific gene regulation and could be the basis of cell-type
specific effects of p53 responses. Over 100 genes can be modulated (both
activation and repression) by p53, as revealed by genome-wide expression
profiles [220].
In vivo binding of the p53 protein to specific promoter sequences has
been demonstrated for a limited number of p53-regulated genes using
chromatin immunoprecipitation (ChIP) assays [12, 101]. For other p53
targets, sequence response elements that are bound in vitro by p53 in gel
shift assays have been identified in promoter or intronic regions [49]. These
sequences are sufficient for transcripional activation by p53 of reporter
genes during transient transfection of mammalian cells. A degenerate p53
consensus response element (RE) has been identified that contains two DNA
repeats each of which contains a head-to-head inverted sequence (5′-
RRRCWWGYYYN(0-13)RRRCWWGYYY-3′ where R = purine, Y =
Pyrimidine; W = A or T) [50]. The single inverted repeat can bind a p53
protein dimer, so that the two inverted repeats provide for the binding of a
p53 tetramer (see diagram of a p53 tetramer bound to DNA in Figure 1).
There are many variations which are expected to affect intrinsic RE binding
affinity and differential transactivation. The p53 dimer-binding sites can be
separated by up to 13 nucleotides with neutral sequence, and p53 REs
identified in regulatory regions of p53-target genes can deviate considerably
from the consensus sequence. Also, there are variations in the number or
repeats, position relative to the ATG start site, and the arrangement of REs.
In vitro DNA-binding experiments with naked DNA indicate that the
ability of p53 to act as a transcription factor depends on posttranslational
modifications that activate the protein, enabling it to bind DNA [82]. In
particular, Cter modifications have been proposed to relieve its inhibitory
effect by triggering a conformational switch. Models whereby covalent
modifications activate p53 DNA-binding and transactivation function are
consistent with the observation that p53 is induced by stress responses.
Moreover, different posttranslational modifications might have differential
effects on p53 activity, thus providing a direct link between a particular
activating signal and a specific response [48].
Conclusions from gel shift assays, however, should be taken with caution
since it is questionable how reliably this in vitro assay reproduces the
nuclear environment. Recently the role of chromosome structure in p53
transactivation potential has been examined in vitro using chromatinized
DNA preparations and in vivo using ChIP assays [12, 52, 101, 196]. Inter-
estingly, acetylation of p53 does not appear to affect its DNA-binding affinity,
yet the histone acetylase activity of p300/CBP is important in p53-mediated
transactivation [12]. It is possible that by virtue of its specific DNA-binding
capacity and protein–protein interaction with p300/CBP, p53 can stimulate
chromatin modifications at promoter sites that stimulate transcription. The
acetylation of p53 might be a by-product of these molecular interactions
[165]. Thus, p53 intrinsic DNA-binding affinity might be an important
component in the regulation of p53-dependent transcriptional modulation.
Since p53 is active as a tetramer, its intranuclear concentration may
strongly affect binding kinetics and transactivation. The importance of p53
levels is often overlooked (discussed below), especially for mutants with
altered function, since assays in mammalian cells usually involve high
expression. In fact, both the chromatin structure of the reporter plasmid and
the high levels of p53 protein expressed from constitutive viral promoters
result in assessments of transactivation activity under conditions that are not
physiological.
1.3 Identification of p53 mutations in tumor samples
Several techniques have been used to determine p53 status in fresh,

frozen, or archived tumor specimens [132]. These include immuno-
histochemistry (IHC), DNA sequencing, single-stranded conformational
polymorhphism (SSCP), denaturing gradient gel electrophoresis (DGGE),
loss of heterozygosity (LOH), and functional assessment in yeast. The levels
of p53 expression in individual cells from tumor sections can be determined

by IHC. Since tumors are generally highly heterogeneous, single cell
microscopic analysis is useful. High expression of p53, which is detectable
by antibody staining in individual cells, can be indicative of p53 mutations.
p53 missense mutants often lack the control of downstream pathways which
regulate p53 levels, as is the case for p53 mutants defective in MDM2
transactivation. However, IHC positivity has only limited correlation (~60%)
with the appearance of p53 mutations [10, 66, 80, 131].
Methods are also available for the direct detection of mutations in the p53
gene based on the conformational changes they produce in isolated DNA
fragments. However, these are limited by the heterogeneity of cells within
tumors with respect to p53 status. These electrophoresis-based approaches
(SSCP, DGGE) are time-consuming, since short PCR-generated DNA
fragments corresponding to at best a single exon are examined separately in
the gels. Fragments with altered mobility can be detected only when a mutant
allele is present in at least 10–20% of the cells in the specimen [29, 81].
Given the high frequency of p53 mutations in tumors and the modest size
of the p53 gene, direct DNA sequencing has been used as an alternative
method. This approach has, however, the same sensitivity issues as SSCP
and DGGE and is relatively time-consuming. Recently, an oligonucleotide-
based array (p53 GeneChip) was developed that contains almost all possible
p53 point mutations, but not deletions/insertions. DNA fragments obtained
by multiplex PCR of p53 exons from tumor samples are fragmented,
fluorescein-labeled and then hybridized to the chip. p53 mutations are
detected and identified by the presence and position of hybridization signals
that differ from WT sequences. This method is very sensitive (detecting as
few as 1–2% of mutant alleles in the cell population) and can also be applied
to microdissected tumor sections [2, 121, 213]. However, the high cost of
this approach is a limiting factor. An arrayed primer extension (APEX) assay
for detection and identification of p53 mutations has also been developed
recently [203]. Similar to the GeneChip, a DNA sample is amplified,
fragmented enzymatically, but it is then annealed to arrayed primers on a
chip. There are two primers to probe p53 coding nucleotides (both sense and
antisense strands). Four fluorescently labeled dideoxynucleotides are used
for template-dependent DNA polymerase extension reactions, and mutants
are detected as novel extension reactions.
One limitation which is common with all the methods described above is
that they do not provide information on the functional consequences of a
given amino acid change in the p53 protein.
1.4 Characterization of p53 alleles that retain function

in mammalian cells
Expression of p53 alleles under constitutive viral promoters from

expression vectors in mammalian cell lines have provided means to evaluate
the tumor suppressor potential of WT and mutant forms of the protein in
different cell types. Generally, ectopic expression in tumor cell lines of WT
p53 leads to growth suppression through cell cycle arrest and apoptosis.
Loss-of-function p53 mutants usually lose this activity. However, tumor-
associated amino acid changes in the p53 DNA-binding domain do not
always result in complete loss of growth suppressing activity and biological
function. For example, some p53 mutants retain the ability to induce cell
cycle arrest, but cannot induce apoptosis when expressed in tumor cell lines.
This biological effect correlates with the ability to induce the cyclin-
dependent kinase inhibitor p21 but not the apoptotic gene bax [19, 122].
Transactivation activity of p53 mutants in human cells can also be assessed
using luciferase reporter assays in transient transfection. Other p53 mutants
that are temperature-sensitive, show loss-of-function at 39°C but normal or
partial activity at 32°C [116, 161].
Some p53 mutants acquire oncogenic gain-of-function features such as the
ability to deregulate chromosome segregation, upregulate genes conferring
resistance to chemotherapeutic agents and promote angiogenesis [20, 58, 64,
142]. A group of tumor-associated mutants was identified that appeared to
retain WT levels of activity toward growth suppression, apoptosis, and
transactivation [187]. Interestingly, they were observed in familial cases of
breast cancer associated with BRCA1 germline mutations where the incidence
of p53 mutations is much higher than those found for sporadic breast cancer
[72]. Furthermore the spectrum of p53 mutations associated with familial
breast cancer is different from that of sporadic breast cancer.
The actual functional status of p53 alleles, not simply whether or not
there is a mutation, may correlate with clinical features of a tumor and
represent a relevant prognostic marker for patient management in
conventional chemo- and radiotherapy protocols. There are, however, severe
limitations on the functional analysis of p53 mutations in mammalian cells
including, but not limited to, the nonphysiological high level of ectopic
expression of p53 obtained with viral promoters.
1.5 Cancer-therapy approaches based on p53 functional

status
Although the development of neoplasia involves a global change in the

complex architecture of signaling pathways that define and regulate the
growth characteristics of a cell, the extensive interconnections between these

pathways suggest that targeting a specific branch may lead to global effects
[129, 206]. Given the high frequency of p53 alterations in almost every
tumor type and the tumor suppressive role of the protein, p53 is an appealing
target for cancer therapy. Various approaches have been used to restore p53
function in tumor cells or exploit the loss of p53 activity to selectively
destroy cancer cells [128].
The observation that some p53 alleles can retain partial function has
stimulated investigations into the biophysical/biochemical features of p53
mutant alleles and the possibility of reactivating mutant proteins using small
molecules (chemicals or peptides) [27, 61, 147, 171, 183]. The same
approach, but with the opposite aim to partially inactivate WT p53, has been
explored with the idea that normal cells surrounding a tumor may be
protected from the inactivating effects of conventional therapy [108]. The
viral delivery to tumor cells of WT copies of p53 capable of inducing
apoptosis has also been evaluated [38, 195, 214].
Another strategy for treatment of p53 defective tumor cells is replication
of viruses that only propagate in p53-defective cells. Promising results have
been reported both with a modified adenovirus, that has a mutation in the
protein responsible for WT p53 inactivation [18, 128] and with a WT adeno-
associated virus that induces cell cycle arrest in normal WT p53 cells but
apoptosis in p53-deficient tumor cells [167].
Given the many differences in p53 mutations, a detailed knowledge of
the functional status of p53 mutants could have clinical value, especially for
therapies tailored to specific tumors. Several methods for functionally
classifying p53 mutants have been used that are based on physical/chemical,
or immunological/structural parameters [1, 26, 35, 146]. However, many of
these approaches to functional analysis have had limited utility.
2 YEAST-BASED p53 FUNCTIONAL ASSAYS
There are now many examples where the yeast Saccharomyces cerevisiae
has proven an invaluable in vivo test tube for examining diverse human gene
functions and disease including, for example, cell signaling, DNA
metabolism, and mitochondrial function [102, 169].
No p53 homologue has been reported in yeast. In fact, the evolution of
the p53 gene seems to coincide with the development of multicellular
organisms, possibly because of the need for tissue remodeling via
programmed cell death and also because the elimination of individual cells
in an organism in response to DNA damage can be beneficial. However,
damage recognition and cell cycle responses induced by signaling pathways
in yeast are very sensitive and elaborate and they involve protein kinases and
complexes that are well conserved during evolution [208]. The high
evolutionary conservation between yeast and mammals is also evident in
basic cellular processes such as DNA replication and repair, chromosome
segregation, transcription, and translation [14]. Because of these features and
its genetic tractability, we have referred to yeast as honorary mammal [169].
As described in the following sections, yeast-based assays have provided
the means for structure/function characterization of p53, functional
classification of p53 mutant alleles, identification and evaluation of p53
response elements, and screening of chemicals capable of reactivating p53
mutants. These assays currently provide the only practical means to rapidly
identify p53 mutations from individual patients and to assess functional
status. In a broader sense, the development of a p53 mutant functionality
database along with linkage to the vast IARC human p53 tumor mutation
database will be valuable in understanding the correlation between p53
functional status, tumor aggressiveness, and responsiveness to therapy.
2.1 Human p53 can function as a transcription factor

in yeast
The evolutionary conservation of transcriptional machinery prompted

early investigations of human p53 as a sequence-specific transcription factor
in yeast. Schärer and Iggo [180] constructed a hybrid yeast promoter where
the yeast upstream activating sequences were replaced by a p53 RE. They
demonstrated that expression of WT, full-length p53 but not tumor-
associated mutants (four were tested) could induce a reporter gene down-
stream of the promoter. Interestingly, a murine p53 mutant that appeared
temperature-sensitive in mammalian cells, was also temperature-sensitive for
transactivation in yeast [175]. This work showed that the key function of the
p53 tumor suppressor gene could be rapidly assessed in yeast.
2.1.1 p53 functional assays for screening cancer cell lines and tumor
samples
The observations in yeast prompted the development of assays using

cDNAs that could more rapidly evaluate p53 functional status in tumors or
cell lines. The general features are summarized in Figure 3A. p53 cDNAs
can be expressed in yeast from constitutive or inducible promoters (Figure
3A). Most investigations have employed the strong, constitutive promoter of
ADH1. WT and mutant p53s do not inhibit the growth of yeast at the level of
expression obtained with this promoter. The inducible GAL1 promoter has
also been used. Its expression is repressed on glucose media but is induced
to very high levels on media containing galactose as carbon source.
Figure 3A. General features of the p53 functional assay in yeast. Expression of p53 in yeast
and transactivation by p53. The complete cDNA of a given p53 allele can be expressed under
either a constitutive or an inducible promoter from a selectable plasmid. The centromeric
plasmids are stably transmitted at low copy number. WT p53 can act as a transcription factor
in yeast stimulating the activity of a promoter whose upstream activating sequences have been
replaced with a p53 response element. Mutant p53 would change the level of transcription of
the reporter gene thus leading to phenotypic changes. If the p53 is not active, no
transactivation is observed. If the p53 allele is less active, higher levels of GAL1 induction are
needed; if more active, less induction is required for a phenotypic change.
Overexpression of WT p53 under the GAL1 promoter leads to severe

growth inhibition [17, 144]. The mechanism awaits further investigation, but
the phenotype has been exploited in different types of selection screens [89]
(see sections 2.1.4 and 2.3). Recently, we developed a protocol using the
GAL1 promoter that allows for both inducible and rheostatable expression of
p53 alleles. This method further expands the sensitivity of the transactivation
assay as a means to classify p53 tumor alleles [87] (see sections II.3 and II.6)
since it allows quantitative assessment of mutant activity compared to WT.
The p53-dependent reporter gene can be transcribed from a plasmid or an
integrated copy in the yeast genome. p53 transactivation is assessed using
different phenotypic assays according to the reporter gene [24, 54, 95] (see
Figure 3B). For example, HIS3 gene expression allows growth on plates
lacking histidine (i.e., only WT p53 expressing cells will grow). The URA3
gene can be similarly used on plates lacking uracil, but it also allows for
+
counterselection of colonies where p53 function is lost since Ura cells are
sensitive to 5-fluoro-orotic acid (5-FOA) [21].
Phenotypic characterization of p53 null alleles based on transactivation of a reporter gene
Reporter gene:
HIS3 URA3 ADE2
- -
Assay: growth on his growth on ura growth on plates with low adenine
or 5-FOA
Response:
Wild type p53: growth growth on ura- and 5-FOA sensitive growth, white color
-
p53 mutant: no growth no growth on ura and growth on 5-FOA slow growth, red color
Figure 3B. Summary of reporter genes and the assays used for phenotypic analysis of p53
mutant alleles under conditions of high expression.
Transcription of the ADE2 gene provides for a simple color assay on

plates containing low amounts of adenine. Cells expressing WT p53 and
nonfunctional p53 mutants will grow but the colonies have different sizes
and colors. As shown in Figure 3C, colonies expressing nonfunctional alleles
are small and red, while WT p53 colonies are normal size and white.
Blockage of adenine biosynthesis upstream of ADE2 gene leads to the
accumulation of a red pigment. On plates containing a low amount of
adenine, the ade2 mutants
+
appear as small red colonies. Expression of WT
p53 leads to an Ade2 phenotype resulting in white colonies.
Three other reporters of p53 activity have been used: the beta-
galactosidase gene [200, 202], Green fluorescent protein (GFP) [184], and
the firefly luciferase gene [222]. While their levels of expression can be
quantified, they are not as useful in genetic screens of p53 mutations, since
they do not provide for rapid identification of colonies expressing p53
mutants.
Figure 3C. Example of phenotypic assays using the ADE2 red/white reporter gene. Under
conditions of high constitutive expression of WT p53, transactivation of ADE2 results in
white colonies. Loss-of-function p53 mutants will result in red colonies since they cannot turn
on ADE2. When the GAL1-based inducible expression system is used, transactivation is
proportional to the levels of WT (or mutant) p53 protein expressed. Presented are yeast
transformants with WT p53 grown on plates containing different amounts of galactose
inducer (plus raffinose carbon source). Also shown are western blots of protein extracts from
cells grown under the same conditions. The appearance of colonies extends from red to pink
to white as the amount of p53 is increased.
2.1.2 Characterization of tumors with single and multiple p53

mutations
Tumors containing p53 mutations may have cells that are heterozygous
or homozygous for WT p53 or have more than one p53 mutation. As
indicated above, the yeast-based approaches provide for the isolation and
functional analysis of separated alleles (FASAY) [28, 41, 185]. The
procedure utilizes the exquisite recombination abilities of yeast as described
in Figure 3D. First, total cDNAs are prepared by reverse transcription of
total RNA extracted from fresh or frozen tumor biopsies. The p53 cDNA is
then amplified using a pair of specific primers, typically for amino acids 43
through 374 [54]. This region corresponds to part of exon 4 through the
beginning of exon 11 and includes the entire sequence-specific DNA-binding
domain where most tumor-associated mutations are found (see Figure 2).
Unpurified PCR products are directly transformed into yeast together with
an expression plasmid that contains a selectable marker, an origin of
replication, a centromere for stable propagation at low copy number, and
consecutive sequences corresponding to the Nter and Cter regions of p53,
coding for amino acids 1–64 and 351–393, respectively. The vector is
linearized between these regions. Thus, the PCR product and plasmid have
63 and 69 nucleotides of common sequence at each end.
Figure 3D. Diagram of the gap repair assay for isolation of individual p53 alleles directly
from tumor samples. Total RNA is extracted from fresh or frozen tumor biopsies and total
cDNA is prepared by RT-PCR. The p53 cDNA is PCR amplified using specific primers.
Unpurified PCR products are transformed into yeast together with a linearized p53 expression
vector whose ends are identical to the ends of the PCR fragment. The highly efficient
homologous recombination system of yeast generates a complete p53 expression vector. The
functionalily of the p53 allele is then assessed using a transactivation assay.
Cotransformation of PCR product and linearized vector leads to efficient

gap repair of the plasmid by the PCR fragments yielding a circular vector
capable of expressing a complete p53 cDNA. Using 50 ng of PCR product
and 10 ng of vector, more than 1,000 colonies can be obtained in a typical
transformation experiment. Each colony is selected for the presence of the
reconstituted vector and corresponds to the isolation of a single p53 allele.
Thus, the intrinsic heterogeneity of tumor materials is not a major limitation
with this approach and mixed samples containing various mutant or wild
type alleles can be analyzed effectively. In the case of the ADE2 p53 reporter
gene, WT p53 alleles expressed in normal cells or in heterozygous tumor
cells would result in variable percentages of white colonies and red colonies
corresponding to the frequency of the loss-of-function p53 alleles. The
nature of the mutations can be easily determined by recovering the plasmid
from the red colonies or using a colony PCR approach followed by DNA
sequencing.
There are two limitations with the yeast functional assay. It requires
extraction from fresh or frozen samples and errors may result during
transcription and amplification. These limitations hold true for many
analyses of clinical samples by PCR. Typically there are 2–5% red colonies,
possibly due to RT-PCR-generated errors. Amplification fidelity can be
assessed by parallel RT-PCR of each sample [34]. The critical requirement
for RNA can be avoided by modifying the functional assay into an exon-by-
exon DNA analysis that uses formalin-fixed, paraffin-embedded archival
samples [41].
When compared to other systems for p53 mutant identification, the yeast
functional assay has greater sensitivity than electrophoresis-based methods
(SSCP, DGGE) [113, 132, 197, 198] and is comparable to the GeneChip
approach [213]. In addition, the yeast assay is rapid and relatively
inexpensive and, furthermore, it enables functional defects in p53 to be
assessed. Functional analysis based on transactivation capacity in yeast is
robust and appears more sensitive than transient transfection gene reporter
assays in mammalian cells. Over 100 different mutant p53 alleles isolated
from clinical samples were found to lack transactivation function in the
red/white ADE2-based assays [31, 55, 172]. The tissues have included fresh
and frozen specimens from brain (glioblastoma and astrocytoma), upper
aero-digestive tract, breast, colon, bladder, prostate, lymphocytes, and skin
[29, 30, 34, 46, 65, 93, 103, 123, 125, 134, 145, 151, 159, 160, 168, 172,
186, 204, 211, 215]. Included in these analyses are dysplasias, primary
carcinomas, and advanced tumors.
The physical and functional separation of p53 alleles also provides an
assay to monitor intra-tumor WT p53 delivery by viral vectors for cancer
gene therapy approaches to treat brain, head and neck, ovarian, and lung
cancer [38, 106, 195, 199, 214]. Specific primers can be used to amplify the
mRNA obtained from endogenous p53 alleles or the virally encoded WT
cDNA to estimate the contribution of viral WT p53 mRNA in tumor
samples.
2.1.3 Identification of p53 mutants with differential effects

on the transactivation of specific promoters
The yeast-based systems provide opportunities to evaluate p53 mutants

for ability to transactivate at various REs. As described above, the consensus
sequence of the p53 response element is degenerate and REs from
mammalian p53-regulated genes exhibit many deviations from the consensus
[42, 49, 50]. This observation prompted the evaluation of the transactivation
activity of p53 mutants with different p53 REs. Most screenings of tumor
samples have utilized high p53 expression and the RGC RE to determine if
p53 is capable of transactivation. The RGC sequence was originally found
within the human ribosome gene cluster and was shown to be a bona fide
p53 RE based on both in vitro binding and transactivation
WAF1
assays [11]. The
REs of the key p53-regulated genes such as p21 and bax have also been
used [55]. Highly expressed WT p53 is capable of transactivation from these
REs and results in white colonies in the ADE2 based assay. Flaman et al.
[54] examined 51 different tumor-associated mutations observed in human
tumors. Nine of them scored as WT with P21, while none were able to
transactive the BAX or the RGC RE. Another 77 mutants were examined by
Campomenosi et al. [30] using REs of RGC, BAX, and P21. Consistent with
the previous report, about 15% of the mutants were WT toward the P21,
but none of them was able to transactivate BAX. This finding suggests
that inactivation of the apoptotic function of p53 is critical for tumor
development.
DiComo and Prives [42] further expanded the investigation of p53
transactivation ability using nine different REs upstream of the HIS3 reporter.
Nineteen tumor-derived mutants were analyzed at different temperatures.
While p53 hot spot mutants appeared transcriptionally inactive, four mutants
in the DNA-binding domain scored as WT with P21 and a strong consensus
RE at 37°C, and one of them [R283H) appeared fully active with several
REs. An additional group of four mutants was active with the same REs at
lower temperatures. These mutations affected amino acids belonging to the
beta-sheet scaffold of the p53 DNA-binding domain. All the mutations were
inactive with RGC. Consistent with this, WT p53 was less active with the
RGC and BAX REs compared to P21 and the strong consensus sequence.
A sensitive screen was recently developed to specifically isolate p53
mutants retaining partial function (in preparation). The assay was based on
the observation that in the ADE2 reporter system white and red colonies
represent the extreme phenotypes in a range of p53 transactivation
capabilities from WT to complete loss-of-function. With the view that it
might be possible to identify mutants with reduced function, p53 mutations
were generated by random mutagenesis and directly cloned into yeast using
gap repair (see Figure 3). Examination of colony color and morphology
allowed the identification of intermediate phenotypic classes (pink, sectored,
and speckled colonies) that were due to the expression of p53 mutants with
reduced transactivation activity. Approximately 60 different single amino
acid changes were found that could result in proteins with reduced activity.
The changes in activity were quantified with a luciferase reporter assay that
was developed in yeast. A group of mutants that were tested for
transactivation and growth suppression in human cells validated the yeast
assay (in preparation). Interestingly, the mutations were spread uniformly

across the entire mutagenized region (amino acids 50–350) which extends
beyond the DNA-binding domain. Since there were no repeats, the screen
was far from saturation. Based on the number of these mutants that
correspond to p53 tumor mutations in the IARC p53 database, we estimate
that up to 20% of tumor-associated p53 mutants retain partial function.
The functional evaluation in yeast of many p53 alleles associated with
cancer has revealed that a large fraction of mutants retain residual activity.
Interestingly, transactivation assays in mammalian cells along with in vitro
measurements of conformation stability and DNA-binding potential have
confirmed the yeast results [26, 28], although the number of p53 mutants
examined is small. We have concluded that it is not possible a priori to
predict the functional effect of most mutations based on sequence conserva-
tion, topology, or structural models, although loss-of-function alleles appear
to affect amino acids with a higher degree of evolutionary conservation [31].
These results further support the need for a detailed rational classification of
p53 mutants associated with cancer based on in vivo transactivation at many
promoters.
2.1.4 p53 functional assays to evaluate dominance
Many of the p53 mutants appear to be dominant, which helps explain

their frequent occurrence in tumors. The levels of expression of p53 alleles
and the ratio between WT and mutant proteins within a cell can significantly
influence the dominant-negative potential of p53 mutants. In fact, since p53
is active as a tetramer, dominance could result from the formation of mixed
tetramers that would be conformationally unstable and incapable of DNA
binding and transactivation. Yeast-based functional assays have provided for
rapid assessment of the dominance potential of p53 mutants when equally
expressed with WT p53. Brachmann et al. [24] developed a mutation
selection screen for dominant-negative p53 mutants that was based on the
counterselectable URA3 reporter gene. Briefly, WT p53 can induce URA3
transcription leading both to uracil prototrophy and sensitivity to 5-FOA. If a
p53 mutant is co-expressed with WT p53 and acts in a dominant fashion,
cells will grow on 5-FOA plates. The screen identified 31 dominant mutants
of which 13 conferred at least partial growth even in the presence of 2 copies
of WT p53. The mutations clustered in the p53 mutational hot spots,
supporting the view that dominant-negative interactions are important in the
selection of p53 missense mutations in human cancers.
The ADE2 color assay has also been used to evaluate the dominance of
71 different p53 alleles [85, 136]. In this assay, a recessive p53 mutant in the
presence of WT p53 would lead to white colonies, while a dominant mutant
would lead to pink/red colonies. Approximately 30% of the mutations

exhibited dominance. These mutants affected residues that are highly
conserved and that are more frequently associated with cancer; they
appeared to cluster in the L3 loop and H1 and H2 helices of the DNA-
binding domain (for an extensive description of p53 structure, see [36, 189]).
The possibility that the mutants exhibited different degrees of dominance
according to the p53 RE used in the transactivation assay was also explored.
A significantly higher fraction of mutants was dominant over WT p53 with
the BAX compared to the P21 RE. This result is likely due to the intrinsic
difference in activity of these two REs and suggests that dominance in vivo
could be p53 gene-target specific. Interestingly, a recent study in mammalian
cells using bi-cistronic vectors that allows for equal expression of WT and
mutant p53 from a strong viral promoter revealed that dominant-negative
effects were visible only with the bax gene [8].
A recent investigation employed the ADE2-based yeast functional assay
to evaluate transactivation and dominance of p53 alleles expressed in
endometrial carcinomas. Both the frequency of p53 mutations and the ratio
of dominant-negative mutants was higher among endometrioid-type com-
pared to serous-type tumors [177].
2.1.5 Reactivation of p53 mutants using yeast-based screens
The restoration of cellular pathways in tumor cells that lead to cell death
by apoptosis is an appealing cancer therapy approach. In this respect p53
represents a potentially powerful target. The identification of a large group
of p53 mutants that appear to retain some transactivation function and the
observation that the majority of loss-of-function alleles are recessive when
heterozygous with WT p53 [136] suggest that many tumor-associated
mutants retain native conformation or do not dramatically perturb protein
conformation.
Different types of yeast assays have been used to address the possibility
that small molecules could partially reactivate p53 function either by
stabilizing the structure of the mutants and/or by enhancing DNA-binding
capability. Brachmann et al. [25] used an elegant screen to identify p53
mutations that act as intragenic suppressors of common hot spot p53 tumor
mutations. The assay utilizes random PCR mutagenesis or degenerate
oligonucleotides [13] to induce mutations in defined regions of the p53
cDNA. Gap repair was used to clone the mutagenized fragments within a
p53 cDNA that contains a specific mutation corresponding to a p53 hot spot.
Compensating mutations were identified that restore p53 function based on
transcription of the p53 URA3 reporter. Single amino acid changes were
identified that were capable either of restoring function to specific p53
mutations or had a broad reactivating effect on several hots pots. The

intragenic suppressors affected amino acids that belonged either to the L1
loop (T123A, T123P) or the L3 loop (N239Y, S240N). Another suppressor
(N268D) was found in the S10 beta-strand. This mutation might have a
stabilizing effect on the DNA-binding domain. It is interesting that intragenic
suppressors were identified in both L1 and L3 loops. According to the
crystal structure, these loops are exposed regions when the DNA-binding
domain is bound to a consensus RE [36]. Thus, the loops might be good
targets for small molecules that could provide in trans functional
reactivation of p53 hot spot mutants. Expression of the reactivated double
mutants in mammalian cells resulted in restoration of transactivation and
apoptotic function in transient assays.
No suppressor for the common tumor p53 mutation R175H has been
identified with the yeast screen [13]. According to studies with confor-
mational sensitive antibodies and also in vitro analysis with purified core
domains, R175H has a denatured conformation that might prevent this type
of rescue [36]. However, a recent report based on a mammalian cell-based
screen of molecules capable of reactivating p53 function reported the
identification of a compound that can activate R175H [27].
Another approach can also reveal suppressor mutations. We developed a
screen for p53 mutants exhibiting enhanced transactivation function
compared to WT p53 [87]. Unlike other assays described above, our
approach was based on rheostatable expression of p53 under the GAL1
promoter (see Figure 3A & C). At low-expression levels WT p53 cannot
activate transcription of the ADE2 reporter gene and yeast colonies appear as
p53 mutants (i.e., red colonies). Thus, p53 alleles with enhanced function
can be easily selected (i.e., white colonies) at low levels of p53 expression.
Interestingly, several single amino acid changes produced by PCR
mutagenesis were identified both in the L1 and L3 loops, in the H2 helix and
in exposed regions of the beta-strands located on the opposite side of the
protein:DNA surface. This supertrans group of mutants included the
intragenic suppressors reported by Brachmann et al. [25]. Consistent with
the enhanced function, the supertrans alleles were not affected by dominant-
negative p53 mutants when heterozygous.
A recent study showed that the radio/chemoprotective agent amifostine
could activate WT p53 in mammalian cells [148]. Maurici et al. [127] used
the yeast functional assay to evaluate whether amifostine had a direct effect
on p53 activity. A temperature-sensitive p53 mutant (V272M) was activated
by amifostine at the nonpermissive temperature and a small number of p53
mutants that retain function with the P21 but not the RGC or BAX REs were
partially reactivated by the compound. These results suggest that the yeast
functional assay could be applied to screening of compounds to identify
leads for p53 reactivating drugs. However, the results were based on the
color assay (red/pink/white) that might also be influenced by the effects of
the drug on the growth of yeast. A quantitative assessment of the effect of
amifostine both on WT and mutant p53 activity is needed.
2.2 p53 mutational fingerprints identified with the yeast

functional assay: a tool for molecular epidemiology
As indicated above, p53 mutation spectra associated with various cancers

in specific populations may be helpful in identifying carcinogens and
understanding underlying events [205]. Exposure to some environmental
agents has been associated with the features of p53 mutation spectra in
tumors [15, 83].
The yeast-based p53 functional assay has been adapted to identify p53
mutations induced in vitro or in vivo by mutagens and carcinogens [86, 140].
p53 DNA has been treated directly with several mutagens and carcinogens
and DNA changes were determined using in vivo yeast functional assays
after transformation with the treated DNA. p53 functional mutants identified
by the lack of reporter gene expression (color or growth assay) were
sequenced and p53 mutational fingerprints were developed. The following
agents have been examined: UV, 8-methoxy-psoralen, CCNU, Me-lex, nitric
oxide, and reactive oxygen species [23, 62, 84, 86, 90, 105, 137, 138, 141].
The UV-induced p53 mutation spectra identified by the yeast system
included tandem mutations at dipyrimidine sites, a hallmark of UV
mutagenesis [90, 137, 140]. Further analysis revealed that the yeast p53
spectra and the reported p53 mutations associated with nonmelanoma skin
cancer are indistinguishable. This result exemplifies the utility of
determining mutation spectra in defined experimental conditions using the
same in vivo target of mutagenesis that is used in molecular epidemiological
studies [62]. Similarly mutation spectra induced by bifunctional alkylating
agents were consistent with earlier mutagenesis studies [86].
The correlation between lesion hot spots and mutation hot spots has also
been evaluated using a minor-groove specific methylating agent as well as
oxidative damage [23, 105]. Based on results with UV the functional assay
can also be used to examine the effects on p53 of DNA damage induced
in vivo [139]. So far, only direct-acting mutagens have been used. The study
of chemicals that require metabolic activation may also be feasible if yeast are
provided with a repertoire of human or mammalian cytochrome genes [156].
2.3 Yeast assays to characterize p53 amino- and

carboxy-terminal functional domains
The Nter and Cter domains play important roles in p53 structural
organization, interaction with other proteins and transactivation. The p53
functional assays can also be used to address changes in these regions.
Ishioka et al. [94] reported that the p53 Cter is generally refractory to
mutations that inactivate transactivation functions and p53 deleted for the
last 40 amino acids (Q354Stop) retains WT activity. However, single amino
acid changes in the tetramerization domain (L344P, K351E) can nearly
inactivate p53 transactivation. A germline p53 mutation in the tetrameri-
zation domain (R337C) that is found in cancer prone families [119] results in
yeast colonies that are pink in the ADE2 assay, suggesting partial retention
of transactivation activity. It is possible that this amino acid change affects
the stability of the p53 tetramer; however, the mutant protein is unaffected
by temperature changes (23°–34°C), unlike many pink colony mutants that
we have examined (unpublished). A second germline mutation has been
identified at codon 337 (R337H) in Brazilian families that were charac-
terized by increased incidence of childhood adrenal carcinoma (ACC) [170].
Surprisingly, there was no effect on the incidence of other types of cancer. A
recent study showed that R337H retained almost normal function in in vitro
DNA-binding assays, but exhibited temperature and pH-sensitivity [43]. The
reason why this specific functional defect is highly tissue-specific remains to
be determined, although it has been proposed that the adrenocortical cellular
environment would lead to functional inactivation of this p53 mutant thus
increasing the risk of cancer development. It will be interesting to compare
the R337C and R337H phenotypes in the yeast functional assay.
Candau et al. [32] used a yeast-based assay to analyze Nter functions of
p53. Instead of expressing the complete p53 cDNA, chimeras were created
between p53 and the DNA-binding domain of the GAL4 transcription factor.
The use of p53 missense and deletion mutants revealed that the Nter region
of p53 contains a second transactivation domain mapping between amino
acids 40–83 with amino acids W53 and F54 playing a critical role. The
activity of this transactivation domain has been confirmed in mammalian
cell experiments [221]. It has also been proposed that this element might
display target gene specificity, thereby contributing to p53-induced
differential transactivation. However, the underlying mechanisms for this
effect remains to be established.
Coincident changes at amino acids 22 and 23 (L22E and W23S) in the
first p53 trnsactivation domain [42] can inhibit binding to MDM2,
p300/CBP, and TAF proteins [53, 118, 152, 163]. Consistent with this, the
double mutant was not functional in mammalian cells nor in transgenic

mice even though it had no effect on p53 DNA-binding affinity [97].
Unexpectedly, the mutant retained transactivation similar to WT when tested
with a strong consensus RE in yeast [42]. By way of comparison, this
suggested that the transactivation domain at amino acids 53 and 54 is
relatively more important to p53 activity in yeast than in mammalian cells.
However, the functional effect of mutating amino acids 22 and 23 as well as
deleting the last 40 amino acids [94] might have been masked since p53 was
expressed at high levels from a constitutive ADH1 promoter. Experiments at
low expression levels indicate that both transactivation domains have similar
contributions to p53 activity (unpublished). In addition, we have found that
deletion of the last 40 amino acid at the carboxy terminus also reduces p53
transactivation in yeast (unpublished). This is contrary to the results of gel
shift experiments with naked DNA, but consistent with DNA-binding
experiments with chromatinized DNA that may mimic more closely the
nuclear environment [52].
2.4 Yeast assays to characterize and identify p53

interacting proteins
The contribution of yeast transcriptional adaptor proteins in p53

transactivation can also be explored with yeast functional assays. The yeast
ADA2, ADA3, and GCN5 genes, whose product are involved in multiprotein
complexes that have histone acetylating activities, were shown to contribute
to transactivation mediated by the p53 Nter [32]. A recent study showed that
the histone H4 and H2A acetyltransferase activity of the NuA4 complex can
contribute to p53 activity in yeast [149]. Transcriptional activation by p53
was associated with targeted NuA4-dependent histone H4 hyperacetylation.
A genetic screen for human proteins that interfere with p53
transactivation function identified the human homologue of ADA3 as an
important cofactor in p53 activity [210]. This “yeast dissociator screen” is
based on the URA3 reporter gene and selection against p53 activity on
5-FOA plates (described in section 2.1.4; see also Figure 3). A human
protein that is capable of binding to p53 might reduce transactivation activity
thereby enhancing growth on 5-FOA plates. This protein–protein interaction
could identify proteins that are truly negative regulators of p53 activity or
that instead cooperate with p53 in human cells but appear as inhibitors in the
heterologous system in yeast since additional protein components are
missing. Experiments in mammalian cells showed that hADA3, which is part
of histone acetyltransferase complexes, was capable of stimulating p53
activity and that its physical interaction with p53 is possibly modulated by
stress responses leading to posttranslational modifications of p53.
A similar system was developed in the fission yeast Schizosaccharomyces

pombe [207]. As in the budding yeast, p53 can stimulate transcription in this
organism and its overexpression leads to severe growth inhibition [17]. An
S. pombe strain was constructed containing two p53-dependent reporter
genes, one of which (ura4+) is counterselected on 5-FOA plates and
positively selected on uracil-deficient plates. The assay was validated using
SV40 large-T antigen and MDM2, both known interactors and inhibitors of
p53 activity. It was also shown that tumor-derived p53 mutants are
functionally distinguishable from WT p53 based on transactivation. Finally,
the assay revealed that the p53 homologue from Drosophila could also be
functionally tested in S. pombe [207].
p53 protein–protein interactions have also been identified in yeast 2-
hybrid screens [96]. However, since p53 also can activate transcription with
its transactivation domains, the entire p53 protein cannot be used. The
screening relies on the construction of two chimeric proteins, one containing
a DNA-binding domain and the other a transactivation domain [109]. The
GAL4 DNA-binding domain is fused to the desired p53 protein fragment for
which interacting proteins are sought. This chimeric protein is presented as a
“bait” to a library of proteins, each of which is fused to the transactivation
domain of the GAL4 protein. Physical interaction between the bait and an
unknown protein would bridge the DNA-binding and the transactivation
domain of GAL4 leading to selectable transactivation of a reporter gene. The
2-hybrid screen was performed using a murine p53 sequence lacking the
Nter domain leaving amino acids 73–390 [96]. Two p53 interacting proteins
were discovered, 53BP1 and 53BP2. Binding of p53 with either protein
inhibited its binding to REs in vitro. 53BP1 contains two Brca1 C-terminal
(BRCT) domains, a common protein–protein interaction motif found in
proteins involved in the DNA damage and repair responses [98]. Structural
analysis confirmed that 53BP1 binds p53 at a region that overlaps with the
DNA-binding surface of p53 and involves p53 residues that are mutated in
cancer. Analysis of intracellular localization of 53BP1 in human cells
revealed discrete nuclear accumulation within 5–15 min after exposure to
ionizing radiation (IR) [4]. These results suggest 53BP1 functions early in
the cellular response to DNA DSBs and may activate p53 responses [182].
The 53BP2 contains an SH3 domain and also ankyrin repeats that may
mediate protein–protein interactions. 53BP2 is a 528 amino acid fragment of
the carboxy terminal region of the ASPP2 protein [179]. Interestingly,
ASPP2 and a related protein ASPP1 can specifically enhance p53 trans-
activation function but only of specific genes leading to p53-induced
apoptosis but not cell cycle arrest. The expression of ASPP2 is subject to
stress signals and frequently appears downregulated in breast carcinomas
expressing WT but not mutant p53 [179]. This result provides additional
evidence that functional alteration of the p53 pathway may occur through
upstream regulation of p53 protein activity.
Differential transactivation is important in the biological responses to
stress, particularly apoptosis vs cell cycle arrest. The finding that ASPP2 acts
as cofactor for p53 but possibly only for the activation of specific target
genes provides support to a proposed mechanism explaining how differential
transactivation by p53 and the specificity of the p53-induced cellular
responses are achieved [110, 208]. Namely, cell- type-dependent availability of
cofactors may decide which pathway, cell cycle arrest or apoptosis, is
induced by p53 in response to a stress signal. Consistent with this, a recent
2-hybrid screen identified the homeodomain-interacting protein kinase-2
(HIPK2) as a p53 cofactor. HIPK2 phosphorylates p53 at serine 46,
stimulates p53 transactivation and mediates the induction of apoptosis [40].
In addition to p53-binding factors and modifying proteins, the intrinsic
DNA-binding affinity of p53 toward the many related REs in target genes
can play an important role in differential transactivation. As discussed below
(see section 2.6), yeast-based functional assays have provided tools to assess
this possibility.
2.5 Identification of p53 mutants exhibiting altered

transactivation specificity
Several mammalian transcription factors including p53 recognize a broad

variety of REs that belong to a rather loose consensus [133]. Little is known
about the mechanisms determining recognition and levels of binding. The
proteins interact with only some of the nucleotides in the response elements
and also have contacts with the sugar/phosphate backbone. The DNA-
binding affinity is affected by conformational changes of the protein:DNA
complex and this is particularly important when the transcription factor is a
multimer (as hetero- and homodimers or tetramers) [130, 143]. The effects
of DNA sequence context in determining structural features of the DNA
elements, such as bending of the double helix, are not well understood. In
the case of the p53 transcription factor which coordinates multiple biological
responses, the loose consensus sequence suggests that p53 variants with
altered DNA sequence binding specificity might lead to selective down-
stream responses among the multiple p53 pathways. For example, changes in
the amino acids 120, 248, 277 (including C277R), and 283 that contact the
DNA have been examined with in vitro DNA-binding assays and trans-
activation in yeast [200]. The impact of base changes in the p53 consensus
was also examined. Interestingly, while all mutations showed a defect in
binding to the canonical DNA element, some mutants exhibited WT or even
greater activity toward specific sequences.
Yeast-based selection screens have been developed to isolate p53

mutants with altered DNA sequence binding specificity. Freeman et al. [60]
selected p53 mutants with enhanced activity using only one of the two
inverted dimers within a RE (i.e., a half-site consisting of 5′ RRR-CWWG-
YYY) to mediate transactivation. Two mutations in the conserved domain II
of the DNA-binding domain (L1 loop, S121F, and T123A) were identified.
They also varied the content of the Gs and As in the purine (R) tract and
found that both 121F and 123A were more active than WT with a G-rich RE
(5′ GGGCW-3′). With other sequences, the mutants were less than or equal
to WT p53 in acitivity.
An alternative approach to identifying p53 mutants has involved reducing
the amount of p53 protein to a low level using the GAL1 rheostatable
promoter and using complete REs [87]. Using this approach we have
identified p53 toxic mutants, that prevent growth at moderate to high levels
of expression and can lead to inviability [89]. This screen was based on
previous results that showed a direct correlation between the growth
inhibition phenotype and the ability of p53 to act as transcription factor in
yeast. We hypothesized that mutants with a greater impact than WT p53 on
growth of yeast may possess enhanced DNA binding/transactivation. Among
the many mutants some (including T123A) appeared more active with all the
p53 REs examined at low levels of p53 and were termed supertrans (see
section 2.1.5). Other mutants exhibited altered DNA-binding selectivity
including enhanced, reduced, and loss-of-function depending on the RE
used. These mutants had amino acid changes in the L1 loop (positions 122
and 125) and in the helix II (the DNA contact sites 277 and 279). Most
mutants were at amino acids that do not directly contact DNA.
Saller et al. [178] investigated the impact of S121F and C277R when
expressed in mammalian primary and tumor cell lines. Interestingly, the
altered DNA-binding specificity in yeast was also observed in mammalian
cells both with gene reporter assays and endogenous gene expression. This
suggests that the intrinsic DNA-binding affinities of p53 mutants toward
specific REs assessed by the yeast functional assay can predict the behavior
of a p53 mutant in mammalian cells. In a subsequent study this group
compared the in vivo DNA binding of WT p53, S121F, and C277R at six
promoters using chromatin immunoprecipitation assays in mammalian cells
[101]. Also they correlated DNA binding with transactivation determined by
real-time PCR of four genes. S121F appeared defective for binding and
transactivation of p21, MDM2, and PIG3, but was slightly more active than
WT p53 at the bax promoter. 277R was defective or partially defective at
p21 and bax, but active at MDM2 and PIG3. These results are again
consistent with predictions of the yeast functional assays. Furthermore, the
ChIP data showed differences in promoter occupancy by WT p53 as well as

the different p53 mutants.
Overall, these results suggest that differences in intrinsic DNA binding of
p53 toward the many REs are important factors in p53 differential
transactivation and can play a significant role in the biological consequences
of p53 mutants. This is exemplified by our recent analysis of the murine
T122L p53 mutation hot spot [88]. This mutation was found in 40% of UV-
induced skin tumors in Xpc–/– p53+/– mice but was never detected in UV-
induced skin cancers in WT mice and was rarely observed in Xpc+/– p53+/–
Xpc–/– p53+/+, or Xpa–/– mice. The yeast functional assay at low levels of
T122L expression revealed altered transactivation specificity that included
enhanced, reduced, and loss-of-function for specific REs. Interestingly,
transactivation was impaired with some REs from apoptotic genes but it was
normal or even slightly enhanced with REs from cell cycle control and DNA
repair genes. The altered DNA-binding affinity and specificity of the T122L
mutant protein suggest that it would lead to an altered pattern of expression
of downstream p53-regulated genes in vivo in skin cells and that this could
contribute to its selection. The balance between the defect in apoptosis
induction and the retained activity in DNA repair and cell cycle control may
become advantageous specifically in XPC–/– cells that are proficient for
transcription coupled repair and exhibit a WT like p53 response to DNA
damage. In WT and Xpa–/– cells dominant-negative loss-of-function p53
mutants would instead be more advantageous to reduce p53-dependent and
independent apoptosis.
2.6 Functional classification of p53 response elements

in yeast reveals a wide range of transactivation
capacities
The yeast functional assay provides the means to specifically address

the role of p53 protein levels and p53 intrinsic DNA-binding affinity in
differential transactivation of target genes. Recent work using in vitro
preparation of chromatinized DNA and in vivo ChIP assays suggested that
p53 protein in human cells could be active for DNA binding even in the
absence of posttranslational modifications [9, 12, 52, 196]. Furthermore,
changes in promoter occupancy resulting from stress-induced signals
reflected more directly the changes in p53 levels rather than modifications
in relative binding affinity [101]. However, chromatin modification
induced by p53 binding to promoter sequences and recruitment of histone
acetylases is likely very important in transactivation although effects are
locus dependent [165].
We have developed a set of isogenic yeast strains that differ only for a
single p53 response element cloned upstream of a minimal CYC1promoter-
ADE2 open reading frame at the ADE2 locus [91]. Thus, all the possible
factors affecting p53 transactivation are kept equal and the role of binding
affinity and p53 protein amount can be precisely evaluated. To construct
these strains we took advantage of a recently developed in vivo mutagenesis
approach (discussed below) that allows for rapid modifications of yeast
sequences using oligonucleotides that are transformed directly into yeast
[192]. Thirty strains, each with a different p53 RE derived from mammalian
p53-responsive genes, were constructed [91]. Genes involved in cell cycle
control, apoptosis, DNA repair/replication, and p53 regulation were included
in this broad panel of REs to examine various features of WT and mutant
p53s.
To understand the importance of p53 levels in differential transactivation,
we defined conditions for the rheostatable induction of p53 under the
GAL1 promoter. Nearly linear increase of p53 over a 100-fold range could
be accomplished using a range of galactose concentrations from 0.001% to
0.12% added to medium containing 2% raffinose [91]. Transactivation of the
ADE2 reporter gene could be assessed using the phenotypic (red/pink/white)
assay and also by quantification of ADE2 mRNA with real-time PCR
measurements. The p53 REs were ranked according to their relative activity.
Surprisingly, there was a 1000-fold range in transcriptional activation bet-
ween the various RE sequences. Statistical models that estimate binding
energy based on nucleotide usage and the degree of heterology of a RE from
the consensus [16] predicted only small variations and these did not correlate
with the functional results. We found that the DNA sequence CATG at the
junction of p53 monomer binding sites greatly affects p53 transactivation
capacity, possibly due to structural properties such as bending [143]. The
absence of spacing between dimmer-binding sites is also crucial for high
transactivation capacity by WT p53. This result contrasts with in vitro DNA-
binding assays [50] but confirms previous observation with yeast-based
transactivation [202].
Our results suggest that intrinsic DNA-binding affinity as well as p53
protein levels are important contributors to p53-induced differential trans-
activation. WT p53 had weak activity toward half the apoptotic REs,
particularly those active on the mitochondrial pathway of programmed cell
death. Activation of these targets requires high levels of p53. Possibly
specific coactivators that are missing in yeast or the combinatorial action of
multiple transcription factors play an important role in transactivation of
these REs.
Qian et al. [166] recently reported the construction of an even larger
panel of REs and surrounding promoter regions into a yeast expression
plasmid as well as a luciferase vector for transactivation experiments in

mammalian cells. While they only used high, constitutive expression of p53
and a different p53-dependent reporter, their results are consistent with ours.
Interestingly, luciferase assays in mammalian cells were completely con-
sistent with the yeast results, suggesting that the yeast-based observation can
predict p53 transactivation capacity in transient assays in cell lines [166].
2.7 Functional assays for p53 family members

and their interactions with p53 mutants
Given the key role of p53 protein in the stress-induced cellular responses
it was surprising that lack of this gene does not result in severe defects
during embryonic development in mice. However, inappropriate expression
of p53 can lead to embryonic lethality or increased risk of malformations
[37, 44, 76]. Two p53 homologues, p63 and p73, have been identified [99,
115]. The p63 and p73 genes might compensate for the p53 defects in
germline and somatic cells. p73 was initially proposed to be a tumor
suppressor gene in neuroblastoma due to its chromosomal location at 1p36.2
[191]. This region is frequently associated with loss of heterozygosity (LOH)
in other types of cancer.
Although there is limited evidence of an alternative spliced form of p53
[56], multiple spliced variants at the Cter and an alternative promoter at the
Nter have been described for p63 and p73. Six variants of p73 with differing
Cter structures (alpha, beta, gamma, delta, epsilon, and xi) and three of p63
have been reported [191]. The relative abundance of these protein variants
varies according to the cell type and in tumor cells. For example, the Νter
variants of both p63 and p73 are highly expressed (as much as 10 times more
than p53) in epithelial and brain tumors where they appear to have
oncogenic properties [39, 45]. The structural organization of the p63 and p73
proteins resembles p53 and comprises a transactivation domain (~20–30%
homology to p53), a DNA-binding domain (~60–70% homology), a
tetramerization domain (~30–40% homology), and a long carboxy terminus
of uncertain function [6]. KO mice have been obtained both for p63 and p73.
Unlike the p53 KO, growth defects were observed. The p73 KO showed
chronic inflammation, infections, and hippocampal dysgenesis [218]. It has
been proposed that the Nter variants act as neuroprotective factors possibly
by inhibiting p53-dependent apoptosis. Interestingly, no increase in tumor
incidence was reported in these mice. The p63 KO had a lack of mammary
glands and defects in limb formation [217].
Mutations in the DNA-binding mutations of the p63 gene have been
observed in families affected by the autosomal dominant disorder EEC
characterized by ectrodactyly, ectodermal dysplasia, and facial clefts [33].

These and other observations suggest that defects in p63 are associated with
alteration of the asymmetric cell division of epithelial stem cells [158].
Recent evidence suggests that p63 and p73 play a role in stress-responses;
both genes exhibit changes in expression levels and posttranslational
modifications [57, 117]. For example, UV-radiation can lead to a reduction
in the expression of the Nter p63 variants and this correlates with p53
stabilization. This suggested that p63- as well as p73-variants that lack the
transactivation domain may function to override and restrain p53 function.
However, there appears to be cooperation of p63 and p73 with p53 in the
induction of apoptosis. p63 and p73 variants that contain the transactivation
domain can transactivate p53 target genes in experimental systems [157,
216]. Different transactivation capacities were reported for the various Cter
variants. It is possible that while the Nter variants inhibit apoptosis
physiologically during development, in defined stages or cell types and upon
aberrant expression in somatic tumor cells, the other variants could exhibit
tumor suppressive functions and be particularly active in stress-induced
apoptotic responses [92, 100, 190]. In this respect, it is also possible that
abundantly expressed mutant forms of p53 could inhibit p63 and p73 (as
well as p53 when heterozygous) through physical interactions that are not
mediated by the tetramerization domains [63, 193, 194].
Yeast-based functional assays have been developed to address the
transactivation function of p63 and p73 isoforms, to identify functional
mutants in tumor samples and to evaluate the effects on functional inter-
action between p73 isoforms and hotspots p53 mutants. A functional assay
for p63 transactivation function has been developed based on the HIS3
reporter gene and a quantifiable assay based on expression of GFP [104,
184]. p63 can use p53 REs to stimulate transcription but the degree of
transactivation differs from that of p53. Although creation of p53 equivalent
hot spot mutants led to loss of p63 function, the rare p63 missense changes
identified in tumors do not appear to affect protein activity. This confirms
that p63 is not a target for mutational inactivation in tumors. Nozaki et al.
[150] developed an ADE2 - based functional assay for p73 splice variants.
They reported that different p73 variants were active to different degrees
with p53 response elements. Functional analysis showed that p73 is not
mutated in meningiomas, although LOH at 1p36 is common in these tumors.
2.8 Future directions: determining functional

fingerprints of tumor-associated p53 alleles using
quantitative yeast-based assays
There is considerable evidence suggesting that knowledge of the p53

status of tumors and of the functional defect of a specific p53 mutation may
be relevant to prognosis and to tailoring cancer therapy to individual patient
needs. The precise functional status of p53 mutations may influence tumor
responsiveness to chemo- and radiotherapy, the effect of viral particles
targeting p53-defective cells or reintroducing WT p53 and the efficacy of
small molecules in reactivating p53 alleles. Furthermore, the residual
capacity of each mutant to induce a set of responses, to influence the activity
of the remaining WT p53 allele or that of the p73 and p63 protein variants
may drive tumor development through different selective pressures. In fact,
p53-dependent modulation of transcription is broad and flexible and can lead
to different biological outcomes, and mutants may preferentially affect the
expression of downstream genes involved in specific pathways (see section
2.1.3 and 2.1.4). It is, therefore, important to evaluate transactivation
capacity of p53 mutants using many REs under conditions where protein
expression can be varied. The same holds true for investigations of
dominance, the ability of a mutant to interfere with p53 family members or
the reactivation of p53 mutants. As transient transfection experiments in
mammalian cells have shown, transactivation assays with overexpressed p53
alleles under viral promoters may poorly represent physiological conditions.
The yeast assay we described has several features that make it appealing
for p53 studies. It is rapid, relatively inexpensive, versatile, and highly
reproducible; it can assess p53 missense as well as nonsense mutant proteins,
and loss-of-function as well as subtle mutations. Here we present a strategy
that improves the throughput of the screen.
To take into account the levels of p53 expression in the transactivation
assay, we have developed a protocol that provides for functional classify-
cation of any p53 mutant using quantitative yeast assays with tight control of
p53 expression [91]. The scheme is presented in Figure 4A and 4B. p53 can
be expressed from a selectable centromeric plasmid. For rapid modification
of the p53 sequence we have also constructed a yeast host strain by
integrating a single copy of the GAL1:p53 expression cassette into the
genome in order to utilize our recently developed delitto perfetto in vivo
mutagenesis system [192]. A CORE cassette (CO = counterselectable
KLURA3; RE = reporter kanMX4 gene) is inserted at a specific location
within the p53 cDNA sequence. Four isogenic derivatives of the host strain,
each with a single CORE cassette positioned at 200 nucleotides intervals of
Figure 4A. Development of a p53 mutant functionality database. Construction of any

missense p53 mutant can be accomplished rapidly with the delito perfetto in vivo mutagenesis
system [192]. The GAL1:p53 expression cassette is cloned into a yeast chromosomal locus to
obtain a p53-host strain. A number of isogenic derivatives of this strain are created that have a
CORE cassette (containing a counterselectable gene and a reporter gene that identifies
cassette integration) [192] inserted at different sites (amino acid 250 in the example, Jordan
and Resnick, unpublished). By placing a CORE cassette at 200 nucleotide intervals, the entire
p53 can be subjected to mutagenesis. Introduction of oligonucleotides that surround the
CORE allows for creation of specific mutants (R273H in the example) or the generation of
many different mutants in a small region if degenerate oligonucleotides are used. Only DNA
sequencing of the region surrounding the replacement site is needed to confirm the nature of
the induced mutation(s) [192].
the p53 cDNA, enables easy production of all possible single missense
mutants between amino acids 50 and 350. The mutant construction is
achieved by simple replacement of the CORE cassette (selected on 5-FOA
and confirmed by kanamycin sensitivity) with oligonucleotides. The
oligonucleotide can be specific for a single desired mutation or degenerate in
order to randomly mutate the region of interest. Limited DNA sequencing of
the replaced fragment can confirm and/or identify the mutants [192]. The
host strain with a given p53 allele is then mated (Figure 4B) to a set of 20
isogenic strains representing a p53 functional array. Each of these strains
can test p53 transactivation using a reporter gene construct that only differs
for the sequence of a p53 RE. Cell cycle checkpoint, DNA repair/replication,
apoptosis (both receptor and mitochondrial pathway), and p53-regulatory

genes are included. The functional array also covers a wide range of intrinsic
DNA-binding affinities (see section 2.6). Diploids are selected and the
transactivation capacity of the p53 mutants is assessed at different levels of
protein expression using replica-plating of purified diploids on different
concentrations of the galactose inducer. The same procedure can be used to
evaluate p53 dominance toward WT p53, p63, or p73 when expressed by an
identical cassette from a centromeric plasmid. Pharmacological reactivation
of p53 mutants can be similarly evaluated and also more precisely quantified
using the luciferase reporter gene.
Strategy for the determination of p53 allele functional fingerprints using the p53 Functional Array
x
Mate
or
MATα
MATa
p53 Functional Array:
Yeast “p53-host” strain contains an > 20 isogenic strains that differ only
integrated p53 allele or a p53 in the sequence of a p53 response
expression vector element (e.g., P21, BAX, MDM2,
PIG3, AIP1, NOXA, GADD45, etc.)
cloned upstream of the ADE2 reporter
Select diploids gene
Evaluate transactivation capacity using the color (red/pink/white) assay

by replica plating onto plates containing different amounts of galactose
to induce variable p53 expression.
Figure 4B. Transactivation assays using the functional array of p53 response elements. Over
20 isogenic yeast strains that differ only in the sequence of a p53 response element upstream
of the p53 reported gene ADE2 (the luciferase reporter gene can also be used to quantify
transactivation) are mated with the p53-host strain expressing a given p53 mutant. Diploids
are selected on suitable media and then tested with the phenotypic transactivation assay to
determine the functional fingerprint of the p53 allele being tested. The analysis of the
dominance potential of p53 mutants can be performed when the p53-host strain is transformed
with a second GAL1:WT p53 (or p63, or p73) expression cassette.
We have analyzed 20 tumor-associated p53 alleles for transactivation

[222] and dominance (unpublished) using this protocol. The alleles represent
different functional classes including loss-of-function, WT, and promoter-
selective. In particular, we tested a group of p53 mutations associated with
familial breast cancer from patients affected by BRCA1 germline mutations.
Under conditions of high expression these p53 mutants appeared as active as
WT p53, both in mammalian and yeast functional assays [31, 187].
Interestingly, analysis with many p53 response elements at low and variable
p53 expression clearly distinguished all mutants from WT p53. Several

mutations showed residual function with at least half of the REs. The group
of mutants associated with familial breast cancer exhibited subtle defects
comprising both enhanced and reduced function with different promoters.
Promoter-selective mutants that were classified as WT with a P21 RE (but
mutant with BAX) instead showed reduced activity compared to WT p53 at
low levels of expression. Similar reduction, but not loss of activity, was
observed with other REs that are highly responsive to p53. The dominance
assay at various low p53 expression levels was sensitive to p53 gene dosage in
that one and two copies of the GAL1:WT p53 expression cassette were
phenotypically distinguishable. Different degrees of dominance were revealed
with a group of loss-of-function p53 mutants when heterozygous. Among the
p53 alleles retaining partial function some were completely recessive (i.e., it
appeared as if two WT copies of p53 were expressed) while others led to a
phenotype corresponding to a reduction in gene dosage (i.e., hemizygous for
WT p53).
2.9 Concluding comments: development of a p53-mutant

functionality database and relevance to other
transcription factors
While many p53 mutations associated with cancer may retain partial
transactivation function and although several classification methods for p53
mutants have been attempted, it is not presently possible to predict a priori
the behavior of a mutant protein. The yeast functional assays provide an
important contribution to the functional classification of p53 alleles. It
appears that the combination of low and variable expression of p53 alleles
and the assessment of the transactivation capacity using many p53 response
elements provides a greater sensitivity for the functional classification of p53
mutants than the standard yeast assays using high-constitutive p53 expres-
sion. When combined with the efficient method for constructing p53 alleles
using in vivo mutagenesis and with the high-throughput selection and
screening system using diploids formed between “p53-host” and “the p53
functional array” strains (see Figure 4) these tools allow for rapid develop-
ment of p53 functional fingerprints. Hence, the yeast functional assay offers
a practical means to develop a p53 mutant functionality database that
enables the functional fingerprints for all the p53 mutants relevant to cancer
to be linked with the IARC p53 mutation database. This information will
become valuable in understanding the correlation between p53 functional
status and tumor aggressiveness and responsiveness to therapy, thus
providing directions for effective patient management in the clinical setting.
An example of functional classification of 24 p53 mutants with 15 p53

REs is presented in Figure 5 (222; see also Recent Developments). As a
general trend, transactivation capacity toward REs from apoptotic genes is
more severely affected compared to cell cycle arrest and DNA repair genes.
Five functional classes of p53 mutants can be identified [222]. For example,
supertrans mutants, such as 121F and 123A, showed enhanced capacity with
all or most of the REs. The V122A mutation showed instead a preferential
increase in activity with a subset of Res. The mutant S215C that has been
reported in familial breast cancer associated with BRCA1-germline
mutations, showed subtle differences compared to WT p53. Interestingly, it
showed a slight increase in transactivation capacity toward a few REs. The
mutant R282Q is weak and shows a reduction in transactivation for all REs
that range from ~50% to ~5%. Two mutants at codon 277 have split
functions with enhanced activity toward some REs of the cell cycle arrest
group, but loss-of-function with all apoptotic REs. Finally, three mutants
appear to have almost completely lost transactivation potential.
The system developed for assessment of p53 function can be applied to
many sequence-specific transactivation factors. The combination of tight
regulation of protein expression, rapid construction of responsive promoters,
and phenotypic/quantitative assessment of transcription levels offers oppor-
tunities to investigate transactivation specificity for several families of trans-
cription factors that recognize diverged promoter elements. The approach
provides the means to study altered selectivity, intrinsic DNA binding, and
differential transactivation in vivo, to isolate and characterize enhancer
elements as well as cofactors, corepressors, and external modifiers of the
transcription complex assembly.
ACKNOWLEDGMENTS
Our thanks to Drs. Robbert Slebos, Daniel Menendez, Dmitry Gordenin,

Tom Darden, Gilberto Fronza for advice, helpful discussions and comments
on the manuscript. We also want to acknowledge that there are many
additional valuable contributions and directions that have been made in the
vast p53 field which we were unable to include in this review because of
space limitations.
REFERENCES
1. Aas, T., A. L. Borresen, S. Geisler, B. Smith-Sorensen, H. Johnsen, J. E.
Varhaug, L. A. Akslen, and P. E. Lonning. 1996. Specific p53 mutations are
associated with de novo resistance to doxorubicin in breast cancer patients. Nat.
Med. 2:811–814.
2. Ahrendt, S. A., S. Halachmi, J. T. Chow, L. Wu, N. Halachmi, S. C. Yang,
S. Wehage, J. Jen, and D. Sidransky. 1999. Rapid p53 sequence analysis in
primary lung cancer using an oligonucleotide probe array. Proc. Natl. Acad. Sci.
USA 96:7382–7387.
3. Albrechtsen, N., I. Dornreiter, F. Grosse, E. Kim, L. Wiesmuller, and
W. Deppert. 1999. Maintenance of genomic integrity by p53: complementary roles
for activated and non-activated p53. Oncogene 18:7706–7717.
4. Anderson, L., C. Henderson, and Y. Adachi. 2001. Phosphorylation and rapid
relocalization of 53BP1 to nuclear foci upon DNA damage. Mol. Cell. Biol.
21:1719–1729.
5. Appella, E., and C. W. Anderson. 2001. Post-translational modifications and
activation of p53 by genotoxic stresses. Eur. J. Biochem. 268:2764–2772.
6. Arrowsmith, C. H. 1999. Structure and function in the p53 family. Cell Death
Differ. 6:1169–1173.
7. Ashcroft, M., Y. Taya, and K. H. Vousden. 2000. Stress signals utilize multiple
pathways to stabilize p53. Mol. Cell. Biol. 20:3224–3233.
8. Aurelio, O. N., X. T. Kong, S. Gupta, and E. J. Stanbridge. 2000. p53 Mutants
have selective dominant-negative effects on apoptosis but not growth arrest in
human cancer cell lines. Mol. Cell. Biol. 20:770–778.
9. Ayed, A., F. A. Mulder, G. S. Yi, Y. Lu, L. E. Kay, and C. H. Arrowsmith.
2001. Latent and active p53 are identical in conformation. Nat. Struct. Biol.
8:756–760.
10. Baas, I. O., F. M. van den Berg, J. W. Mulder, M. J. Clement, R. J. Slebos,
S. R. Hamilton, and G. J. Offerhaus. 1996. Potential false-positive results with
antigen enhancement for immunohistochemistry of the p53 gene product in
colorectal neoplasms. J. Pathol. 178:264–267.
11. Bargonetti, J., I. Reynisdottir, P. N. Friedman, and C. Prives. 1992. Site-
specific binding of wild-type p53 to cellular DNA is inhibited by SV40 T antigen
and mutant p53. Genes Dev. 6:1886–1898.
12. Barlev, N. A., L. Liu, N. H. Chehab, K. Mansfield, K. G. Harris, T. D. Halazonetis,
and S. L. Berger. 2001. Acetylation of p53 activates transcription through recruit-
ment of coactivators/histone acetyltransferases. Mol. Cell. 8:1243–1254.
13. Baroni, T. E., T. Wang, H. Qian, L. R. Dearath, L. Truong, J. Zeng, A. E.
Denes, S. W. -Y. Chen, and R. K. Brachmann. 2004. A global suppressor motif
for p53 cancer mutants. Proc. Natl. Acad. Sci. USA 101:4930–4935.
14. Bassett, D. E., Jr., M. S. Boguski, and P. Hieter. 1996. Yeast genes and human
disease. Nature 379:589–590.
15. Bennett, W. P., S. P. Hussain, K. H. Vahakangas, M. A. Khan, P. G. Shields,
and C. C. Harris. 1999. Molecular epidemiology of human cancer risk: gene-
environment interactions and p53 mutation spectrum in human lung cancer. J.
Pathol. 187:8–18.
16. Berg, O. G., and P. H. von Hippel. 1987. Selection of DNA binding sites by
regulatory proteins. Statistical-mechanical theory and application to operators and
promoters. J. Mol. Biol. 193:723–750.
17. Bischoff, J. R., D. Casso, and D. Beach. 1992. Human p53 inhibits growth in
Schizosaccharomyces pombe. Mol. Cell. Biol. 12:1405–1411.
18. Bischoff, J. R., D. H. Kirn, A. Williams, C. Heise, S. Horn, M. Muna, L. Ng, J.
A. Nye, A. Sampson-Johannes, A. Fattaey, and F. McCormick. 1996. An
adenovirus mutant that replicates selectively in p53deficient human tumor cells.
Science 274:373–376.
19. Blagosklonny, M. V., P. Giannakakou, L. Y. Romanova, K. M. Ryan, K. H.
Vousden, and T. Fojo. 2001. Inhibition of HIF-1- and wild-type p53-stimulated
transcription by codon Arg175 p53 mutants with selective loss of functions.
20. Blandino, G., A. J. Levine, and M. Oren. 1999. Mutant p53 gain of function:
differential effects of different p53 mutants on resistance of cultured cells to
chemotherapy. Oncogene 18:477–485.
21. Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants
lacking orotidine-5’-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid
resistance. Mol. Gen. Genet. 197:345–346.
22. Bonk, T., A. Humeny, C. Sutter, J. Gebert, M. von Knebel Doeberitz, and
C. M. Becker. 2002. Molecular diagnosis of familial adenomatous polyposis (FAP):
genotyping of adenomatous polyposis coli (APC) alleles by MALDI-TOF mass
spectrometry. Clin. Biochem. 35:87–92.
23. Bowman, K. J., Y. Guichard, L. Langford, T. O’Connor, M. N. Routledge,
G. Scott, P. A. Burns, and D. D. Jones. 2002. Correlation of in vitro oxidative
damage formation and mutation at nucleotide resolution in p53. Proc. Am. Assoc.
for Cancer Res. 43:699.
24. Brachmann, R. K., M. Vidal, and J. D. Boeke. 1996. Dominant-negative p53
mutations selected in yeast hit cancer hot spots. Proc. Natl. Acad. Sci. USA
93:4091–4095.
25. Brachmann, R. K., K. Yu, Y. Eby, N. P. Pavletich, and J. D. Boeke. 1998.
Genetic selection of intragenic suppressor mutations that reverse the effect of
common p53 cancer mutations. EMBO J. 17:1847–1859.
26. Bullock, A. N., J. Henckel, and A. R. Fersht. 2000. Quantitative analysis of
residual folding and DNA binding in mutant p53 core domain: definition of mutant
states for rescue in cancer therapy. Oncogene 19:1245–1256.
27. Bykov, V. J., N. Issaeva, A. Shilov, M. Hultcrantz, E. Pugacheva,
P. Chumakov, J. Bergman, K. G. Wiman, and G. Selivanova. 2002. Restoration of
the tumor suppressor function to mutant p53 by a low-molecular-weight compound.
Nat. Med. 8:282–288.
28. Camplejohn, R. S., and J. Rutherford. 2001. p53 Functional assays: detecting
p53 mutations in both the germline and in sporadic tumours. Cell Prolif. 34:1–14.
29. Campomenosi, P., M. Conio, M. Bogliolo, S. Urbini, P. Assereto, A. Aprile,
P. Monti, H. Aste, G. Lapertosa, A. Inga, A. Abbondandolo, and G. Fronza. 1996.
p53 is frequently mutated in Barrett’s metaplasia of the intestinal type. Cancer
Epidemiol. Biomarkers Prev. 5:559–565.
30. Campomenosi, P., G. Fronza, L. Ottaggio, S. Roncella, A. Inga, M. Bogliolo,
P. Monti, P. Assereto, F. Moro, G. Cutrona, S. Bozzo, N. Chiorazzi, A.
Abbondandolo, and M. Ferrarini. 1997. Heterogeneous p53 mutations in a
Burkitt lymphoma from an AIDS patient with monoclonal c-myc and VDJ
rearrangements. Int. J. Cancer 73:816–821.
31. Campomenosi, P., P. Monti, A. Aprile, A. Abbondandolo, T. Frebourg,
B. Gold, T. Crook, A. Inga, M. A. Resnick, R. Iggo, and G. Fronza. 2001. p53
Mutants can often transactivate promoters containing a p21 but not Bax or PIG3
responsive elements. Oncogene 20:3573–3579.
32. Candau, R., D. M. Scolnick, P. Darpino, C. Y. Ying, T. D. Halazonetis, and S. L.
Berger. 1997. Two tandem and independent sub-activation domains in the amino
terminus of p53 require the adaptor complex for activity. Oncogene 15:807–816.
33. Celli, J., P. Duijf, B. C. Hamel, M. Bamshad, B. Kramer, A. P. Smits,
R. Newbury-Ecob, R. C. Hennekam, G. Van Buggenhout, A. van Haeringen,
G. Woods, A. J. van Essen, R. de Waal, G. Vriend, D. A. Haber, A. Yang,
F. McKeon, H. G. Brunner, and H. van Bokhoven. 1999. Heterozygous germline
mutations in thep53 homolog p63 are the cause of EEC syndrome. Cell 99:143–153.
34. Chappuis, P. O., A. Estreicher, B. Dieterich, H. Bonnefoi, M. Otter, A. P.
Sappino, and R. Iggo. 1999. Prognostic significance of p53 mutation in breast
cancer: frequent detection of non-missense mutations by yeast functional assay. Int.
J. Cancer 84:587–593.
35. Chen, J. M., R. Rosal, S. Smith, M. R. Pincus, and P. W. Brandt-Rauf. 2001.
Common conformational effects of p53 mutations. J. Protein Chem. 20:101–105.
36. Cho, Y., S. Gorina, P. D. Jeffrey, and N. P. Pavletich. 1994. Crystal structure of
a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations.
Science 265:346–355.
37. Choi, J., and L. A. Donehower. 1999. p53 in embryonic development:
maintaining a fine balance. Cell Mol. Life Sci. 55:38–47.
38. Clayman, G. L., A. K. el-Naggar, S. M. Lippman, Y. C. Henderson,
M. Frederick, J. A. Merritt, L. A. Zumstein, T. M. Timmons, T. J. Liu,
L. Ginsberg, J. A. Roth, W. K. Hong, P. Bruso, and H. Goepfert. 1998.
Adenovirus-mediated p53 gene transfer in patients with advanced recurrent head
and neck squamous cell carcinoma. J. Clin. Oncol. 16:2221–2232.
39. Crook, T., J. M. Nicholls, L. Brooks, J. O’Nions, and M. J. Allday. 2000. High
level expression of deltaNp63: a mechanism for the inactivation of p53 in
undifferentiated nasopharyngeal carcinoma (NPC)? Oncogene 19:3439–3444.
40. D’Orazi, G., B. Cecchinelli, T. Bruno, I. Manni, Y. Higashimoto, S. Saito,
M. Gostissa, S. Coen, A. Marchetti, G. Del Sal, G. Piaggio, M. Fanciulli,
E. Appella, and S. Soddu. 2002. Homeodomain-interacting protein kinase-2
phosphorylates p53 at Ser 46 and mediates apoptosis. Nat. Cell Biol. 4:11–19.
41. deVere White, R. W., A. D. Deitch, P. H. Gumerlock, and X. B. Shi. 1999. Use
of a yeast assay to detect functional alterations in p53 in prostate cancer: review and
future directions. Prostate 41:134–142.
42. Di Como, C. J., and C. Prives. 1998. Human tumor-derived p53 proteins exhibit
binding site selectivity and temperature sensitivity for transactivation in a yeast-
based assay. Oncogene 16:2527–2539.
43. DiGiammarino, E. L., A. S. Lee, C. Cadwell, W. Zhang, B. Bothner, R. C.
Ribeiro, G. Zambetti, and R. W. Kriwacki. 2002. A novel mechanism of
tumorigenesis involving pH-dependent destabilization of a mutant p53 tetramer.
Nat. Struct. Biol. 9:12–16.
44. Donehower, L. A., M. Harvey, B. L. Slagle, M. J. McArthur, C. A. Montgomery,

Jr., J. S. Butel, and A. Bradley. 1992. Mice deficient for p53 are developmentally
normal but susceptible to spontaneous tumours. Nature 356:215–221.
45. Douc-Rasy, S., M. Barrois, M. Echeynne, M. Kaghad, E. Blanc, G. Raguenez,
D. Goldschneider, M. J. Terrier-Lacombe, O. Hartmann, U. Moll, D. Caput,
and J. Benard. 2002. DeltaN-p73alpha accumulates in human neuroblastic tumors.
Am. J. Pathol. 160:631–639.
46. Duddy, P. M., A. M. Hanby, D. M. Barnes, and R. S. Camplejohn. 2000.
Improving the detection of p53 mutations in breast cancer by use of the FASAY, a
functional assay. J. Mol. Diagn. 2:139–144.
47. Dudenhoffer, C., G. Rohaly, K. Will, W. Deppert, and L. Wiesmuller. 1998.
Specific mismatch recognition in heteroduplex intermediates by p53 suggests a role
in fidelity control of homologous recombination. Mol. Cell. Biol. 18:5332–5342.
48. Dumaz, N., and D. W. Meek. 1999. Serine15 phosphorylation stimulates p53
transactivation but does not directly influence interaction with HDM2. EMBO J.
18:7002–7010.
49. El-Deiry, W. S. 1998. Regulation of p53 downstream genes. Semin. Cancer Biol.
8:345–357.
50. El-Deiry, W. S., S. E. Kern, J. A. Pietenpol, K. W. Kinzler, and B. Vogelstein
1992. Definition of a consensus binding site for p53. Nat. Genet. 1:45–49.
51. Elkhuizen, P. H., H. J. van Slooten, P. C. Clahsen, J. Hermans, C. J. van de
Velde, L. C. van den Broek, and M. J. van de Vijver. 2000. High local
recurrence risk after breast-conserving therapy in node-negative premenopausal
breast cancer patients is greatly reduced by one course of perioperative
chemotherapy: A European organization for research and treatment of cancer,
Breast Cancer Cooperative Group Study. J. Clin. Oncol. 18:1075–1083.
52. Espinosa, J. M., and B. M. Emerson. 2001. Transcriptional Regulation by p53
through intrinsic DNA/chromatin binding and site-directed cofactor recruitment.
Mol. Cell 8:57–69.
53. Farmer, G., J. Colgan, Y. Nakatani, J. L. Manley, and C. Prives. 1996.
Functional interaction between p53, the TATA-binding protein (TBP), andTBP-
associated factors in vivo. Mol. Cell Biol. 16:4295–4304.
54. Flaman, J. M., T. Frebourg, V. Moreau, F. Charbonnier, C. Martin,
P. Chappuis, A. P. Sappino, I. M. Limacher, L. Bron, J. Benhattar, et al. 1995.
A simple p53 functional assay for screening cell lines, blood, and tumors. Proc.
Natl. Acad. Sci. USA 92:3963–3967.
55. Flaman, J. M., V. Robert, S. Lenglet, V. Moreau, R. Iggo, and T. Frebourg.
1998. Identification of human p53 mutations with differential effects on the bax and p21
promoters using functional assays in yeast. Oncogene 16:1369–1372.
56. Flaman, J. M., F. Waridel, A. Estreicher, A. Vannier, J. M. Limacher,
D. Gilbert, R. Iggo, and T. Frebourg. 1996. The human tumour suppressor gene p53
is alternatively spliced in normal cells. Oncogene 12:813–818.
57. Flores, E. R., K. Y. Tsai, D. Crowley, S. Sengupta, A. Yang, F. McKeon, and
T. Jacks. 2002. p63 and p73 are required for p53-dependent apoptosis in response to
DNA damage. Nature 416:560–564.
58. Frazier, M. W., X. He, J. Wang, Z. Gu, J. L. Cleveland, and G. P. Zambetti.
1998. Activation of c-myc gene expression by tumor-derived p53 mutants requires a
discrete C-terminal domain. Mol. Cell Biol. 18:3735–3743.
59. Frebourg, T., M. Sadelain, Y. S. Ng, J. Kassel, and S. H. Friend. 1994. Equal
transcription of wild-type and mutant p53 using bicistronic vectors results in the wild-
type phenotype. Cancer Res. 54:878–881.
60. Freeman, J., S. Schmidt, E. Scharer, and R. Iggo. 1994. Mutation of conserved
domain II alters the sequence specificity of DNA binding by the p53 protein. EMBO J.
13:5393–5400.
61. Friedler, A., L. O. Hansson, D. B. Veprintsev, S. M. Freund, T. M. Rippin,
P. V. Nikolova, M. R. Proctor, S. Rudiger, and A. R. Fersht. 2002. A peptide that
binds and stabilizes p53 core domain: chaperone strategyfor rescue of oncogenic
mutants. Proc. Natl. Acad. Sci. USA 99:937–942.
62. Fronza, G., A. Inga, P. Monti, G. Scott, P. Campomenosi, P. Menichini,
L. Ottaggio, S. Viaggi, P. A. Burns, B. Gold, and A. Abbondandolo. 2000. The
yeast p53 functional assay: a new tool for molecular epidemiology. Hopes and facts.
Mutat. Res. 462:293–301.
63. Gaiddon, C., M. Lokshin, J. Ahn, T. Zhang, and C. Prives. 2001. A subset of
tumor-derived mutant forms of p53 down-regulate p63 and p73 through a direct
interaction with the p53 core domain. Mol. Cell Biol. 21:1874–1887.
64. Gallagher, W. M., M. Argentini, V. Sierra, L. Bracco, L. Debussche, and
E. Conseiller. 1999. MBP1: a novel mutant p53-specific protein partner with
oncogenic properties. Oncogene 18:3608–3616.
65. Gao, C., R. Ohashi, H. Pu, Y. Inoue, T. Tsuji, M. Miyazaki, and M. Namba
1999. Yeast functional assay of the p53 gene status in 11 cell lines and 26 surgical
specimens of human hepatocellular carcinoma. Oncol. Rep. 6:1267–1271.
66. Gao, J. P., T. Uchida, C. Wang, S. X. Jiang, K. Matsumoto, T. Satoh, S. Minei,
S. Soh, T. Kameya, and S. Baba. 2000. Relationship between p53 gene mutation and
protein expression: clinical significance in transitional cell carcinoma of the bladder. Int.
J. Oncol. 16: 469–475.
67. Geisler, J. P., M. A. Hatterman-Zogg, J. A. Rathe, T. A. Lallas, P. Kirby, and
R. E. Buller. 2001. Ovarian cancer BRCA1 mutation detection: protein truncation test
(PTT) outperforms single strand conformation polymorphism analysis (SSCP). Hum.
Mutat. 18:337–344.
68. Giaccia, A. J., and M. B. Kastan. 1998. The complexity of p53 modulation:
emerging patterns from divergent signals. Genes Dev. 12:2973–2983.
69. Gostissa, M., A. Hengstermann, V. Fogal, P. Sandy, S. E. Schwarz,
M. Scheffner, and G. Del Sal. 1999. Activation of p53 by conjugation to the
ubiquitin-like protein SUMO-1. EMBO J. 18:6462–6471.
70. Goyette, M. C., K. Cho, C. L. Fasching, D. B. Levy, K. W. Kinzler,
C. Paraskeva, B. Vogelstein, and E. J. Stanbridge. 1992. Progression of colorectal
cancer is associated with multiple tumor suppressor gene defects but inhibition of
tumorigenicity is accomplished by correction of any single defect via chromosome
transfer. Mol. Cell Biol. 12:1387–1395.
71. Greenblatt, M. S., W. P. Bennett, M. Hollstein, and C. C. Harris. 1994.
Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular
pathogenesis. Cancer Res. 54: 4855–4878.
72. Greenblatt, M. S., P. O. Chappuis, J. P. Bond, N. Hamel, and W. D. Foulkes.
2001. TP53 mutations in breast cancer associated with BRCA1 or BRCA2 germline
mutations: distinctive spectrum and structural distribution. Cancer Res. 61:4092–4097.
73. Gualberto, A., K. Aldape, K. Kozakiewicz, and T. D. Tlsty. 1998. An oncogenic
form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle
checkpoint control. Proc. Natl. Acad. Sci. USA 95:5166–5171.
74. Hainaut, P. 2002. Tumor-specific mutations in p53: the acid test. Nat. Med. 8:21–23.
75. Hainaut, P., and M. Hollstein. 2000. p53 and human cancer: the first ten thousand
mutations. Adv. Cancer Res. 77:81–137.
76. Hall, P. A., and D. P. Lane. 1997. Tumor suppressors: a developing role for p53.
Curr. Biol. 7: R144–147.
77. Hanahan, D., and R. A. Weinberg. 2000. The hallmarks of cancer. Cell 100:57–70.
78. Hartmann, A., H. Blaszyk, J. S. Kovach, and S. S. Sommer. 1997. The molecular
epidemiology of p53 gene mutations in human breast cancer. Trends Genet. 13:27–33.
79. Hernandez-Boussard, T., P. Rodriguez-Tome, R. Montesano, and P. Hainaut.
1999. IARC p53 mutation database: a relational database to compile and analyze p53
mutations in human tumors and cell lines. International Agency for Research on Cancer.
Hum. Mutat. 14:1–8.
80. Hoos, A., M. J. Urist, A. Stojadinovic, S. Mastorides, M. E. Dudas, D. H.
Leung, D. Kuo, M. F. Brennan, J. J. Lewis, and C. Cordon-Cardo. 2001.
Validation of tissue microarrays for immunohistochemical profiling of cancer specimens
using the example of human fibroblastic tumors. Am. J. Pathol. 158:1245–1251.
81. Hovig, E., B. Smith-Sorensen, A. G. Uitterlinden, and A. L. Borresen. 1992.
Detection of DNA variation in cancer. Pharmacogenetics 2:317–328.
82. Hupp, T. R., and D. P. Lane. 1994. Allosteric activation of latent p53 tetramers. Curr.
Biol. 4:865–875.
83. Hussain, S. P., and C. C. Harris. 1998. Molecular epidemiology of human cancer.
Recent Results Cancer Res. 154:22–36.
84. Inga, A., F. X. Chen, P. Monti, A. Aprile, P. Campomenosi, P. Menichini,
L. Ottaggio, S. Viaggi, A. Abbondandolo, B. Gold, and G. Fronza. 1999. N-(2-
chloroethyl)-N-nitrosourea tethered to lexitropsin induces minor groove lesions at the
p53 cDNA that are more cytotoxic than mutagenic. Cancer Res. 59:689–695.
85. Inga, A., S. Cresta, P. Monti, A. Aprile, G. Scott, A. Abbondandolo, R. Iggo, and G.
Fronza. 1997. Simple identification of dominant p53 mutants by a yeast functional
assay. Carcinogenesis 18:2019–2021.
86. Inga, A., R. Iannone, P. Monti, F. Molina, M. Bolognesi, A. Abbondandolo, R. Iggo,
and G. Fronza. 1997. Determining mutational fingerprints at the human p53 locus with a
yeast functional assay: a new tool for molecular epidemiology. Oncogene 14:1307–1313.
87. Inga, A., P. Monti, G. Fronza, T. Darden, and M. A. Resnick. 2001. p53 Mutants
exhibiting enhanced transcriptional activation and altered promoter selectivity are
revealed using a sensitive, yeast-based functional assay. Oncogene 20:501–513.
88. Inga, A., D. Nahari, S. Velasco-Miguel, E. C. Friedberg, and M. A. Resnick. 2002.
A novel p53 mutational hotspot in skin tumors from UV-irradiated Xpc Mutant mice
alters transactivation functions. Oncogene 21:5704–5715.
89. Inga, A., and M. A. Resnick. 2001. Novel human p53 mutations that are toxic to yeast
can enhance transactivation of specific promoters and reactivate tumor p53 mutants.
Oncogene 20:3409–3419.
90. Inga, A., G. Scott, P. Monti, A. Aprile, A. Abbondandolo, P. A. Burns, and G. Fronza.
1998. Ultraviolet-light induced p53 mutational spectrum in yeast is indistinguishable from
p53 mutations in human skin cancer. Carcinogenesis 19:741–746.
91. Inga, A., F. Storici, T. Darden, and M. A. Resnick. 2002. Differential transactivation
by the p53 transcription factor is highly dependent on p53 level and promoter target
sequence. Mol. Cell. Biol. 22:8612–8625.
92. Irwin, M. S., and W. G. Kaelin, Jr. 2001. Role of the newer p53 family proteins in
malignancy. Apoptosis 6:17–29.
93. Ishii, N., Y. Sawamura, M. Tada, D. M. Daub, R. C. Janzer, M. Meagher-
Villemure, N. de Tribolet, and E. G. Van Meir. 1998. Absence of p53 gene mutations
in a tumor panel representative of pilocytic astrocytomadiversity using a p53 functional
assay. Int. J. Cancer 76:797–800.
94. Ishioka, C., C. Englert, P. Winge, Y. X. Yan, M. Engelstein, and S. H. Friend. 1995.
Mutational analysis of the carboxy-terminal portion of p53 using both yeast and
mammalian cell assays in vivo. Oncogene 10:1485–1492.
95. Ishioka, C., T. Frebourg, Y. X. Yan, M. Vidal, S. H. Friend, S. Schmidt, and R.
Iggo. 1993. Screening patients for heterozygous p53 mutations using a functional assay
in yeast. Nat. Genet. 5:124–129.
96. Iwabuchi, K., P. L. Bartel, B. Li, R. Marraccino, and S. Fields. 1994. Two cellular
proteins that bind to wild-type but not mutant p53. Proc. Natl. Acad. Sci. USA
91:6098–6102.
97. Jimenez, G. S., M. Nister, J. M. Stommel, M. Beeche, E. A. Barcarse, X. Q. Zhang,
S. O’Gorman, and G. M. Wahl. 2000. A transactivation-deficient mouse model
provides insights into Trp53 regulation and function. Nat. Genet. 26:37–43.
98. Joo, W. S., P. D. Jeffrey, S. B. Cantor, M. S. Finnin, D. M. Livingston, and N. P.
Pavletich. 2002. Structure of the 53BP1 BRCT region bound to p53 and its comparison
to the Brca1 BRCT structure. Genes Dev. 16:583–593.
99. Kaelin, W. G., Jr. 1999. The emerging p53 gene family. J. Natl. Cancer Inst.
91:594–598.
100. Kaelin, W. G., Jr. 1999. The p53 gene family. Oncogene 18:7701–7705.
101. Kaeser, M. D., and R. D. Iggo. 2002. From the cover: chromatin immunoprecipitation
analysis fails to support the latency model for regulation of p53 DNA binding activity
invivo. Proc. Natl. Acad. Sci. USA 99:95–100.
102. Karthikeyan, G., L. K. Lewis, and M. A. Resnick. 2002. The mitochondrial protein
frataxin prevents nuclear damage. Hum. Mol. Genet. 11:1351–1362.
103. Kashiwazaki, H., H. Tonoki, M. Tada, I. Chiba, M. Shindoh, Y. Totsuka, R. Iggo,
and T. Moriuchi. 1997. High frequency of p53 mutations in human oral epithelial
dysplasia and primary squamous cell carcinoma detected by yeast functional assay.
Oncogene 15:2667–2674.
104. Kato, H., S. Kato, T. Kumabe, Y. Sonoda, T. Yoshimoto, S. Y. Han, T. Suzuki, H.
Shibata, R. Kanamaru, and C. Ishioka. 2000. Functional evaluation of p53 and PTEN
gene mutations in gliomas. Clin. Cancer Res. 6:3937–3943.
105. Kelly, J. D., A. Inga, F. X. Chen, P. Dande, D. Shah, P. Monti, A. Aprile, P. A.
Burns, G. Scott, A. Abbondandolo, B. Gold, and G. Fronza. 1999. Relationship
between DNA methylation and mutational patterns induced by a sequence selective
minor groove methylating agent. J. Biol. Chem. 274:18327–18334.
106. Kim, I. A., Y. J. Yang, S. C. Yoon, I. B. Choi, C. S. Kay, H. C. Kwon, C. M. Kim,
Y. A. Joe, J. K. Kang, and Y. K. Hong. 2001. Potential of adenoviral p53 gene therapy
and irradiation for the treatment of malignant gliomas. Int. J. Oncol. 19:1041–1047.
107. Ko, L. J., and C. Prives. 1996. p53: puzzle and paradigm. Genes Dev. 10:1054–1072.
108. Komarov, P. G., E. A. Komarova, R. V. Kondratov, K. Christov-Tselkov, J. S.

Coon, M. V. Chernov, and A. V. Gudkov. 1999. A chemical inhibitor of p53 that
protects mice from the side effects of cancer therapy. Science 285:1733–1737.
109. Kumar, A., and M. Snyder. 2001. Emerging technologies in yeast genomics. Nat. Rev.
Genet. 2:302–312.
110. Lane, D. 2001. How cells choose to die. Nature 414:25, 27.
111. Lee, S., L. Cavallo, and J. Griffith. 1997. Human p53 binds holliday junctions strongly
and facilitates their cleavage. J. Biol. Chem. 272:7532–7539.
112. Lee, S. B., S. H. Kim, D. W. Bell, D. C. Wahrer, T. A. Schiripo, M. M. Jorczak, D.
C. Sgroi, J. E. Garber, F. P. Li, K. E. Nichols, J. M. Varley, A. K. Godwin, K. M.
Shannon, E. Harlow, and D. A. Haber. 2001. Destabilization of CHK2 by a missense
mutation associated with Li-Fraumeni Syndrome. Cancer Res. 61:8062–8067.
113. Leung, C. S., and M. L. Lung. 1999. Detection of p53 mutations in Hong Kong
colorectal carcinomas by conventional PCR-SSCP analysis versus p53 yeast functional
assays. Anticancer Res. 19:625–628.
114. Levine, A. J. 1997. p53, the cellular gatekeeper for growth and division. Cell 88:323–331.
115. Levrero, M., V. De Laurenzi, A. Costanzo, J. Gong, J. Y. Wang, and G. Melino
2000. The p53/p63/p73 family of transcription factors: overlapping and distinct
functions. J. Cell Sci. 113:1661–1670.
116. Li, R., P. D. Sutphin, D. Schwartz, D. Matas, N. Almog, R. Wolkowicz, N. Goldfinger,
H. Pei, M. Prokocimer, and V. Rotter. 1998. Mutant p53 protein expression interferes
with p53-independent apoptotic pathways. Oncogene 16:3269–3277.
117. Liefer, K. M., M. I. Koster, X. J. Wang, A. Yang, F. McKeon, and D. R. Roop.
2000. Down-regulation of p63 is required for epidermal UV-B-induced apoptosis.
Cancer Res. 60:4016–4020.
118. Lin, J., A. K. Teresky, and A. J. Levine. 1995. Two critical hydrophobic amino acids
in the N-terminal domain of the p53 protein are required for the gain of function
phenotypes of human p53 mutants. Oncogene 10:2387–2390.
119. Lomax, M. E., D. M. Barnes, R. Gilchrist, S. M. Picksley, J. M. Varley, and R. S.
Camplejohn. 1997. Two functional assays employed to detect an unusual mutation in the
oligomerisation domain of p53 in a Li-Fraumeni like family. Oncogene 14:1869–1874.
120. Lowe, S. W., S. Bodis, A. McClatchey, L. Remington, H. E. Ruley, D. E. Fisher, D.
E. Housman, and T. Jack. 1994. p53 Status and the efficacy of cancer therapy in vivo.
Science 266:807–810.
121. Lu, M. L., F. Wikman, T. F. Orntoft, E. Charytonowicz, F. Rabbani, Z. Zhang, G.
Dalbagni, K. S. Pohar, G. Yu, and C. Cordon-Cardo. 2002. Impact of alterations
affecting the p53 pathway in bladder cancer onclinical outcome, assessed by
conventional and array-based methods. Clin. Cancer Res. 8:171–179.
122. Ludwig, R. L., S. Bates, and K. H. Vousden. 1996. Differential activation of target
cellular promoters by p53 mutants with impaired apoptotic function. Mol. Cell. Biol.
16:4952–4960.
123. Lung, M. L., Y. Hu, Y. Cheng, M. F. Li, C. M. Tang, S. K. O, and R. D. Iggo. 1998.
p53 inactivating mutations in Chinese nasopharyngeal carcinomas. Cancer Lett. 133:89–
94.
124. Mandard, A. M., Y. Denoux, P. Herlin, F. Duigou, M. J. van De Vijver, P. C.
Clahsen, L. van Den Broek, T. M. Sahmoud, M. Henry-Amar, and C. J. van De
Velde. 2000. Prognostic value of DNA cytometry in 281premenopausal patients with
lymph node negative breast carcinoma randomized in a control trial: multivariate analysis
with Ki-67 index, mitotic count, and microvessel density. Cancer 89:1748–1757.
125. Martin, A., J. M. Flaman, T. Frebourg, F. Davi, S. El Mansouri, J. Amouroux, and
M. Raphael. 1998. Functional analysis of the p53 protein in AIDS-related non-
Hodgkin’s lymphomas and polymorphic lymphoproliferations. Br. J. Haematol.
101:311–317.
126. Martin, A. C., A. M. Facchiano, A. L. Cuff, T. Hernandez-Boussard, M. Olivier, P.
Hainaut, and J. M. Thornton. 2002. Integrating mutation data and structural analysis
of the TP53 tumor-suppressor protein. Hum. Mutat. 19:149–164.
127. Maurici, D., P. Monti, P. Campomenosi, S. North, T. Frebourg, G. Fronza, and
P. Hainaut. 2001. Amifostine (WR2721) restores transcriptional activity of specific p53
mutant proteins in a yeast functional assay. Oncogene 20:3533–3540.
128. McCormick, F. 1999. Cancer therapy based on p53. Cancer J. Sci. Am. 5:139–144.
129. McCormick, F. 1999. Signalling networks that cause cancer. Trends Cell Biol. 9:M53–56.
130. McLure, K. G., and P. W. Lee. 1998. How p53 binds DNA as a tetramer. EMBO J.
17:3342–3350.
131. McShane, L. M., R. Aamodt, C. Cordon-Cardo, R. Cote, D. Faraggi, Y. Fradet, H.
B. Grossman, A. Peng, S. E. Taube, and F. M. Waldman. 2000. Reproducibility of
p53 immunohistochemistry in bladder tumors. National Cancer Institute, Bladder Tumor
Marker Network. Clin. Cancer Res. 6:1854–1864.
132. Meinhold-Heerlein, I., E. Ninci, H. Ikenberg, T. Brandstetter, C. Ihling, I.
Schwenk, A. Straub, B. Schmitt, H. Bettendorf, R. Iggo, and T. Bauknecht. 2001.
Evaluation of methods to detect p53 mutations in ovarian cancer. Oncology 60:176–188.
133. Merika, M., and D. Thanos. 2001. Enhanceosomes. Curr. Opin. Genet. Dev. 11:205–208.
134. Montes de Oca Luna, R., L. L. Amelse, A. Chavez-Reyes, S. C. Evans, J.
Brugarolas, T. Jacks, and G. Lozano. 1997. Deletion of p21 cannot substitute for p53
loss in rescue of mdm2 null lethality. Nat. Genet. 16:336–337.
135. Montes de Oca Luna, R., D. S. Wagner, and G. Lozano. 1995. Rescue of early
embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378:203–206.
136. Monti, P., P. Campomenosi, Y. Ciribilli, R. Iannone, A. Inga, A. Abbondandolo, M.
A. Resnick, and G. Fronza. 2002. Tumour p53 mutations exhibit promoter selective
dominance over wild type p53. Oncogene 21:1641–1648.
137. Monti, P., A. Inga, A. Aprile, P. Campomenosi, P. Menichini, L. Ottaggio, S.
Viaggi, G. Ghigliotti, A. Abbondandolo, and G. Fronza. 2000. p53 Mutations
experimentally induced by 8-methoxypsoralen plus UVA (PUVA) differ from those
found in human skin cancers in PUVA-treated patients. Mutagenesis 15:127–132.
138. Monti, P., A. Inga, G. Scott, A. Aprile, P. Campomenosi, P. Menichini, L. Ottaggio,
S. Viaggi, A. Abbondandolo, P. A. Burns, and G. Fronza. 1999. 5-Methylcytosine at
HpaII sites in p53 is not hypermutable after UVC irradiation. Mutat. Res. 431:93–103.
139. Moshinsky, D. J., and G. N. Wogan. 2000. UV-induced mutagenesis of human p53:
analysis using a double-selection method in yeast. Environ. Mol. Mutagen. 35:31–38.
140. Moshinsky, D. J., and G. N. Wogan. 1997. UV-induced mutagenesis of human p53
in a vector replicated in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA
94:2266–2271.
141. Murata, J., M. Tada, R. D. Iggo, Y. Sawamura, Y. Shinohe, and H. Abe. 1997.
Nitric oxide as a carcinogen: analysis by yeast functional assay of inactivating p53
mutations induced by nitric oxide. Mutat. Res. 379:211–218.
142. Murphy, K. L., A. P. Dennis, and J. M. Rosen. 2000. A gain of function p53 mutant
promotes both genomic instability and cell survival in a novel p53-null mammary
epithelial cell model. FASEB J. 14:2291–2302.
143. Nagaich, A. K., E. Appella, and R. E. Harrington. 1997. DNA bending is essential for
the site-specific recognition of DNA response elements by the DNA binding domain of
the tumor suppressor protein p53. J. Biol. Chem. 272:14842–1489.
144. Nigro, J. M., R. Sikorski, S. I. Reed, and B. Vogelstein. 1992. Human p53 and
CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae. Mol.
Cell Biol. 12:1357–1365.
145. Niklinska, W., L. Chyczewski, J. Laudanski, B. Sawicki, and J. Niklinski. 2001.
Detection of P53 abnormalities in non-small cell lung cancer by yeast functional assay.
Folia Histochem. Cytobiol. 39:147–148.
146. Nikolova, P. V., J. Henckel, D. P. Lane, and A. R. Fersht. 1998. Semirational design
of active tumor suppressor p53 DNA binding domain with enhanced stability. Proc.
Natl. Acad. Sci. USA 95:14675–14680.
147. Nikolova, P. V., K. B. Wong, B. DeDecker, J. Henckel, and A. R. Fersht. 2000.
Mechanism of rescue of common p53 cancer mutations by second-site suppressor
mutations. EMBO J. 19:370–378.
148. North, S., F. El-Ghissassi, O. Pluquet, G. Verhaegh, and P. Hainaut. 2000. The
cytoprotective aminothiol WR1065 activates p21waf-1 and down regulates cell cycle
progression through a p53-dependent pathway. Oncogene 19:1206–1214.
149. Nourani, A., Y. Doyon, R. T. Utley, S. Allard, W. S. Lane, and J. Cote. 2001. Role
of an ING1 growth regulator in transcriptional activation and targeted histone
acetylation by the NuA4 complex. Mol. Cell Biol. 21:7629–7640.
150. Nozaki, M., M. Tada, H. Kashiwazaki, M. F. Hamou, A. C. Diserens, Y. Shinohe,
Y. Sawamura, Y. Iwasaki, N. de Tribolet, and M. E. Hegi. 2001. p73 is not mutated
in meningiomas as determined with a functional yeast assay but p73 expression
increases with tumor grade. Brain Pathol. 11:296–305.
151. Nozaki, M., M. Tada, R. Matsumoto, Y. Sawamura, H. Abe, and R. D. Iggo. 1998.
Rare occurrence of inactivating p53 gene mutations in primary non-astrocytic tumors of
the central nervous system: reappraisal by yeast functional assay. Acta Neuropathol.
95:291–296.
152. O’Connor, M. J., H. Zimmermann, S. Nielsen, H. U. Bernard, and T. Kouzarides.
1999. Characterization of an E1A-CBP interaction defines a novel transcriptional
adapter motif (TRAM) in CBP/p300. J. Virol. 73:3574–3581.
153. O’Connor, P. M., J. Jackman, I. Bae, T. G. Myers, S. Fan, M. Mutoh, D. A.
Scudiero, A. Monks, E. A. Sausville, J. N. Weinstein, S. Friend, A. J. Fornace, Jr.,
and K. W. Kohn. 1997. Characterization of the p53 tumor suppressor pathway in cell
lines of the National Cancer Institute anticancer drug screen and correlations with the
growth-inhibitory potency of 123 anticancer agents. Cancer Res. 57:4285–4300.
154. Oren, M. 1999. Regulation of the p53 tumor suppressor protein. J. Biol. Chem.
274:36031–36034.
155. Origone, P., A. De Luca, C. Bellini, A. Buccino, R. Mingarelli, S. Costabel, C. La
Rosa, C. Garre, D. A. Coviello, F. Ajmar, B. Dallapiccola, and E. Bonioli. 2002. Ten
novel mutations in the human neurofibromatosis type 1 (NF1) gene in Italian patients.
Hum. Mutat. 20:74–75.
156. Paladino, G., B. Weibel, and C. Sengstag. 1999. Heterocyclic aromatic amines
efficiently induce mitotic recombination in metabolically competent Saccharomyces
cerevisiae strains. Carcinogenesis 20:2143–2152.
157. Park, J. S., E. J. Kim, J. Y. Lee, H. S. Sin, S. E. Namkoong, and S. J. Um. 2001.
Functional inactivation of p73, a homolog of p53 tumor suppressor protein, by human
papillomavirus E6 proteins. Int. J. Cancer 91:822–827.
158. Pellegrini, G., E. Dellambra, O. Golisano, E. Martinelli, I. Fantozzi, S. Bondanza,
D. Ponzin, F. McKeon, and M. De Luca. 2001. p63 identifies keratinocyte stem cells.
159. Pfister, C., J. M. Flaman, F. Dunet, P. Grise, and T. Frebourg. 1999. p53 Mutations
in bladder tumors inactivate the transactivation of the p21 and Bax genes, and have a
predictive value for the clinical outcome after bacillus Calmette–Guerin therapy. J. Urol.
162:69–73.
160. Pfister, C., J. M. Flaman, C. Martin, P. Grise, and T. Frebourg. 1999. Selective
detection of inactivating mutations of the tumor suppressor gene p53 in bladder tumors.
J. Urol. 161:1973–1975.
161. Ponchel, F., and J. Milner. 1998. Temperature sensitivity of human wild-type and
mutant p53 proteins expressed in vivo. Br. J. Cancer 77:1555–1561.
162. Preuss, U., R. Kreutzfeld, and K. H. Scheidtmann. 2000. Tumor-derived p53 mutant
C174Y is a gain-of-function mutant which activates the fos promoter and enhances
colony formation. Int. J. Cancer 88:162–171.
163. Prives, C. 1998. Signaling to p53: breaking the MDM2-p53 circuit. Cell 95:5–8.
164. Prives, C., and P. A. Hall. 1999. The p53 pathway. J. Pathol. 187:112–126.
165. Prives, C., and J. L. Manley. 2001. Why is p53 acetylated? Cell 107:815–818.
166. Qian, H., T. Wang, L. Naumovski, C. D. Lopez, and R. K. Brachmann. 2002.
Groups of p53 target genes involved in specific p53 downstream effects cluster into
different classes of DNA binding sites. Oncogen 21:7901–7911.
167. Raj, K., P. Ogston, and P. Beard. 2001. Virus-mediated killing of cells that lack p53
activity. Nature 412:914–917.
168. Rand, A., K. S. Glenn, C. P. Alvares, M. B. White, S. M. Thibodeau, and W. E.
Karnes, Jr. 1996. p53 Functional loss in a colon cancer cell line with two missense
mutations (218leu and 248trp) on separate alleles. Cancer Lett. 98:183–191.
169. Resnick, M. A., and B. S. Cox. 2000. Yeast as an honorary mammal. Mutat Res.
451:1–11.
170. Ribeiro, R. C., F. Sandrini, B. Figueiredo, G. P. Zambetti, E. Michalkiewicz, A. R.
Lafferty, L. DeLacerda, M. Rabin, C. Cadwell, G. Sampaio, I. Cat, C. A. Stratakis,
and R. Sandrini. 2001. An inherited p53 mutationthat contributes in a tissue-specific
manner to pediatric adrenal cortical carcinoma. Proc. Natl. Acad. Sci. USA 98:9330–9335.
171. Rippin, T. M., V. J. Bykov, S. M. Freund, G. Selivanova, K. G. Wiman, and A. R.
Fersht. 2002. Characterization of the p53-rescue drug CP-31398 in vitro and in living
cells. Oncogene 21:2119–2129.
172. Robert, V., P. Michel, J. M. Flaman, A. Chiron, C. Martin, F. Charbonnier, B.
Paillot, and T. Frebourg. 2000. High frequency in esophageal cancers of p53
alterations inactivating the regulation of genes involved in cell cycle and apoptosis.
Carcinogenesis. 21:563–565.
173. Rodriguez, M. S., J. M. Desterro, S. Lain, C. A. Midgley, D. P. Lane, and R. T.
Hay. 1999. SUMO-1 modification activates the transcriptional response of p53. EMBO
J. 18:6455–6461.
174. Romanov, S. R., B. K. Kozakiewicz, C. R. Holst, M. R. Stampfer, L. M. Haupt, and

T. D. Tlsty. 2001. Normal human mammary epithelial cells spontaneously escape
senescence and acquire genomic changes. Nature 409:633–637.
175. Ruaro, E. M., L. Collavin, G. Del Sal, R. Haffner, M. Oren, A. J. Levine, and
C. Schneider. 1997. A proline-rich motif in p53 is required for transactivation-
independent growth arrest as induced by Gas1. Proc. Natl. Acad. Sci. USA 94:4675–4680.
176. Saintigny, Y., D. Rouillard, B. Chaput, T. Soussi, and B. S. Lopez. 1999. Mutant p53
proteins stimulate spontaneous and radiation-induced intrachromosomal homologous
recombination independently of the alteration of the transactivation activity and of the
G1 checkpoint. Oncogene 18:3553–3563.
177. Sakuragi, N., A. Hirai, M. Tada, H. Yamada, R. Yamamoto, S. Fujimoto, and
T. Moriuchi. 2001. Dominant-negative mutation of p53 tumor suppressor gene in
endometrial carcinoma. Gynecol. Oncol. 83:485–490.
178. Saller, E., E. Tom, M. Brunori, M. Otter, A. Estreicher, D. H. Mack, and R. Iggo
1999. Increased apoptosis induction by 121F mutant p53. EMBO J. 18:4424–4437.
179. Samuels-Lev, Y., D. J. O’Connor, D. Bergamaschi, G. Trigiante, J. K. Hsieh, S.
Zhong, I. Campargue, L. Naumovski, T. Crook, and X. Lu. 2001. ASPP proteins
specifically stimulate the apoptotic function of p53. Mol. Cell. 8:781–794.
180. Scharer, E., and R. Iggo. 1992. Mammalian p53 can function as a transcription factor
in yeast. Nucleic Acids Res. 20:1539–1545.
181. Schiller, J. H., S. Adak, R. H. Feins, S. M. Keller, W. A. Fry, R. B. Livingston,
M. E. Hammond, B. Wolf, L. Sabatini, J. Jett, L. Kohman, and D. H. Johnson.
2001. Lack of prognostic significance of p53 and K-rasmutations in primary resected
non-small-cell lung cancer on E4592: a Laboratory Ancillary Study on an Eastern
Cooperative Oncology Group Prospective Randomized Trial of Postoperative Adjuvant
Therapy. J. Clin. Oncol. 19:448–457.
182. Schultz, L. B., N. H. Chehab, A. Malikzay, and T. D. Halazonetis. 2000. p53 binding
protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand
breaks. J. Cell. Biol. 151:1381–1390.
183. Selivanova, G., V. Iotsova, I. Okan, M. Fritsche, M. Strom, B. Groner, R. C.
Grafstrom, and K. G. Wiman. 1997. Restoration of the growth suppression function of
mutant p53 by a synthetic peptide derived from the p53 C–terminal domain. Nat. Med.
3:632–638.
184. Shimada, A., S. Kato, K. Enjo, M. Osada, Y. Ikawa, K. Kohno, M. Obinata,
R. Kanamaru, S. Ikawa, and C. Ishioka. 1999. The transcriptional activities of p53
and its homologue p51/p63: similarities and differences. Cancer Res. 59:2781–2786.
185. Smardova, J. 1999. FASAY: a simple functional assay in yeast for identification of p53
mutation in tumors. Neoplasma 46:80–88.
186. Smardova, J., V. Vagunda, E. Jandakova, M. Vagundova, H. Koukalova,
J. Kovarik, and J. Zaloudik. 1999. p53 Status in breast carcinomas revealed by
FASAY correlates well with p53 protein accumulation determined by
immunohistochemistry. Neoplasma 46:384–389.
187. Smith, P. D., S. Crossland, G. Parker, P. Osin, L. Brooks, J. Waller, E. Philp, M. R.
Crompton, B. A. Gusterson, M. J. Allday, and T. Crook. 1999. Novel p53 mutants
selected in BRCA-associated tumours which dissociate transformation suppression from
other wild-type p53 functions. Oncogene 18:2451–2459.
188. Soussi, T., and C. Beroud. 2001. Assessing TP53 status in human tumours to evaluate
clinical outcome. Nature Rev. Cancer 1:233–240.
189. Soussi, T., and P. May. 1996. Structural aspects of the p53 protein in relation to gene
evolution: a second look. J. Mol. Biol. 260:623–637.
190. Stiewe, T., and B. M. Putzer. 2001. p73 in apoptosis. Apoptosis 6:447–452.
191. Stiewe, T., and B. M. Putzer. 2002. Role of p73 in malignancy: tumor suppressor or
oncogene? Cell Death Differ. 9:237–245.
192. Storici, F., L. K. Lewis, and M. A. Resnick. 2001. In vivo site-directed mutagenesis
using oligonucleotides. Nat. Biotechnol. 19:773–776.
193. Strano, S., G. Fontemaggi, A. Costanzo, M. G. Rizzo, O. Monti, A. Baccarini,
G. Del Sal, M. Levrero, A. Sacchi, M. Oren, and G. Blandino. 2002. Physical inter-
action with human tumor-derived p53 mutants inhibits p63 activities. J. Biol. Chem. 13:13.
194. Strano, S., E. Munarriz, M. Rossi, B. Cristofanelli, Y. Shaul, L. Castagnoli, A. J.
Levine, A. Sacchi, G. Cesareni, M. Oren, and G. Blandino. 2000. Physical and
functional interaction between p53 mutants and different isoforms of p73 [In Process
Citation]. J. Biol. Chem. 275:29503–29512.
195. Swisher, S. G., J. A. Roth, J. Nemunaitis, D. D. Lawrence, B. L. Kemp, C. H.
Carrasco, D. G. Connors, A. K. El-Naggar, F. Fossella, B. S. Glisson, W. K. Hong,
F. R. Khuri, J. M. Kurie, J. J. Lee, J. S. Lee, M. Mack, J. A. Merritt, D. M. Nguyen,
J. C. Nesbitt, R. Perez-Soler, K. M. Pisters, J. B. Putnam, Jr., W. R. Richli,
M. Savin, M. K. Waugh, et al. 1999. Adenovirus-mediated p53 gene transfer in
advanced non-small-cell lung cancer. J. Natl. Cancer Inst. 91:763–771.
196. Szak, S. T., D. Mays, and J. A. Pietenpol. 2001. Kinetics of p53 binding to promoter
sites in vivo. Mol. Cell Biol. 21:3375–3386.
197. Tada, M., R. D. Iggo, N. Ishii, Y. Shinohe, S. Sakuma, A. Estreicher, Y. Sawamura,
and H. Abe. 1996. Clonality and stability of the p53 gene in human astrocytic tumor
cells: quantitative analysis of p53 gene mutations by yeast functional assay. Int. J.
Cancer 67:447–450.
198. Tada, M., R. D. Iggo, F. Waridel, M. Nozaki, R. Matsumoto, Y. Sawamura,
Y. Shinohe, J. Ikeda, and H. Abe. 1997. Reappraisal of p53 mutations in human
malignant astrocytic neoplasms by p53 functional assay: comparison with conventional
structural analyses. Mol. Carcinog. 18:171–176.
199. Tada, M., S. Sakuma, R. D. Iggo, H. Saya, Y. Sawamura, T. Fujiwara, and J. A.
Roth. 1998. Monitoring adenoviral p53 transduction efficiency by yeast functional
assay. Gene Ther. 5:339–344.
200. Thukral, S. K., Y. Lu, G. C. Blain, T. S. Harvey, and V. L. Jacobsen. 1995.
Discrimination of DNA binding sites by mutant p53 proteins. Mol. Cell Biol. 15:5196–
202.
201. Tlsty, T. D. 2001. Stromal cells can contribute oncogenic signals. Semin Cancer Biol.
11:97–104.
202. Tokino, T., S. Thiagalingam, W. S. el-Deiry, T. Waldman, K. W. Kinzler, and
B. Vogelstein. 1994. p53 tagged sites from human genomic DNA. Hum. Mol. Genet.
3:1537–1542.
203. Tonisson, N., J. Zernant, A. Kurg, H. Pavel, G. Slavin, H. Roomere, A. Meiel,
P. Hainaut, and A. Metspalu. 2002. Evaluating the arrayed primer extension
resequencing assay of TP53 tumor suppressor gene. Proc. Natl. Acad. Sci. USA
99:5503–5508.
204. Varley, J. M., P. Chapman, G. McGown, M. Thorncroft, G. R. White, M. J.
Greaves, D. Scott, A. Spreadborough, K. J. Tricker, J. M. Birch, D. G. Evans,
R. Reddel, R. S. Camplejohn, J. Burn, and J. M. Boyle. 1998. Genetic and functional
studies of a germline TP53 splicing mutation in a Li-Fraumeni-like family. Oncogene
16:3291–3298.
205. Vogelstein, B., and K. W. Kinzler. 1992. Carcinogens leave fingerprints. Nature
355:209–210.
206. Vogelstein, B., D. Lane, and A. J. Levine. 2000. Surfing the p53 network. Nature
408:307–310.
207. Waddell, S., J. R. Jenkins, and T. Proikas-Cezanne. 2001. A “no-hybrids” screen for
functional antagonizers of human p53 transactivator function: dominant negativity in
fission yeast. Oncogene 20:6001–6008.
208. Wahl, G. M., and A. M. Carr. 2001. The evolution of diverse biological responses to
DNA damage: insights from yeast and p53. Nat. Cell Biol. 3:E277–286.
209. Walker, D. R., J. P. Bond, R. E. Tarone, C. C. Harris, W. Makalowski, M. S.
Boguski, and M. S. Greenblatt. 1999. Evolutionary conservation and somatic mutation
hotspot maps of p53: correlation with p53 protein structural and functional features.
Oncogene 18:211–218.
210. Wang, T., T. Kobayashi, R. Takimoto, A. E. Denes, E. L. Snyder, W. S. el-Deiry,
and R. K. Brachmann. 2001. hADA3 is required for p53 activity. EMBO J.
20:6404–6413.
211. Waridel, F., A. Estreicher, L. Bron, J. M. Flaman, C. Fontolliet, P. Monnier,
T. Frebourg, and R. Iggo. 1997. Field cancerisation and polyclonal p53 mutation in the
upper aero- digestive tract. Oncogene 14:163–169.
212. Weinstein, I. B., M. Begemann, P. Zhou, E. K. Han, A. Sgambato, Y. Doki,
N. Arber, M. Ciaparrone, and H. Yamamoto. 1997. Disorders in cell circuitry
associated with multistage carcinogenesis: exploitabletargets for cancer prevention and
therapy. Clin. Cancer Res. 3:2696–2702.
213. Wikman, F. P., M. L. Lu, T. Thykjaer, S. H. Olesen, L. D. Andersen, C. Cordon-
Cardo, and T. F. Orntoft. 2000. Evaluation of the performance of a p53 sequencing
microarray chip using 140 previously sequenced bladder tumor samples. Clin. Chem.
46:1555–1561.
214. Wolf, J. K., G. B. Mills, L. Bazzet, R. C. Bast, Jr., J. A. Roth, and D. M.
Gershenson. 1999. Adenovirus-mediated p53 growth inhibition of ovarian cancer cells
is independent of endogenous p53 status. Gynecol. Oncol. 75:261–266.
215. Yamamoto, S., M. Tada, C. C. Lee, C. Masuda, H. Wanibuchi, R. Yoshimura,
S. Wada, K. Yamamoto, T. Kishimoto, and S. Fukushima. 2000. p53 Status in
multiple human urothelial cancers: assessment for clonality by the yeast p53 functional
assay in combination with p53 immunohistochemistry. Jpn. J. Cancer Res. 91:181–189.
216. Yang, A., M. Kaghad, Y. Wang, E. Gillett, M. D. Fleming, V. Dotsch, N. C.
Andrews, D. Caput, and F. McKeon. 1998. p63, a p53 homolog at 3q27-29, encodes
multiple products with transactivating, death-inducing, and dominant-negative activities.
Mol. Cell. 2:305–316.
217. Yang, A., R. Schweitzer, D. Sun, M. Kaghad, N. Walker, R. T. Bronson, C. Tabin,
A. Sharpe, D. Caput, C. Crum, and F. McKeon. 1999. p63 is essential for
regenerative proliferation in limb, craniofacial and epithelial development. Nature
398:714–718.
218. Yang, A., N. Walker, R. Bronson, M. Kaghad, M. Oosterwegel, J. Bonnin,
C. Vagner, H. Bonnet, P. Dikkes, A. Sharpe, F. McKeon, and D. Caput. 2000. p73-
Deficient mice have neurological, pheromonal and inflammatory defects but lack
spontaneous tumours. Nature 404:99–103.
219. Zhang, Y., and Y. Xiong. 2001. A p53 amino-terminal nuclear export signal inhibited
by DNA damage-induced phosphorylation. Science 292:1910–1915.
220. Zhao, R., K. Gish, M. Murphy, Y. Yin, D. Notterman, W. H. Hoffman, E. Tom, D.

H. Mack, and A. J. Levine. 2000. Analysis of p53-regulated gene expression patterns
using oligonucleotide arrays. Genes Dev. 14:981–993.
221. Zhu, J., S. Zhang, J. Jiang, and X. Chen. 2000. Definition of the p53 functional
domains necessary for inducing apoptosis. J. Biol. Chem. 275:39927–39934.
ADDENDUM
RECENT DEVELOPMENTS
Since the original submission, there have been several additional

publications related to this topic. Rather than greatly extending this paper,
we have chosen to incorporate a small number of recent, yeast-based studies
relevant to our review.
Using variable expression of p53 and measuring transactivation at many
REs, we have shown that single amino acid changes in the p53 DNA-binding
domain can have diverse effects on the ability to induce transcription from
many target promoters [222] – see also section 2.9 and Figure 5. Based on
these results, we have proposed to include p53 in a newly defined group of
genes which we have called master genes of diversity. We defined a master
gene for genetically derived phenotypic diversity in terms of “a single
transcriptional activator (or repressor) that regulates many genes through
different REs. Mutations of the master gene can lead to a variety of
simultaneous changes in both the selection of targets and the extent of
transcriptional modulation at the individual targets, resulting in a vast
number of potential phenotypes that can be created with minimal mutational
changes without altering existing protein–protein interactions” [222].
We have expanded our functional analysis of p53 REs [91] to establish
response element rules that can be used for in silico searches of genomes. In
particular, we have focused on the impact of the spacer between dimmer-
binding sites and on sequence features in the core element of the RE (i.e.,
CWWG – see section 1.2.2) as well as local sequence-context effects in the
homopurine (RRR) and homopyrimidine (YYY) repeats flanking the core.
Contrary to predictions based on in vitro binding assay, our results indicated
that the spacer is critical for RE transactivation potential, with a 2 nt spacer
reducing activity of a strong RE down to ~10%. Spacers of 5 and 10 nt led to
even stronger reduction in activity to ~1% of a full site (Inga, Menendez, and
Resnick, in preparation). Our analyses also revealed that half-site REs (i.e.,
dimmer - binding sites) with consensus sequence and the CATG core
signature can mediate low level of p53 transactivation and that a dimmer -
binding site with an adjacent monomer site (i.e., 3/4 sites) can mediate
moderate transactivation. These findings have implications for addressing
the functional impact of sequence variation in p53 REs and for identifying
target genes in the p53 transcriptional network.
Figure 5. Relative transactivation capacity of p53 alleles with mutations in the DNA binding
domain [222]. Twenty-four human p53 mutations were examined in 15 isogenic yeast strains
each containing a different p53 RE that regulates expression of the ADE2 reporter gene. The
REs are ordered based on decreasing transactivation capacity with wild type p53 [91]. The
p53 mutants are ordered according to their position in the primary sequence. The trans-
activation capacity of each allele toward each RE was determined using variable expression
of p53 under the GAL1 promoter and compared to the activity of wild type p53. The relative
transactivation capacities of mutants with respect to wt is presented in a form similar to that
for expression arrays, with red greatly increased, green greatly decreased, blue loss-of-
function, and black equal to wild type. The quantification is based on the amounts of p53
protein required for transactivation with wt or with a mutant allele. This was derived from the
minimal amount of galactose required for transactivation to occur – see section 2.6.
Besides mutations in p53 or in proteins involved in the p53 response

pathway, genetic variation in promoter REs of individual p53 target genes
can be expected to alter biological responses to stress. Based on our set of
response elements rules, we have developed custom bioinformatics tools to
address the evolution of p53 networks between human and mouse due to
variation in RE sequences. We had previously observed that mouse and
human p53 proteins exhibited identical transactivation capacities toward a
large panel of REs [222]. We focused on 26 p53 target genes that belong to
different functional groupings (including cell cycle control, apoptosis, and
DNA repair). First, we examined the conservation of established p53 target
REs in humans in the homologous promoter regions in mouse. Then we
searched in both species for additional p53 functional REs in a 10 kb region
encompassing the transcriptional start site. We found that ~60% of the REs
are functionally conserved. Surprisingly, most of the genes involved in DNA
metabolism and repair showed no conservation of RE sequence nor compens-
atory REs in the 10 kb promoter regions examined (Inga, Menendez,
Jegga, Aronow, and Resnick, in preparation).
A similar strategy was developed to identify single nucleotide
polymorphisms (SNPs) in p53 REs that can lead to functional variation in
the p53 regulatory network between individuals [223]. An approach was
developed that combines a custom bioinformatics search to identify
candidate SNPs and functional assays to assess their effect on p53
transactivation. Among ~2 million human SNPs examined, we identified
over 200 located within putative p53 REs that are located in the proximity of
transcriptional start site of an established gene. We then ranked the putative
p53 REs based on predicted transactivation potential and functional impact
of the SNP. Forty REs were predicted to mediate p53 transactivation in one
allele and to be functionally altered by the SNP. Eight of these were
evaluated in functional assays to determine both the activity of the putative
REs and the impact of the SNPs on transactivation. All eight candidate p53
REs were functional, and in every case the SNP pair exhibited differential
transactivation capacities. Additionally, six of the eight genes adjacent to
these SNPs were induced by genotoxic stress or activated directly by
transfection with p53 cDNA in human cells. Thus, the overall strategy
efficiently identifies SNPs that may affect gene expression responses in the
p53 regulatory pathway [223]. We have proposed that these SNPs in p53
response elements could contribute to human differences in responses to
stress.
The group led by Ishioka has reported the completion of a
comprehensive analysis of the functional impact of single amino acid
changes in the p53 protein [224]. Using site-directed mutagenesis they
constructed 2314 p53 mutants (an average of nearly six amino acid changes
at each codon) and measured transactivation in yeast using quantitative
reporter systems. To improve the throughput of the functional assay they

chose fluorescent reporter genes (EGFP and Ds-Red) and utilized eight
different p53-binding sequences. To increase the readout from the fluore-
scent reporters, multicopy plasmids (based on a 2 µm replication origin)
were used, although this choice may have led to high experimental vari-
ability (see Figure 4 [224]). The general conclusion from this impressive
study was that nearly two-third of the p53 mutants in the DNA-binding
domain were inactive; activity was instead mostly unaffected by amino acid
changes both at the Nter and Cter of the protein, with the exception of the
tetramerization domain [224]. Unfortunately, this publication contained
mostly summary data for the whole library; hence a comparison of the
results for individual mutants with other yeast-based assays was not possible.
In a subsequent study [225], the same group reported that 142 of the
2314 p53 mutants exhibited temperature sensitivity (ts) toward at least one
of the eight p53-binding sequences examined. Fifty-four mutants were tested
in mammalian cell-based assays and nearly 90% of them were ts [225].
Standard quantitative criteria were adopted to classify ts mutants, based on
the difference in activity between 30°C and 37°C. However, the screening
identified as ts only 11 of 59 p53 mutations that had been described as ts by
other groups and were available in the collection [225]. This discrepancy is
indicative of significant differences in sensitivity or classification parameters
between the various functional assays and emphasizes the need to compare
transactivation data for individual mutants obtained with the different
methodologies available.
An effort to standardize and compare results of the various functional
assays has been taken by the curators of the IARC p53 mutation database
(http://www-p53.iarc.fr/index.html). The group has recently released a p53
mutant function database that can be downloaded and also searched online
(http://www-p53.iarc.fr/p53MUTfunction.html). Data from overexpression
of p53 mutant proteins in human cells and from yeast assays have been
included. Results have been grouped into five categories: (1) retained wild-
type activity; (2) loss-of-function; (3) gain of function; (4) dominant-negative
effect; and (5) temperature sensitivity. Once all the available data are
deposited and standardized, this database will be a valuable tool to assess
whether functionality data contribute prognostic value to the analysis of p53
mutation status in cancer.
Soussi et al., have instead used only the data set from the library of
2314 p53 mutants [224] to compare the functional impact of the mutations
with the frequency of p53 mutations in the tumor database (http://p53.curie.fr)
[226]. They concluded that more than 50% of the rare p53 mutants in the
database display significant activity, while the most frequent mutations have
lost function. The apparent heterogeneity of the functional consequences of
p53 mutations associated with cancer further emphasizes the value of a

comprehensive, accessible, and fully searchable p53 mutant function
database to address the clinical relevance of p53 status in tumors.
REFERENCES
222. Resnick, M. A., and A. Inga. 2003. Functional mutants of the sequence-specific
transcription factor p53 and implications for master genes of diversity. Proc. Natl. Acad.
Sci. USA 100:9935–9939.
223. Tomso, D. J., A. Inga, D. Menendez, G. S. Pittman, M. R. Campbell, F. Storici,
D. A. Bell, and M. A. Resnick. 2005. Functionally distinct polymorphic sequences in
the human genome that are targets for p53 transactivation. Submitted.
224. Kato, S., S.Y. Han, W. Liu, K. Otsuka, H. Shibata, R. Kanamaru, and C. Ishioka
2003. Understanding the function–structure and function–mutation relationships of p53
tumor suppressor protein by high-resolution missense mutation analysis. Proc. Natl.
Acad. Sci. USA 100: 8424–8429.
225. Shiraishi, K., S. Kato, S. Y. Han, W. Liu, K. Otsuka, M. Sakayori, T. Ishida,
M. Takeda, R. Kanamaru, N. Ohuchi, and C. Ishioka. 2004. J. Biol. Chem. 272:348–355.
226. Soussi, T., S. Kato, P. P. Levy, and C. Ishioka. 2005. Reassessment of the tp53
mutation database in human disease by data mining with a library of tp53 missense
mutations. Hum. Mut. 25:6–17.
227. Zacchi, P., M. Gostissa, T. Uchida, C. Salvagno, F. Avolio, S. Volinia, Z. Ronai,
G. Blandino, C. Schneider, and G. Del Sal. 2002. The prolyl isomerase Pin1 reveals a
mechanism to control p53 functions after genotoxic insults. Nature 419: 853–857.
Chapter 11
ABC TRANSPORTERS IN YEAST – DRUG
RESISTANCE AND STRESS RESPONSE
IN A NUTSHELL
Karl Kuchler and Christoph Schüller

Max F. Perutz Laboratories, Department of Medical Biochemistry; Division of Molecular
Genetics, Medical University Vienna, Campus Vienna Biocenter, Vienna, Austria
Key words: Yeast, ABC protein, antifungal resistance, drug screening, detoxification,
stress response
1 SUMMARY
ATP-binding cassette (ABC) proteins have been implicated in multidrug

resistance phenomena and some are also intimately connected to prominent
genetic diseases. Thus, many members of the ubiquitous superfamily of
ABC proteins act at crossroads of vital cellular processes. For example,
mammalian ABC proteins such as P-glycoproteins (P-gp) or multidrug
resistance-associated proteins (MRPs) are associated with multidrug
resistance (MDR) in various cancers. Likewise, homologues of mammalian
ABC transporters in bacteria, fungi, or parasites are tightly linked to
pleiotropic drug and multiple antibiotic resistance phenomena. Because
yeast orthologues of mammalian MDR genes operate in this unicellular
eukaryote, it has been acclaimed as a prime model system for studies on
mammalian ABC proteins. Moreover, many efforts have been made during
the past decade to exploit yeast as a model system for drug development and
drug screening. In this chapter, we shall provide a comprehensive overview
on yeast ABC transporters implicated in drug resistance and cellular
detoxification. We shall discuss the regulatory network of pleiotropic drug
resistance (PDR) in the budding yeast Saccharomyces cerevisiae. Finally, we
289
© 2007 Springer.
290 K. Kuchler and C. Schüller
shall reiterate the advantages and pitfalls associated with using baker’s yeast
as a model system for the molecular analysis of mammalian ABC trans-
porters, in particular its suitability and amenability for drug screening or even
drug discovery related to the function of ABC transporters with medical
importance.
2 INTRODUCTION
Although intrinsic drug resistance has been known for quite some time
[15], MDR may develop under selective conditions such as those caused by
drug exposure or stepwise selection of cells for resistance to xenobiotics or
toxic small molecules [84]. MDR is often characterized by cross-resistance
to many unrelated compounds [14], and while this is a matter of intense
discussion, MDR of tumor cells or tissues may impair efficacy or even
prohibit anticancer therapy in vivo. At least in great many in vitro studies
using cultured cells, MDR phenotypes clearly cause cells to become
refractory to chemotherapy [14, 84].
Complex MDR phenotypes may result from several mechanisms operating
simultaneously or consecutively in a developmental process [84]. Mechanisms
of MDR include decreased drug uptake, plasma membrane permeability
changes, intracellular drug inactivation, transcriptional activation/deactivation,
gene amplification, drug target gene mutations, or even compartment-
specific sequestration of certain drugs (Figure 1). Notably, similar mecha-
nisms may counteract inhibitory effects of endogenous metabolites in case
they are toxic or detrimental for growth. Importantly, ATP-dependent drug
efflux from cells mediated by dedicated membrane transporters is considered
a major cause of MDR [14]. Such drug efflux can be accomplished by so-
called ABC transporters, which comprise one of the largest protein families
with more than 3,000 known representatives operating in all living cells
from bacteria to man [24]. Most ABC proteins are membrane-associated and
display transport activities for great many different cytotoxic drugs or
antibiotics. Substrate transport is powered by ATP hydrolysis, but ATP may
also have important roles in regulating ABC protein function or substrate
specificity. Although the domain organization of most if not all ABC
proteins is conserved throughout evolution, the molecular basis for their
broad drug substrate specificity has remained an unsolved mystery [24].
Chapter 11: ABC Transporters in Yeast 291
Figure 1. Principle mechanisms of pleiotropic drug resistance in living cells. Complex PDR
phenotypes can arise from several molecular mechanisms. PDR is typically characterized by
cross-resistance to many structurally and functionally unrelated drugs. Each mechanism may
operate on its own or in combination with another one at the same time or in a developmental
process. Yellow balls indicate environmental xenobiotics or drugs; green balls, endogenous
metabolites; N, nucleus; V, vacuole.
Similar to the situation in mammals, overexpression of ABC transporters

in yeast or fungal pathogens leads to PDR [11]. MDR phenomena have also
been described in parasites [106] and even in bacteria [156]. In fact, PDR in
fungi [152] is at least in principle quite similar to MDR, since it can be
invoked by imposing drug selection on S. cerevisiae cells [6]. Again,
parasites [106] and bacteria [81] exposed to sublethal doses of xenobiotics
will eventually aquire a multiple antibiotic resistance phenotype in vitro and
in vivo.
Genome sequencing uncovered 30 ABC proteins in yeast [26, 127, 138],
some of which are structural and functional homologues of mammalian ABC
pumps. The molecular basis and mechanisms of ABC-mediated drug
resistance have thus been under intense investigation for many years. Studies
in numerous laboratories disclosed the functions of most yeast ABC
proteins, but also uncovered a complex network of transcription factors
required for the dynamic regulation of ABC genes in response to many
environmental cues. Because certain fungal ABC transporters are also
implicated in drug resistance, stress response, as well as other cellular
processes [10], yeast has been considered a well-suited model system for
studies on ABC proteins of medical importance [11, 138]. Yeast strains
lacking endogenous ABC pumps have been utilized as “test tubes” in
functional cloning approaches. Closely related yeasts such as Pichia pastoris
were also successfully exploited as eukaryotic hosts for the high-level
expression of certain ABC transporters. Cross-complementation studies

yielded important insights on mammalian, plant and parasite ABC transporters
of the P-gp and MRP families. In this chapter, we provide a comprehensive
discussion of how yeast has been beneficial for studies on drug resistance
mechanisms mediated by ABC efflux pumps, and how these studies may aid
future attempts to overcome ABC-mediated diseases.
3 FUNCTIONS OF YEAST ABC TRANSPORTERS

IN DRUG RESISTANCE AND STRESS RESPONSE
According to their evolutionary relationships, yeast ABC proteins are

classified into five distinct groups represented by the MDR, PDR,
MRP/CFTR, ALDP, and YEF3/RLI families [127]. In this chapter, we shall
limit our discussion on yeast ABC proteins (Table 1) implicated in cellular
detoxification, stress response and drug resistance, and refer the reader to a
widely available review literature on the function other yeast ABC proteins
or non-ABC genes [68] of the PDR network [11, 68, 138].
Table 1. Membrane-associated yeast ABC transporters and some relevant transport substrates
ABC protein Relevant substrates Length Topology Location References
Pdr5p/Sts1p Antifungal drugs, 1,511 (ABC-TMS6)2 PM [13, 9]
steroids, myco-toxins,
peptides
Snq2p Drugs, steroids, 1,501 (ABC-TMS6)2 PM [129]
mutagens, 4-NQO
Pdr15p Detergents, chlo- 1,529 (ABC-TMS6)2 PM [150]
ramphenicol, phenolic
herbicides
Pdr10p Membrane 1,511 (ABC-TMS6)2 PM [151]
detergents?
Yor1p Oligomycin, 1,477 TMS11-ABC-TMS6- PM [62]
reveromycin A, drugs ABC
Pdr12p C3-C7 weak acid 1,511 (ABC-TMS6)2 PM [111]
anions, benzoate,
sorbate
Ycf1p GSH-conjugates, 1,515 TMS11-ABC-TMS6- VAC [137]
cadmium, DNB-GS ABC
Bpt1p Cadmium, DNB-GS, 1,515 TMS11-ABC-TMS6- VAC [66, 132]
glucuronides ABC
Ybt1p/Bat1p Taurocholate, UCB, 1,515 TMS11-ABC-TMS6- VAC [105]
bilirubin ABC
Vmr1p Unknown 1,592 TMS11-ABC-TMS6- Unknown SGD
ABC
Nft1p Unknown 1,524 TMS11-ABC-TMS6- Unknown SGD [96]
ABC
ABC, ATP-binding cassette; TMS, transmembrane segment; PM, plasma membrane; VAC, Vacuole; GS,
glutathione S; UCB, unconjugated bilirubin; PM, plasma membrane; 4-NQO, 4-nitroguinoline-A-oxide;
DNB-GS, (2,4-dinitrobenzene)-glutathione; UCB, unconjugated bilirubin; SGD, Saccharomyces Genome
Database (http://www.yeastgenome.org/).
3.1 The plasma membrane ABC transporters – a first defense
Although most PDR transporters are found in the plasma membrane,

most intracellular compartment membranes also harbor at least one or two
ABC transporters (Figure 2). Well-known ABC pumps of the PDR family
are the plasma membrane-localized efflux pumps Pdr5p [9, 13] and Snq2p
[129], whose overexpression mediates pronounced resistance phenotypes.
These transporters recognize a remarkable broad spectrum of xenobiotics
and hydrophobic drugs, comprising hundreds of different compounds that
are extruded to the extracellular space [13, 21, 35, 48, 57, 69, 88]. The first
yeast drug pump identified was Snq2p [129], causing tolerance to mutagens
such as 4-nitroquinoline-N-oxide (4-NQO), although Snq2p also modulates
sensitivity to Na+, Li+, and Mn2+ ions [100]. PDR5/STS1/YDR1/LEM1 was
then identified independently by several groups through its ability to mediate
resistance to cycloheximide [9], cerulenenin and staurosporine [57],
mycotoxins [13] or as glucocorticoid transporter [70]. Other closely related
members of the PDR family include Pdr10p and Pdr15p, sharing more than
70% primary sequence identity with Pdr5p [151]. Their transport functions
or substrates have remained obscure for quite some time. However, recent
evidence suggests that Pdr15p is a stress response gene displaying signify-
cant transport capacity for chloramphenicol [153] and phenolic herbicides
(Mamnun and Kuchler, unpublished data). Notably, a distinct expression
regulation of Pdr15p and Pdr5p suggests a normal role of Pdr15p [153] and
Pdr5p [91] in detoxification under adverse conditions and in actively
growing cells, respectively. Another ABC transporter mediating drug efflux
is represented by Yor1p, a plasma membrane pump of the MRP/CFTR
family [62]. Yor1p was originally identified as a oligomycin transporter, but
it can also transport a large spectrum of different drugs [21, 69]. Therefore,
certain plasma membrane ABC transporters constitute a first defense line
against environmental toxins. Some others may contribute to endogenous
detoxification because they can extrude toxic metabolites that accumulate
during cell growth. For instance, the plasma membrane pump Pdr12p, which
is most closely related to Snq2p, is essential for adaptation to weak organic
acid stress exerted by benzoate or sorbate [111]. Pdr12p also effluxes
metabolites such as short chain carboxylic acid anions, and it is essential for
weak acid stress adaptation [111]. Finally, the PDR transporters Aus1p and
Pdr11p may be involved in sterol uptake under conditions of impaired
ergosterol biogenesis [150], but a distinct drug efflux activity has not been
established for them.
Figure 2. Membrane ABC transporters in yeast. Graphical presentation of characterized ABC

transporters and their cellular localization in yeast. Membrane-associated yeast ABC
transporters of the vacuolar membrane, the plasma membrane, the inner mitochondrial
membrane, and the peroxisome are depicted. Only those membrane-associated ABC
transporters are depicted for which literature data on their function is available. ER,
endoplasmic reticulum; N, nucleus; VB, vacuole; M, mitochondrium; GV, Golgi vesicles;
P. peroxisomes.
A second important mechanism of PDR involves the intracellular

sequestration of metabolites or xenobiotics into the vacuole (Figure 2).
Hence, the MRP/CFTR-like ABC pumps Ycf1p, Ybt1p, and Bpt1p perhaps
constitute a second and equally important intracellular defense line in the
vacuolar membrane against cytotoxic compounds or even stress-derived
breakdown products [127]. Ycf1p mediates vacuolar detoxification of heavy
metals, as well as glutathione S-conjugates [82, 137]. This explains the drug
and heavy metal tolerance phenotypes of strains with increased YCF1 gene
dosage, but also the hypersensitivity of cells lacking Ycf1p. Loss of Ycf1p
also causes defects in vacuolar uptake of As(GS)3 [46, 137]. Similarly, a
lack of Yor1p also causes cadmium hypersensitivity, indicating a functional
overlap of plasma membrane Yor1p with vacuolar Ycf1p [21, 62].
Two additional MRP/CFTR members exist that have been implicated in
vacuolar transport processes. The Ybt1p/Bat1p pump transports taurocholate
[105] and magnetic resonance contrast agents across vacuolar membranes
[109]. The closest Ycf1p homologue Bpt1p accounts for one third of the
total vacuolar uptake of glutathione conjugates. Furthermore, Bpt1p

mediates vacuolar delivery of cadmium and unconjugated bile pigments [66,
110, 132]. Interestingly, Bpt1p levels are strongly elevated in early stationary
phase, suggesting that both Bpt1p and Ycf1p represent key players in
vacuolar sequestration of glutathione conjugates, which is important during
later stages of cell growth [66]. A new MRP/CFTR transporter of as yet
unknown substrate specificity may be encoded by NFT1 [96]. This gene
exists in some strain backgrounds as a fusion of the YKR103 and YKR104
ORFs. In the sequenced strain S288c, however, these ORFs are separated by
a single stop codon, but this may in fact represent mutation or genomic
polymorphism [96]. Finally, Vmr1p (YHL035c) could represent another
MRP/CFTR-like transporter of the vacuolar membrane, but a cellular
transport function remains to be disclosed (http://www.yeastgenome.org/).
Notably, vacuolar detoxification of conjugated metabolites, xenobitics, or
toxins by MRP/CFTR-like transporters, in addition to their cellular extrusion
across the plasma membrane, represents a fundamental detoxification
mechanism in plants [95]. Likewise, xenobiotic and drug detoxification in
mammals is accomplished by their conjugation and subsequent removal
from organs such as liver, kidney, or intestine by several MRP-like
transporters [14].
The yeast MDR family also harbors mitochondrial ABC transporters,
Mdl1p, Mdl2p [25], and Atm1p [79], all of which localize to the inner
mitochondrial membrane [83]. None of these transporters has been associated
with typical drug transport. However, the peptide exporter Mdl1p [155] may
also serve a function in oxidative stress response as its overexpression
suppresses a loss of Atm1p [19]. Atm1p is otherwise mediating
mitochondrial export of Fe/S cluster precursors [65]. The ALD-family
members Pxa1p/Pxa2p [131, 136] are found in the peroxisomal membrane
[5], where they may mediate peroxisomal uptake of very long chain fatty
acids. Interestingly, Mdl1p, Mdl2p, Pxa1p and Pxa2p, and Atm1p, are
homologues of human ABC genes such as the TAP peptide transporters
required for MHC class I antigen presentation [20], the adrenoleuko-
dystrophy genes [5], and ABC7 involved in X-linked sideroblastic anemia
and ataxia [4], respectively.
What then is the physiological role of PDR-genes apart from their ability
to confer basal drug tolerance and hyperresistance upon increasing their
gene dosage? A hypothetical function of yeast ABC pumps might be a role
in the asymmetric distribution of phospholipids in membranes or the
membrane removal of damaged or oxidized lipids. The fact that membrane-
damaging agents such as herbicides, lysophospholipids, and other detergents
strongly induce Pdr15p seems to further support this idea (Mamnun and
Kuchler, in preparation). At least some fungal pumps [134] and their
regulators [63] have indeed been implicated in membrane lipid transport or

even phospholipid flipping (see also section 4). In fact, lipid homeostasis in
the bilayer must undergo rapid and dynamic changes to form transient
regions with distinct functions such as lipid rafts or even non bilayer phases
[33]. However, the current evidence for a role of yeast ABC pumps in
membrane lipid homeostasis, lipid-remodeling, or trans-bilayer transport
[23] is vague at best, due to a lack of related phenotypes of cells lacking
these pumps. Nevertheless, several MRP and MDR-like ABC transporters in
the liver are tightly linked to membrane transport of sterols or phospholipids,
since phenotypes of associated human diseases, mouse models lacking
corresponding ABC transporters [67] or in vitro studies [114] strongly
suggest a role of mammalian ABC transporters in lipid transport [14].
The most prominent and abundant yeast PDR pumps Pdr5p and Snq2p
have the ability to transport hundreds of different compounds, including
lipid-like substrates or even detergents. However, their physiological
substrates, apart from detoxification of environmental xenobiotics, remain
obscure. Notably, the expression regulation of the PDR5-family genes is
quite distinct. Pdr5p is mainly expressed during logarithmic growth [91],
whereas Pdr15p is strongly induced when cells approach stationary phase
[153], and both Pdr10p (Wolfger and Kuchler, unpublished data) and Pdr15p
[153] are highly induced by a variety of adverse conditions or metabolic
stress. Further, Pdr12p effluxes weak organic acids such as C3–C7
carboxylate anions including toxic metabolites such as propionate [111],
thus participating in the defense against metabolic stress. Taken together, a
normal function of PDR transporters in detoxification and stress adaptation
remains as the most plausible one, particularly considering the environ-
mental cues yeast cells encounter in nature. In fact, it is feasible that yeast
ABC transporters have a limited number of endogenous “drug-like” sub-
strates, but have rather evolved to serve defensive functions in the ever-
ongoing “biological” warfare of various microbial species, including plants
and their fungal pathogens that have to share similar ecological niches [143].
4 THE PLEIOTROPIC DRUG RESISTANCE (PDR)

NETWORK – THE TRANSCRIPTION
CONNECTION
Transcription must play a key role in modulating PDR, since cells ought
to be able to discriminate between cellular defense against metabolites or if
there is a need to combat environmental challenges. Indeed, most yeast PDR
and MRP/CFTR genes (Table 1) form a complex genetic network known
as the PDR network. Several related and to some extent promiscuous

transcription factors (Table 2) control expression of target genes to allow for
control of ABC pumps under a variety of growth conditions and environ-
mental cues (Figure 3). For simplicity, Figure 3 only depicts ABC genes
present in the PDR network, while it also includes several non-ABC genes
such as major facilitator permeases and others [10, 30, 32, 68].
Table 2. Transcriptional Regulators of Yeast ABC transporters in the PDR Network

Protein TF family Function Promoter Reference
motif
Pdr1p Zn(II)2Cys6 - TF Regulator of PDR network PDRE [8]
Pdr3p Zn(II)2Cys6 - TF Regulator of PDR network PDRE [29]
Pdr8p Zn(II)2Cys6 - TF Regulation of PDR15, PDRE [56]
YOR1, SNQ2
Yrr1p Zn(II)2Cys6 - TF Regulation of SNQ2, YOR1 PDRE [22]
Yrm1p Zn(II)2Cys6 - TF Regulation of PDR, Yrr1p Unknown [86]
competitor?
Ucp22p Zn(II)2Cys6 - TF Regulator of PDR11 and SRE [145]
AUS1
Ecm22pp Zn(II)2Cys6 - TF Regulator of PDR11 and SRE [145]
AUS1
Rdr1p Zn(II)2Cys6 - TF Negative regulator of PDR5 PDRE?? [54]
Ngg1p Coactivator Pdr1p modulator, HAT Not applicable [94]
complex
Stb5p Zn(II)2Cys6 - TF Regulation of PDR PDRE?? [2]
transporters
War1p Zn(II)2Cys6 - TF Required for stress WARE [71]
induction of PDR12
Yap1p bZip - TF Regulation of YFC1, YRE [101]
cadmium resistance
Cad1p/Yap2p bZip - TF Regulator of cadmium YRE [99]
resistance by Ycf1p
Msn2p/Msn4p Cys2His2 - TF General stress response STRE [36]
regulators
PDR, pleiotropic drug resistance; PDRE, pleiotropic drug resistance element; STRE, stress response
element; YRE, Yeast AP-1 response element; TF, transcription factor; SRE, sterol regulatory element.
The first yeast gene capable of conferring PDR was in fact PDR1 that
encoded a predicted Zn(II)2Cys6 transcription factor [8] rather than a
membrane efflux pump. Further work showed that additional Zn(II)2Cys6
regulators (Table 2), including Pdr3p [29], Yrr1p [22, 157], Pdr8p [56],
Ucm22p and Ecm22p [145, 150], and War1p [71] play crucial roles in the
regulation of yeast ABC pumps during normal cellular growth but also in
response to adverse conditions. While several factors contribute to the
regulation of ABC transporters in the network (Figure 3), Pdr1p and
Pdr3p are still considered the master regulators of PDR [11, 68]. Pdr1p and
Pdr3p constitutively localize to the nucleus, and control PDR genes as
homo- and heterodimers [90] through so-called PDR elements (PDREs,
5′-YCCYCCGR-3′) in target promoters [30, 60]. A single cis-acting PDRE
is necessary and sufficient to attract control by Pdr1p/Pdr3p [60, 61].
Deletion of both genes causes pronounced drug hypersensitivity phenotypes,
since they regulate basal expression of several ABC transporters, including
PDR5 [60], PDR10, PDR15 [151], SNQ2 [89], YOR1 [52], as well as other
non-ABC genes implicated in the PDR phenomenon [30, 68]. In addition,
Yrr1p as well as Pdr8p are involved in the regulation of Snq2p, Pdr15p, and
Yor1p [22, 56, 157]. The presence of two PDREs in the promoters of PDR3
and YRR1 suggest autoregulatory loops in their control [21, 27, 157]. The
scenario is even more complex, since Yrm1p appears to compete with Yrr1p
for promoter occupancy on certain target genes, but a physiological
relevance for such a competition is unclear [86]. Strikingly, Pdr1p and Pdr3p
can exert opposing regulatory effects on target ABC genes that even encode
highly homologous transporters. For example, a lack of PDR1 reduces PDR5
expression, but increases PDR15 mRNA levels. In turn, only Pdr3p, but not
Pdr1p, is required for basal PDR15 expression [151].
Figure 3. ABC gene regulation within the yeast PDR network. The gene names in the
ellipses in the center represent ABC target genes of transcriptional regulators depicted about
and below. Transcription factors exerting regulatory effects are connected with target genes
by an arrow. An arrow can indicate positive or negative control. Note that the cartoon only
includes ABC genes for clarity and simplicity. Several non-ABC genes such as membrane
transporters of the major facilitator family are also regulated by some of the transcription
factors depicted in the cartoon. For details see text and references.
All GAL4-like and fungal-specific Zn(II)2Cys6 transcription factors share

a rather simple molecular architecture: the N-terminal zinc cluster mediates
DNA binding, while the regulatory and activation domains are located in the
large C-terminal part [93]. Constitutive active, and sometimes dominant
alleles of Pdr1p/Pdr3p as well as other Zn(II)2Cys6 regulators arise from
mutations in the activation domain or in the middle homology region that is
considered an inhibitory domain [93]. This is consistent with the fact that
many gain-of-function mutations in PDR1/PDR3 are found in this region or
in the C-terminal activation domains [17, 29, 68, 104, 133]. Hyperactive
alleles of PDR1, PDR3, or YRR1 cause a pronounced PDR phenotype, since
target pumps such as Pdr5p [98] or Snq2p [89] and Yor1p [157] are
dramatically overexpressed. Most constitutive alleles of PDR1 and PDR3
were identified during genetic screens for increased drug tolerance [17, 63,
104, 117, 149]. Equivalent Pdr8p variants were generated by analogy to
Pdr1p/Pdr3p, and in combination with microarray profiling, Pdr15p and
Yor1p were identified as regulatory targets for Pdr8p [56]. Interestingly,
dominant PDR1 and PDR3 alleles can also be specifically obtained by long-
term selection of yeast cultures with the antifungal drug fluconazole [6].
Moreover, mutations of the fluconazole target gene ERG3 are also
preferentially selected during the in vitro evolution of antifungal resistance
[6]. Gain-of-function Pdr1p/Pdr3p variants have also been obtained in a
mutant screen for genes defective in phosphatidylethanolamine endocytosis,
implying a role of some Pdr1p targets in maintaining the phospholipid
asymmetry at the plasma membrane [63]. Pdr1p/Pdr3p have also been
implicated in the control of spingholipid biosynthesis through the regulation
of Ipt1p [51]. Furthermore, the ABC transporters Aus1p and Pdr11p are
regulated by Ucp2p and Ecm22p, two Zn(II)2Cys6 ergosterol pathway
regulators that drive gene expression through sterol regulatory elements
(SRE) in target promoters [145].
However, in all cases, the molecular signaling events leading to the
activation of Zn(II)2Cys6 PDR regulators (Table 2) have remained enigmatic,
and additional components have escaped genetic identification until now.
It is possible that the PDR1/3 system represents a genetic sensing tool
allowing for the rapid selection of hyperactive transcription factor genes
as an alternative to complex drug recognition and signal transduction
pathways. The advantage of such a general drug-sensing tool might be that it
is not restricted to certain molecule classes, thereby providing versatility that
could be beneficial for efficient xenobiotic defense. It is interesting to note,
that several transcription factors of this family seem to respond directly to

small molecules, perhaps inducing conformational changes that lead to their
activation and recruitment of the mediator complex. For instance, Gal4p
[154], Leu3p [50, 147], or Put3p [31] are activated by their cognate ligands
via direct binding. Target pumps of the bacterial mar operon are also
activated to induce antibiotic resistance after binding of several different
antibiotics and chemicals to the Mar regulator [81]. By analogy, several
mammalian MRP and MDR-like ABC genes are tightly regulated by hetero-
and homodimeric members of the RXR, FXR family, which are activated by
ligand-binding. RXR and FXR regulators play important roles in lipid
homeostasis, sterol transport, and hormonal response [39, 92, 124]. Although
Zn(II)2Cys6 genes are restricted to fungal species, they may be viewed at
least as functionally related to nuclear orphan receptors in mammals.
However, activating drug or stress ligands have not been identified for
fungal PDR regulators as yet.
Additional transcription factors increase the complexity of the PDR
network. For example, Ngg1p seems to interact with the C-terminal activation
domains of Pdr1p thus reducing its regulatory activity [94, 121]. Further,
Rdr1p appears to repress PDR5 transcription [54], while Sbt5p seems to
engage PDREs to act as a positive regulator of PDR5 and SNQ2 expression
[2]. Since transcription factors regulating the environmental stress response
are targeting many genes [43], it is not surprising that some overlaps exist
between PDR and stress response. For example, PDR15 regulation involves
Pdr1p and Pdr3p, as well as the general stress response regulators Msn2p
and Msn4p [153]. Moreover, PDR12 is regulated by the stress-specific
transcription factor War1p through cis-acting WARE motifs in PDR12 [71].
Weak acid stress also activates the Msn2p/Msn4p general stress response
[128]. However, Pdr12p induction does not need Msn2p or Msn4p, but only
the War1p regulator, whose posttranslational activation involves a
phosphorylation cycle [71]. Further, the bZip transcription factor Yap1p is a
prime regulator of oxidative stress adaptation [28, 29, 101], and it also
induces Snq2p in response to oxidative stress (Mahé and Kuchler, unpublished
data). This Yap1p-mediated cadmium resistance requires Ycf1p, suggesting
a direct regulation of YCF1 by Yap1p [148]. Notably, Ycf1p-mediated
cadmium tolerance is also controlled through another bZip regulator,
Cad1p/Yap2p [37].
In summary, the above-said illustrates the intense molecular crosstalk
operating between ABC transporter regulation and stress response. Even
mitochondrial dysfunction impacts gene expression within the PDR network
[32, 53], but the physiological relevance needs further clarification. Fact is
that the regulation of the PDR network utilizes a complex array of
functionally distinct but structurally related and sometimes redundant
transcription factors, all of which provide a dynamic expression control

under normal and many adverse conditions. This system might have evolved
to cope with rapidly changing growth conditions, so as to maintain the most
appropriate transporter composition at the right cellular membrane at any
given moment of the yeast life cycle.
5 FUNCTIONAL EXPRESSION OF MAMMALIAN

ABC GENES AND DRUG DISCOVERY – A YEAST
TOOLBOX
The pronounced drug hypersensitivity of certain pump deletion strains

indicated that such strains are useful for susceptibility testing, drug iden-
tification, as well as functional cloning approaches. Simple susceptibility
testing indeed revealed hundreds of potential substrates for Pdr5p, Snq2p,
and Yor1p [69]. Similarly, drug profiling of strains with multiple deletions
of ABC genes (YOR1, SNQ2, PDR5, YCF1, PDR10, PDR11, and PDR15),
as well as the PDR1 and PDR3 regulators, disclosed the extraordinary
hypersensitivity of such strains [119]. Our laboratory has constructed nume-
rous isogenic strains lacking all plasma membrane as well as vacuolar ABC
pumps (Schuetzer-Muehlbauer and Kuchler, unpublished data). We have
also generated many combinations of ABC deletions, yielding dozens of
strains with qualitative and quantitative distinct drug susceptibility spectra
(Schuetzer-Muehlbauer and Kuchler, unpublished data). These host strains
may in fact be useful for setting up cell-based high-throughput screening
(HTS) assays in cases were compound libraries are limited in terms of drug
concentrations. Interestingly, we have generated a strain lacking Pdr5p,
Snq2p, the a-factor mating pheromone transporter Ste6p [73, 97] as well as
the ERG6 gene [41]. This tester strain display an almost three orders of
magnitude increased susceptibility to a vast range of hydrophobic drugs
when compared to its wild-type parent (Pandjaitan and Kuchler, unpublished
data). ERG6 was removed in this pump deletion background, because it has
been known for quite some time that cells lacking Erg6p have abnormal
plasma membrane ergosterol levels, which causes dramatic changes in
overall cell permeability to ions and many drugs [41, 113, 130]. This
supersensitive strain somehow “diffused” into the yeast community and it
has indeed been successfully exploited for drug discovery approaches by
HTS, leading to the identification of novel CDK inhibitors [49].
Because PDR results from efflux pump overexpression, simple gene
dosage approaches allow for cloning of new ABC drug resistance genes.
Indeed, this strategy identified the first Candida albicans ABC transporters,
Cdr1p [112, 123] and Cdr2p [122], both of which mediate clinical antifungal
resistance. The functional expression of C. albicans ABC transporters in
baker’s yeast also uncovered a hitherto unknown caspofungin transport capa-
city of Cdr2p [126]. Importantly, this defined system also established that
antifungal drug resistance can be efficiently reversed through the use of
ABC efflux pump inhibitors [125] or it can be used for the molecular analysis
of fungal drug pump function [103]. Similarly, MDR reversal has also been
attempted in cancer therapy in a clinical setting as a means to restore anti-
cancer drug response, but the success has been limited up to now [84].
To study functional conservation and to clone and identify novel drug
transporters, several mammalian ABC genes have been functionally expressed
in yeast. Cross-complementation studies yielded important information
about the function mammalian, parasite, plant, and other fungal ABC genes.
The first functional expression demonstrated that human Mdr1 P-gp is
functional when expressed in yeast, since it confers resistance to valinomycin
and actinomycin D [74, 142]. Interestingly, mouse mdr3, when expressed in
yeast cells lacking the Ste6p a-factor transporter [72], rescues the mating
defect [115], whereas human Mdr1 is unable to extrude the mating
pheromone [74]. Likewise, pfmdr1 from Plasmodium falciparum appears to
complement a loss of Ste6p, at least in certain strain backgrounds [146].
Mouse mdr3 in yeast also confers resistance to immunosuppressive drugs
such as FK520 [116] or the anticancer drug dactinomycin [55]. Yeast
expression of human Mdr2 demonstrated its physiological function as a
phospholipid translocase [120]. Further, expression of MRPs, homologues of
Ycf1p, revealed that human MRP1 as well as plant AtMRPs can
complement the heavy metal sensitivity of cells lacking Ycf1p [42, 85, 140].
Nevertheless, the signals for correct trafficking of MRPs appear to work
inefficiently in yeast, since MRPs and Mdr1 are also found in other cellular
membranes than their normal cellular location in mammalian cells [74, 140].
Yeast has also been used for the analysis and expression of human ABC
genes implicated in genetic diseases, including the CFTR [118] cystic
fibrosis transmembrane conductance regulator [64, 107, 139], and the TAP
transporters of antigen presentation, which are also functional as peptide
transporters in yeast [144]. Furthermore, the human ABC7 gene [4], which is
mutated in sideroblastic anemia with cerebellar ataxia (XLSA/A), was
produced in yeast and complemented the defect of the yeast homologue
ATM1 [12].
Baker’s yeast and other yeasts have also been exploited as single cell
“bags” for the high-level expression and purification of endogenous [38] and
heterologous ABC pumps [16, 59, 80]. For example, the PMA1 promoter of
the plasma membrane proton pump, significantly boosted MRP1 expression,
allowing for the production of milligram amounts of purified MRP1 [76, 77].
The related species P. pastoris is also suited for production of mammalian
ABC proteins or individual nucleotide binding domains. Purified proteins
can be reconstituted into in vitro systems, enabling studies on the catalytic
cycle of ABC transporters, structural analyses [16, 80] and perhaps even
crystallization trials [38].
Although expression of certain mammalian ABC proteins has been
accomplished in yeast, many attempts to express mammalian or human
transporters in yeast failed or gave inconsistent results. This may be because
of nonfunctional or different trafficking signals, improper protein folding,
aberrant cellular localization, lack of appropriate in vitro or in vivo assays,
unknown drug substrates or just inappropriate strain backgrounds.
Furthermore, the different lipid environment of yeast membranes may
change or even inhibit ABC function. In fact, this is the case for the retinal
ABC transporter, whose activity is even modulated by lipids in the native
membrane environment [1]. Moreover, other ABC pumps even mediate
membrane transport of lipids or sterols [75, 114, 120]. Changes in the
normal lipid composition, like those caused by defects in ergosterol
biosynthesis, dramatically modulate cell permeability [41, 113, 130] and
may therefore also affect transport activity, substrate specificity, or even the
proper folding of ABC pumps [135]. However, once expression is achieved
and verified by appropriate functional assays, yeast does represent a useful
host for the molecular genetic analysis of heterologous ABC transporters.
6 CONCLUSIONS AND PERSPECTIVES
Of all eukaryotic model organisms, yeast is perhaps the most versatile

one, since a wealth of genetic and biochemical tools are available. For instance,
a gene deletion collection is widely available (EUROSCARF, Germany;
http://www.uni-frankfurt.de/fb15/mikro/euroscarf/), all yeast genes have
been tagged with GFP [45], GST fusions are available, and gene arrays
have become almost routine tools by now. Even protein arrays have been
recently described [158, 159]. The yeast community benefits from highly
developed gene ontology at the Saccharomyces Genome Database (http://
www.yeastgenome.org/) that comes along with most up-to-date online
databases [34]. Of course, above all is the genetic tractability of yeast with
its elegant genetics [40] and gene-replacement methods. This makes yeast a
fascinating and irresistible system, enabling functional studies like in no
other eukaryotic model system. In fact, yeast perhaps is the most promising
model for systems biology approaches that are currently receiving consi-
derable attention in the global science community [18].
After entering the post-genomic era, a series of elegant genome-wide
approaches further sparked the general interest in yeast as a tool for drug dis-
covery. For instance, genome-wide drug profiling [108], haplo-insufficiency
screening [47, 87], and global metabolomics [3] indicated that yeast can be
very useful for drug target identification. Haplo-insufficiency screening may
even provide clues about targets of orphane drugs and disclose possible
mechanisms of drug action [87]. The global description of transcription
factor promoter-binding sites undoubtedly indicates incredible complexity,
but at the same time suggest meaningful genetic and biochemical approaches
to better understand transcription in general [78]. We are just beginning to
understand the genetic interactions by exploring genome-wide synthetic
lethality relationships of otherwise nonessential genes, which might be bene-
ficial for future strategies to dissect complex genetic diseases in humans
[141]. The knowledge about global protein interaction maps [7, 44] and
perhaps their dynamic changes during cell growth is closing the circle.
Nevertheless, as elegant as genome-wide approaches seem, one must not
forget that they do not alleviate the need for mechanistic approaches to
deepen the understanding of complex biological phenomena such as PDR
development or ABC protein function both at the molecular level, and in a
whole-cell context.
To come to full circle, yeast is unlikely to loose its attraction as a
valuable test tube for the functional characterization of other eukaryotic or
mammalian ABC proteins. Once physiological substrates of ABC pumps
have been identified, one will be able to produce tailor-made yeast strains
with desired drug susceptibility, facilitating drug discovery through HTS
approaches [49, 102]. The disadvantage of a generally reduced drug
susceptibility of yeast cells compared to mammalian cells can be overcome
by using supersensitive strains lacking certain ABC pumps [49, 58]. Another
argument stems from the financial aspect, since whole cell-based assays in
yeast are more than order of magnitude cheaper than comparable assays in
mammalian cell culture. This way, yeast will perhaps contribute to a better
understanding of other medically relevant ABC proteins, since the closed
nutshell opens a door to a defined genetic system for analysis at the
molecular level. Therefore, yeast must not be neglected as a model system
for functional approaches on eukaryotic ABC transporters of medical
importance, even in cases where no cellular function is known.
ACKNOWLEDGMENTS
Our research is supported by grants from the Austrian Science

Foundation (FWF-15934-B08), by funds from the Austrian National Bank
(OENB #9985), the “Herzfelder Family Foundation”, the Vienna Science
and Technology Funds from the City of Vienna (WWTF-Project LS113),
and in part by COST Action B16 action from the European Commission.
REFERENCES
1. Ahn, J., J. T. Wong, and R. S. Molday. 2000. The effect of lipid environment and
retinoids on the ATPase activity of ABCR, the photoreceptor ABC transporter
responsible for Stargardt macular dystrophy. J. Biol. Chem. 275:20399–20405.
2. Akache, B., and B. Turcotte. 2002. New regulators of drug sensitivity in the family of
yeast zinc cluster proteins. J. Biol. Chem. 277:21254–21260.
3. Allen, J., H. M. Davey, D. Broadhurst, J. K. Heald, J. J. Rowland, S. G. Oliver, and
D. B. Kell. 2003. High-throughput classification of yeast mutants for functional
genomics using metabolic footprinting. Nat. Biotechnol. 21:692–696.
4. Allikmets, R., W. H. Raskind, A. Hutchinson, N. D. Schueck, M. Dean, and D. M.
Koeller. 1999. Mutation of a putative mitochondrial iron transporter gene (ABC7) in X-
linked sideroblastic anemia and ataxia (XLSA/A). Hum. Mol. Genet. 8:743–749.
5. Almashanu, S., and D. Valle. 2003. Peroxisomal ABC transporters, pp. 497–513. In
ABC Proteins: From Bacteria to Man, Susan P. C. Cole, I. Barry Holland, Karl Kuchler,
and Christopher F. Higgins (eds.), Academic Press Elsevier Science, Amsterdam.
6. Anderson, J. B., C. Sirjusingh, A. B. Parsons, C. Boone, C. Wickens, L. E. Cowen,
and L. M. Kohn. 2003. Mode of selection and experimental evolution of antifungal
drug resistance in Saccharomyces cerevisiae. Genetics 163:1287–1298.
7. Bader, G. D., A. Heilbut, B. Andrews, M. Tyers, T. Hughes, and C. Boone. 2003.
Functional genomics and proteomics: charting a multidimensional map of the yeast cell.
Trends Cell Biol. 13:344–356.
8. Balzi, E., W. Chen, S. Ulaszewski, E. Capieaux, and A. Goffeau. 1987. The
multidrug resistance gene PDR1 from Saccharomyces cerevisiae. J. Biol. Chem.
262:16871–16879.
9. Balzi, E., M. Wang, S. Leterme, L. Van Dyck, and A. Goffeau. 1994. PDR5, a novel
yeast multidrug resistance conferring transporter controlled by the transcription
regulator PDR1. J. Biol. Chem. 269:2206–2214.
10. Bauer, B. E., C. Schüller, and K. Kuchler. 2003. Fungal ABC proteins in clinical drug
resistance and cellular detoxification, pp. 295–316. In ABC Proteins: From Bacteria to
Man, Susan P. C. Cole, I. Barry Holland, Karl Kuchler, and Christopher F. Higgins
(ed.), Academic Press Elsevier Science, Amsterdam.
11. Bauer, B. E., H. Wolfger, and K. Kuchler. 1999. Inventory and function of yeast ABC
proteins: about sex, stress, pleiotropic drug and heavy metal resistance. Biochim.
12. Bekri, S., G. Kispal, H. Lange, E. Fitzsimons, J. Tolmie, R. Lill, and D. F. Bishop.
2000. Human ABC7 transporter: gene structure and mutation causing X-linked
sideroblastic anemia with ataxia with disruption of cytosolic iron-sulfur protein
maturation. Blood 96:3256–3264.
13. Bissinger, P. H., and K. Kuchler. 1994. Molecular cloning and expression of the
Saccharomyces cerevisiae STS1 gene product. A yeast ABC transporter conferring
mycotoxin resistance. J. Biol. Chem. 269:4180–4186.
14. Borst, P., and R. O. Elferink. 2002. Mammalian ABC transporters in health and
disease. Annu. Rev. Biochem. 71:537–592.
15. Borst, P., A. H. Schinkel, J. J. Smit, E. Wagenaar, L. Van Deemter, A. J. Smith,
E. W. Eijdems, F. Baas, and G. J. Zaman. 1993. Classical and novel forms of
multidrug resistance and the physiological functions of P-glycoproteins in mammals.
Pharmacol. Ther. 60:289–299.
16. Cai, J., and P. Gros. 2003. Overexpression, purification, and functional
characterization of ATP-binding cassette transporters in the yeast, Pichia pastoris.
Biochim. Biophys. Acta 1610:63–76.
17. Carvajal, E., H. B. van den Hazel, A. Cybularz-Kolaczkowska, E. Balzi, and
A. Goffeau. 1997. Molecular and phenotypic characterization of yeast PDR1 mutants
that show hyperactive transcription of various ABC multidrug transporter genes. Mol.
Gen. Genet. 256:406–415.
18. Castrillo, J. I., and S. G. Oliver. 2004. Yeast as a touchstone in post-genomic
research: strategies for integrative analysis in functional genomics. J. Biochem. Mol.
Biol. 37:93–106.
19. Chloupkova, M., L. S. LeBard, and D. M. Koeller. 2003. MDL1 is a high copy
suppressor of ATM1: evidence for a role in resistance to oxidative stress. J. Mol. Biol.
331:155–165.
20. Cresswell, P., N. Bangia, T. Dick, and G. Diedrich. 1999. The nature of the MHC
class I peptide loading complex. Immunol. Rev. 172:21–28.
21. Cui, Z., D. Hirata, E. Tsuchiya, H. Osada, and T. Miyakawa. 1996. The multidrug
resistance-associated protein (MRP) subfamily (Yrs1/Yor1) of Saccharomyces
cerevisiae is important for the tolerance to a broad range of organic anions. J. Biol.
Chem. 271:14712–14716.
22. Cui, Z., T. Shiraki, D. Hirata, and T. Miyakawa. 1998. Yeast gene YRR1, which is
required for resistance to 4-nitroquinoline N-oxide, mediates transcriptional activation
of the multidrug resistance transporter gene SNQ2. Mol. Microbiol. 29:1307–1315.
23. Daleke, D. L. 2003. Regulation of transbilayer plasma membrane phospholipid
asymmetry. J. Lipid Res. 44:233–242.
24. Dassa, E. 2003. Phylogenetic and functional classification of ABC (ATP-binding
cassette) systems, pp. 3–35. In ABC Proteins: From Bacteria to Man, Susan P. C. Cole,
I. Barry Holland, Karl Kuchler, and Christopher F. Higgins (ed.), Academic Press
Elsevier Science, Amsterdam.
25. Dean, M., R. Allikmets, B. Gerrard, C. Stewart, A. Kistler, B. Shafer, S. Michaelis,
and J. Strathern. 1994. Mapping and sequencing of two yeast genes belonging to the
ATP-binding cassette superfamily. Yeast 10:377–383.
26. Decottignies, A., and A. Goffeau. 1997. Complete inventory of the yeast ABC
proteins. Nat. Genet. 15:137–145.
27. Delahodde, A., T. Delaveau, and C. Jacq. 1995. Positive autoregulation of the yeast
transcription factor Pdr3p, which is involved in control of drug resistance. Mol. Cell.
Biol. 15:4043–4051.
28. Delaunay, A., D. Pflieger, M. B. Barrault, J. Vinh, and M. B. Toledano. 2002. A
thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation. Cell
111:471–481.
29. Delaveau, T., A. Delahodde, E. Carvajal, J. Subik, and C. Jacq. 1994. PDR3, a new
yeast regulatory gene, is homologous to PDR1 and controls the multidrug resistance
phenomenon. Mol. Gen. Genet. 244:501–511.
30. DeRisi, J., B. van den Hazel, P. Marc, E. Balzi, P. Brown, C. Jacq, and A. Goffeau.
2000. Genome microarray analysis of transcriptional activation in multidrug resistance
yeast mutants. FEBS Lett. 470:156–160.
31. Des Etages, S. A., D. Saxena, H. L. Huang, D. A. Falvey, D. Barber, and M. C.
Brandriss. 2001. Conformational changes play a role in regulating the activity of the
proline utilization pathway-specific regulator in Saccharomyces cerevisiae. Mol.
Microbiol. 40:890–899.
32. Devaux, F., E. Carvajal, S. Moye-Rowley, and C. Jacq. 2002. Genome-wide studies
on the nuclear PDR3-controlled response to mitochondrial dysfunction in yeast. FEBS
Lett. 515:25–28.
33. Devaux, P. F., and R. Morris. 2004. Transmembrane asymmetry and lateral domains
in biological membranes. Traffic 5:241–246.
34. Dolinski, K., C. A. Ball, S. A. Chervitz, S. S. Dwight, M. A. Harris, S. Roberts,
T. Roe, J. M. Cherry, and D. Botstein. 1998. Expanding yeast knowledge online.
Yeast 14:1453–1469.
35. Egner, R., F. E. Rosenthal, A. Kralli, D. Sanglard, and K. Kuchler. 1998. Genetic
separation of FK506 susceptibility and drug transport in the yeast Pdr5 ATP-binding
cassette multidrug resistance transporter. Mol. Biol. Cell 9:523–543.
36. Estruch, F., and M. Carlson. 1993. Two homologous zinc finger genes identified by
multicopy suppression in a SNF1 protein kinase mutant of Saccharomyces cerevisiae.
Mol. Cell. Biol. 13:3872–3881.
37. Fernandes, L., C. Rodrigues-Pousada, and K. Struhl. 1997. Yap, a novel family of
eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions. Mol.
Cell. Biol. 17:6982–6993.
38. Ferreira-Pereira, A., S. Marco, A. Decottignies, J. Nader, A. Goffeau, and J. L.
Rigaud. 2003. Three-dimensional reconstruction of the Saccharomyces cerevisiae
multidrug resistance protein Pdr5p. J. Biol. Chem. 278:11995–11999.
39. Fitzgerald, M. L., K. J. Moore, and M. W. Freeman. 2002. Nuclear hormone
receptors and cholesterol trafficking: the orphans find a new home. J. Mol. Med.
80:271–281.
40. Forsburg, S. L. 2001. The art and design of genetic screens: yeast. Nat. Rev. Genet.
2:659–668.
41. Gaber, R. F., D. M. Copple, B. K. Kennedy, M. Vidal, and M. Bard. 1989. The yeast
gene ERG6 is required for normal membrane function but is not essential for
biosynthesis of the cell-cycle-sparking sterol. Mol. Cell Biol. 9:3447–3456.
42. Gaedeke, N., M. Klein, Ü. Kolukisaoglu, et al. 2001. The Arabidopsis thaliana ABC
transporter AtMRP5 controls root development and stomata movement. EMBO J.
20:1875–1887.
43. Gasch, A. P., P. T. Spellman, C. M. Kao, O. Carmel-Harel, M. B. Eisen, G. Storz,
D. Botstein, and P. O. Brown. 2000. Genomic expression programs in the response of
yeast cells to environmental changes. Mol. Biol. Cell 11:4241–4257.
44. Gavin, A. C., M. Bosche, R. Krause, et al. 2002. Functional organization of the yeast
proteome by systematic analysis of protein complexes. Nature 415:141–147.
45. Ghaemmaghami, S., W. K. Huh, K. Bower, R. W. Howson, A. Belle, N. Dephoure,
E. K. O’Shea, and J. S. Weissman. 2003. Global analysis of protein expression in
yeast. Nature 425:737–741.
46. Ghosh, M., J. Shen, and B. P. Rosen. 1999. Pathways of As(III) detoxification in
Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 96:5001–5006.
47. Giaever, G., A. M. Chu, L. Ni, et al. 2002. Functional profiling of the Saccharomyces
cerevisiae genome. Nature 418:387–391.
48. Golin, J., S. V. Ambudkar, M. M. Gottesman, A. D. Habib, J. Sczepanski,
W. Ziccardi, and L. May. 2003. Studies with novel Pdr5p substrates demonstrate a
strong size dependence for xenobiotic efflux. J. Biol. Chem. 278:5963–5969.
49. Gray, N. S., L. Wodicka, A. M. Thunnissen, et al. 1998. Exploiting chemical libraries,
structure, and genomics in the search for kinase inhibitors. Science 281:533–538.
50. Guo, H., and G. B. Kohlhaw. 1996. Regulation of transcription in mammalian cells by
yeast Leu3p and externally supplied inducer. FEBS Lett. 390:191–195.
51. Hallström, T. C., L. Lambert, S. Schorling, E. Balzi, A. Goffeau, and W. S. Moye-
Rowley. 2001. Coordinate control of sphingolipid biosynthesis and multidrug resistance
in Saccharomyces cerevisiae. J. Biol. Chem. 276:23674–23680.
52. Hallström, T. C., and W. S. Moye-Rowley. 1998. Divergent transcriptional control of
multidrug resistance genes in Saccharomyces cerevisiae. J. Biol. Chem. 273:2098–2104.
53. Hallström, T. C., and W. S. Moye-Rowley. 2000. Multiple signals from dysfunctional
mitochondria activate the pleiotropic drug resistance pathway in Saccharomyces
54. Hellauer, K., B. Akache, S. MacPherson, E. Sirard, and B. Turcotte. 2002. Zinc
cluster protein Rdr1p is a transcriptional repressor of the PDR5 gene encoding a
multidrug transporter. J. Biol. Chem. 277:17671–17676.
55. Hemenway, C. S., and J. Heitman. 1996. Immunosuppressant target protein FKBP12
is required for P-glycoprotein function in yeast. J. Biol. Chem. 271:18527–18534.
56. Hikkel, I., A. Lucau-Danila, T. Delaveau, P. Marc, F. Devaux, and C. Jacq. 2003. A
general strategy to uncover transcription factor properties identifies a new regulator of
drug resistance in yeast. J. Biol. Chem. 278:11427–11432.
57. Hirata, D., K. Yano, K. Miyahara, and T. Miyakawa. 1994. Saccharomyces
cerevisiae YDR1, which encodes a member of the ATP-binding cassette (ABC)
superfamily, is required for multidrug resistance. Curr. Genet. 26:285–294.
58. Hughes, T., B. Andrews, and C. Boone. 2004. Old drugs, new tricks: using genetically
sensitized yeast to reveal drug targets. Cell 116:5–7.
59. Julien, M., S. Kajiji, R. H. Kaback, and P. Gros. 2000. Simple purification of highly
active biotinylated P-glycoprotein: enantiomer-specific modulation of drug-stimulated
ATPase activity. Biochemistry 39:75–85.
60. Katzmann, D. J., P. E. Burnett, J. Golin, Y. Mahé, and W. S. Moye-Rowley. 1994.
Transcriptional control of the yeast PDR5 gene by the PDR3 gene product. Mol. Cell.
Biol. 14:4653–4661.
61. Katzmann, D. J., T. C. Hallström, Y. Mahé, and W. S. Moye-Rowley. 1996.
Multiple Pdr1p/Pdr3p binding sites are essential for normal expression of the ATP
binding cassette transporter protein-encoding gene PDR5. J. Biol. Chem. 271:23049–
23054.
62. Katzmann, D. J., T. C. Hallström, M. Voet, W. Wysock, J. Golin, G. Volckaert, and
W. S. Moye-Rowley. 1995. Expression of an ATP-binding cassette transporter-
encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces
cerevisiae. Mol. Cell. Biol. 15:6875–6883.
63. Kean, L. S., A. M. Grant, C. Angeletti, Y. Mahé, K. Kuchler, R. S. Fuller, and
J. W. Nichols. 1997. Plasma membrane translocation of fluorescent-labeled
phosphatidylethanolamine is controlled by transcription regulators, PDR1 and PDR3.
J. Cell Biol. 138:255–270.
64. Kiser, G. L., M. Gentzsch, A. K. Kloser, E. Balzi, D. H. Wolf, A. Goffeau, and J. R.

Riordan. 2001. Expression and degradation of the cystic fibrosis transmembrane
conductance regulator in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 390:195–
205.
65. Kispal, G., P. Csere, C. Prohl, and R. Lill. 1999. The mitochondrial proteins Atm1p and
Nfs1p are essential for biogenesis of cytosolic Fe/S proteins. EMBO J. 18:3981–3989.
66. Klein, M., Y. M. Mamnun, T. Eggmann, C. Schüller, H. Wolfger, E. Martinoia,
and K. Kuchler. 2002. The ATP-binding cassette (ABC) transporter Bpt1p mediates
vacuolar sequestration of glutathione conjugates in yeast. FEBS Lett. 520:63–67.
67. Klett, E. L., K. Lu, A. Kosters, et al. 2004. A mouse model of sitosterolemia: absence
of Abcg8/sterolin-2 results in failure to secrete biliary cholesterol. BMC Med. 2:5.
68. Kolaczkowska, A., and A. Goffeau. 1999. Regulation of pleiotropic drug resistance in
yeast. Drug Res. Updates 2:403–414.
69. Kolaczkowski, M., A. Kolaczowska, J. Luczynski, S. Witek, and A. Goffeau. 1998.
In vivo characterization of the drug resistance profile of the major ABC transporters and
other components of the yeast pleiotropic drug resistance network. Microb. Drug Resist.
4:143–158.
70. Kralli, A., S. P. Bohen, and K. R. Yamamoto. 1995. LEM1, an ATP-binding-cassette
transporter, selectively modulates the biological potency of steroid hormones. Proc.
71. Kren, A., Y. M. Mamnun, B. E. Bauer, et al. 2003. War1p, a novel transcription
factor controlling weak acid stress response in yeast. Mol. Cell. Biol. 23:1775–1785.
72. Kuchler, K. 1993. Unusual routes of protein secretion: the easy way out. Trends Cell
Biol. 3:421–426.
73. Kuchler, K., R. E. Sterne, and J. Thorner. 1989. Saccharomyces cerevisiae STE6
gene product: a novel pathway for protein export in eukaryotic cells. EMBO J. 8:3973–
3984.
74. Kuchler, K., and J. Thorner. 1992. Functional expression of human mdr1 in the yeast
75. Lee, M. H., K. Lu, S. Hazard, et al. 2001. Identification of a gene, ABCG5, important
in the regulation of dietary cholesterol absorption. Nat. Genet. 27:79–83.
76. Lee, S. H., and G. A. Altenberg. 2003. Expression of functional multidrug-resistance
protein 1 in Saccharomyces cerevisiae: effects of N- and C-terminal affinity tags.
Biochem. Biophys. Res. Commun. 306:644–649.
77. Lee, S. H., and G. A. Altenberg. 2003. Transport of leukotriene C4 by a cysteine-less
multidrug resistance protein 1 (MRP1). Biochem. J. 370:357–360.
78. Lee, T. I., N. J. Rinaldi, F. Robert, et al. 2002. Transcriptional regulatory networks in
Saccharomyces cerevisiae. Science 298:799–804.
79. Leighton, J., and G. Schatz. 1995. An ABC transporter in the mitochondrial inner
membrane is required for normal growth of yeast. EMBO J. 14:188–195.
80. Lerner-Marmarosh, N., K. Gimi, I. L. Urbatsch, P. Gros, and A. E. Senior. 1999.
Large scale purification of detergent-soluble P-glycoprotein from Pichia pastoris cells
and characterization of nucleotide binding properties of wild-type, Walker A, and
Walker B mutant proteins. J. Biol. Chem. 274:34711–34718.
81. Levy, S., and M. Alekshun. 1999. The mar regulon: multiple resistance to antibiotics
and other toxic chemicals. Trends Microbiol. 7:410–413.
82. Li, Z. S., M. Szczypka, Y. P. Lu, D. J. Thiele, and P. A. Rea. 1996. The yeast
cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump. J. Biol.
Chem. 271:6509–6517.
83. Lill, R., and G. Kispal. 2003. ABC transporters in mitochondria, pp. 515–531. In ABC
Proteins: From Bacteria to Man, Susan P. C. Cole, I. Barry Holland, Karl Kuchler, and
Christopher F. Higgins (ed.), Academic Press Elsevier Science, Amsterdam.
84. Litman, T., T. E. Druley, W. D. Stein, and S. E. Bates. 2001. From MDR to MXR:
new understanding of multidrug resistance systems, their properties and clinical
significance. Cell Mol. Life Sci. 58:931–959.
85. Liu, G., R. Sanchez-Fernandez, Z. S. Li, and P. A. Rea. 2001. Enhanced
multispecificity of arabidopsis vacuolar multidrug resistance-associated protein-type
ATP-binding cassette transporter, AtMRP2. J. Biol. Chem. 276:8648–8656.
86. Lucau-Danila, A., T. Delaveau, G. Lelandais, F. Devaux, and C. Jacq. 2003.
Competitive promoter occupancy by two yeast paralogous transcription factors
controlling the multidrug resistance phenomenon. J. Biol. Chem. 278:52641–52650.
87. Lum, P. Y., C. D. Armour, S. B. Stepaniants, et al. 2004. Discovering modes of
action for therapeutic compounds using a genome-wide screen of yeast heterozygotes.
Cell 116:121–137.
88. Mahé, Y., Y. Lemoine, and K. Kuchler. 1996. The ATP-binding cassette transporters
Pdr5 and Snq2 of Saccharomyces cerevisiae can mediate transport of steroids in vivo.
J. Biol. Chem. 271:25167–25172.
89. Mahé, Y., A. Parle-McDermott, A. Nourani, A. Delahodde, A. Lamprecht, and
K. Kuchler. 1996. The ATP-binding cassette multidrug transporter Snq2 of Saccharomyces
cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3. Mol. Microbiol.
20:109–117.
90. Mamnun, Y. M., R. Pandjaitan, Y. Mahé, A. Delahodde, and K. Kuchler. 2002. The
yeast zinc finger regulators Pdr1p and Pdr3p control pleiotropic drug resistance (PDR)
as homo- and heterodimers in vivo. Mol. Microbiol. 46:1429–1440.
91. Mamnun, Y. M., C. Schüller, and K. Kuchler. 2004. Expression regulation of the
yeast PDR5 ATP-binding cassette (ABC) transporter suggests a role in cellular
detoxification during the exponential growth phase. FEBS Lett. 559:111–117.
92. Mangelsdorf, D. J., and R. M. Evans. 1995. The RXR heterodimers and orphan
receptors. Cell 83:841–850.
93. Marmorstein, R., M. Carey, M. Ptashne, and S. C. Harrison. 1992. DNA recognition
by GAL4: structure of a protein-DNA complex. Nature 356:408–414.
94. Martens, J. A., J. Genereaux, A. Saleh, and C. J. Brandl. 1996. Transcriptional
activation by yeast PDR1p is inhibited by its association with NGG1p/ADA3p. J. Biol.
Chem. 271:15884–15890.
95. Martinoia, E., M. Klein, M. Geisler, L. Bovet, C. Forestier, U. Kolukisaoglu,
B. Muller-Rober, and B. Schulz. 2002. Multifunctionality of plant ABC transporters-
more than just detoxifiers. Planta 214:345–355.
96. Mason, D. L., M. P. Mallampalli, G. Huyer, and S. Michaelis. 2003. A region within
a lumenal loop of Saccharomyces cerevisiae Ycf1p directs proteolytic processing and
substrate specificity. Eukaryot. Cell 2:588–598.
97. McGrath, J. P., and A. Varshavsky. 1989. The yeast STE6 gene encodes a homologue
of the mammalian multidrug resistance P-glycoprotein. Nature 340:400–404.
98. Meyers, S., W. Schauer, E. Balzi, M. Wagner, A. Goffeau, and J. Golin. 1992.
Interaction of the yeast pleiotropic drug resistance genes PDR1 and PDR5. Curr. Genet.
21:431–436.
99. Miyahara, K., D. Hirata, and T. Miyakawa. 1996. yAP-1- and yAP-2-mediated, heat
shock-induced transcriptional activation of the multidrug resistance ABC transporter
genes in Saccharomyces cerevisiae. Curr. Genet. 29:103–105.
100. Miyahara, K., M. Mizunuma, D. Hirata, E. Tsuchiya, and T. Miyakawa. 1996. The
involvement of the Saccharomyces cerevisiae multidrug resistance transporters Pdr5p
and Snq2p in cation resistance. FEBS Lett. 399:317–320.
101. Moye-Rowley, W. S., K. D. Harshman, and C. S. Parker. 1989. Yeast YAP1
encodes a novel form of the jun family of transcriptional activator proteins. Genes
Dev. 3:283–292.
102. Munder, T., and A. Hinnen. 1999. Yeast cells as tools for target-oriented screening.
Appl. Microbiol. Biotechnol. 52:311–320.
103. Nakamura, K., M. Niimi, K. Niimi, A. R. Holmes, J. E. Yates, A. Decottignies, B. C.
Monk, A. Goffeau, and R. D. Cannon. 2001. Functional expression of Candida
albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in
membrane transporters. Antimicrob. Agents Chemother. 45:3366–3374.
104. Nourani, A., M. Wesolowski-Louvel, T. Delaveau, C. Jacq, and A. Delahodde. 1997.
Multiple-drug-resistance phenomenon in the yeast Saccharomyces cerevisiae:
involvement of two hexose transporters. Mol. Cell. Biol. 17:5453–5460.
105. Ortiz, D. F., M. V. St Pierre, A. Abdulmessih, and I. M. Arias. 1997. A yeast ATP-
binding cassette-type protein mediating ATP-dependent bile acid transport. J. Biol.
Chem. 272:15358–15365.
106. Ouellette, M., and D. Légaré. 2003. Drug resistance mediated by ABC transporters in
parasites of humans, pp. 317–333. In ABC Proteins: From Bacteria to Man, Susan P. C.
Cole, I. Barry Holland, Karl Kuchler, and Christopher F. Higgins (ed.), Academic Press
107. Paddon, C., D. Loayza, L. Vangelista, R. Solari, and S. Michaelis. 1996. Analysis of
the localization of STE6/CFTR chimeras in a Saccharomyces cerevisiae model for the
cystic fibrosis defect CFTR delta F508. Mol. Microbiol. 19:1007–1017.
108. Parsons, A. B., R. L. Brost, H. Ding, et al. 2004. Integration of chemical-genetic and
genetic interaction data links bioactive compounds to cellular target pathways. Nat.
Biotechnol. 22:62–69.
109. Pascolo, L., S. Petrovic, F. Cupelli, C. V. Bruschi, P. L. Anelli, V. Lorusso,
M. Visigalli, F. Uggeri, and C. Tiribelli. 2001. Abc protein transport of MRI contrast
agents in canalicular rat liver plasma vesicles and yeast vacuoles. Biochem. Biophys.
Res. Commun. 282:60–66.
110. Petrovic, S., L. Pascolo, R. Gallo, F. Cupelli, J. D. Ostrow, A. Goffeau, C. Tiribelli,
and C. V. Bruschi. 2000. The products of YCF1 and YLL015w (BPT1) cooperate for
the ATP-dependent vacuolar transport of unconjugated bilirubin in Saccharomyces
cerevisiae. Yeast 16:561–571.
111. Piper, P., Y. Mahé, S. Thompson, R. Pandjaitan, C. Holyoak, R. Egner,
M. Mühlbauer, P. Coote, and K. Kuchler. 1998. The Pdr12 ABC transporter is
required for the development of weak organic acid resistance in yeast. EMBO J.
17:4257–4265.
112. Prasad, R., P. De Wergifosse, A. Goffeau, and E. Balzi. 1995. Molecular cloning and
characterization of a novel gene of Candida albicans, CDR1, conferring multiple
resistance to drugs and antifungals. Curr. Genet. 27:320–329.
113. Prendergast, J. A., R. A. Singer, N. Rowley, A. Rowley, G. C. Johnston, M. Danos,
B. Kennedy, and R. F. Gaber. 1995. Mutations sensitizing yeast cells to the start
inhibitor nalidixic acid. Yeast 11:537–547.
114. Raggers, R. J., A. van Helvoort, R. Evers, and G. van Meer. 1999. The human
multidrug resistance protein MRP1 translocates sphingolipid analogs across the plasma
membrane. J. Cell Sci. 112:415–422.
115. Raymond, M., P. Gros, M. Whiteway, and D. Y. Thomas. 1992. Functional

complementation of yeast ste6 by a mammalian multidrug resistance mdr gene. Science
256:232–234.
116. Raymond, M., S. Ruetz, D. Y. Thomas, and P. Gros. 1994. Functional expression of
P-glycoprotein in Saccharomyces cerevisiae confers cellular resistance to the
immunosuppressive and antifungal agent FK520. Mol. Cell. Biol. 14:277–286.
117. Reid, R. J., E. A. Kauh, and M. A. Björnsti. 1997. Camptothecin sensitivity is
mediated by the pleiotropic drug resistance network in yeast. J. Biol. Chem. 272:12091–
12099.
118. Riordan, J. R., and X. B. Chang. 1992. CFTR, a channel with the structure of a
transporter. Biochim. Biophys. Acta 1101:221–222.
119. Rogers, B., A. Decottignies, M. Kolaczkowski, E. Carvajal, E. Balzi, and
A. Goffeau. 2001. The pleitropic drug ABC transporters from Saccharomyces cerevisiae.
J. Mol. Microbiol. Biotechnol. 3:207–214.
120. Ruetz, S., and P. Gros. 1994. Phosphatidylcholine translocase: a physiological role for
the mdr2 gene. Cell 77:1071–1081.
121. Saleh, A., V. Lang, R. Cook, and C. J. Brandl. 1997. Identification of native
complexes containing the yeast coactivator/repressor proteins NGG1/ADA3 and ADA2.
J. Biol. Chem. 272:5571–5578.
122. Sanglard, D., F. Ischer, M. Monod, and J. Bille. 1997. Cloning of Candida albicans
genes conferring resistance to azole antifungal agents: characterization of CDR2, a new
multidrug ABC transporter gene. Microbiology 143:405–416.
123. Sanglard, D., K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille. 1995.
Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from
AIDS patients involve specific multidrug transporters. Antimicrob. Agents Chemother.
39:2378–2386.
124. Schuetz, E. G., S. Strom, K. Yasuda, et al. 2001. Disrupted bile acid homeostasis
reveals an unexpected interaction among nuclear hormone receptors, transporters, and
cytochrome P450. J. Biol. Chem. 276:39411–39418.
125. Schuetzer-Muehlbauer, M., B. Willinger, R. Egner, G. Ecker, and K. Kuchler.
2003. Reversal of antifungal resistance mediated by ABC efflux pumps from Candida
albicans functionally expressed in yeast. Int. J. Antimicrob. Agents 22:291–300.
126. Schuetzer-Muehlbauer, M., B. Willinger, G. Krapf, S. Enzinger, E. Presterl, and
K. Kuchler. 2003. The Candida albicans Cdr2p ATP-binding cassette (ABC)
transporter confers resistance to caspofungin. Mol. Microbiol. 48:225–235.
127. Schüller, C., B. E. Bauer, and K. Kuchler. 2003. Inventory and evolution of fungal
ABC protein genes, pp. 279–293. In ABC Proteins: From Bacteria to Man, Susan P. C.
Cole, I. Barry Holland, Karl Kuchler, and Christopher F. Higgins (ed.), Academic Press
128. Schüller, C., Y. M. Mamnun, M. Mollapour, G. Krapf, M. Schuster, B. E. Bauer,
P. W. Piper, and K. Kuchler. 2004. Global phenotypic analysis and transcriptional
profiling defines the weak acid stress response regulon in Saccharomyces cerevisiae.
Mol. Biol. Cell 15:706–720.
129. Servos, J., E. Haase, and M. Brendel. 1993. Gene SNQ2 of Saccharomyces cerevisiae,
which confers resistance to 4-nitroquinoline-N-oxide and other chemicals, encodes a
169 kDa protein homologous to ATP-dependent permeases. Mol. Gen. Genet. 236:214–
218.
130. Shah, N., and R. D. Klausner. 1993. Brefeldin A reversibly inhibits secretion in
Saccharomyces cerevisiae. J. Biol. Chem. 268:5345–5348.
131. Shani, N., P. A. Watkins, and D. Valle. 1995. PXA1, a possible Saccharomyces
cerevisiae ortholog of the human adrenoleukodystrophy gene. Proc. Natl. Acad. Sci.
USA 92:6012–6016.
132. Sharma, K. G., D. L. Mason, G. Liu, P. A. Rea, A. K. Bachhawat, and S. Michaelis.
2002. Localization, regulation, and substrate transport properties of Bpt1p, a
Saccharomyces cerevisiae MRP-type ABC transporter. Eukaryot. Cell 1:391–400.
133. Simonics, T., Z. Kozovska, D. Michalkova-Papajova, A. Delahodde, C. Jacq, and
J. Subik. 2000. Isolation and molecular characterization of the carboxy-terminal pdr3
mutants in Saccharomyces cerevisiae. Curr. Genet. 38:248–255.
134. Smriti, S. K., B. L. Dixit, C. M. Gupta, S. Milewski, and R. Prasad. 2002. ABC
transporters Cdr1p, Cdr2p and Cdr3p of a human pathogen Candida albicans are
general phospholipid translocators. Yeast 19:303–318.
135. Smriti, S. K., and R. Prasad. 1999. Membrane fluidity affects functions of Cdr1p, a
multidrug ABC transporter of Candida albicans. FEMS Microbiol. Lett. 173:475–481.
136. Swartzman, E. E., M. N. Viswanathan, and J. Thorner. 1996. The PAL1 gene
product is a peroxisomal ATP-binding cassette transporter in the yeast Saccharomyces
cerevisiae. J. Cell Biol. 132:549–563.
137. Szczypka, M. S., J. A. Wemmie, W. S. Moye-Rowley, and D. J. Thiele. 1994. A yeast
metal resistance protein similar to human cystic fibrosis transmembrane conductance
regulator (CFTR) and multidrug resistance-associated protein. J. Biol. Chem.
269:22853–22857.
138. Taglicht, D., and S. Michaelis. 1998. Saccharomyces cerevisiae ABC proteins and
their relevance to human health and disease. Methods Enzymol. 292:130–162.
139. Teem, J. L., H. A. Berger, L. S. Ostedgaard, D. P. Rich, L. C. Tsui, and M. J.
Welsh. 1993. Identification of revertants for the cystic fibrosis delta F508 mutation
using STE6-CFTR chimeras in yeast. Cell 73:335–346.
140. Tommasini, R., R. Evers, E. Vogt, C. Mornet, G. J. Zaman, A. H. Schinkel,
P. Borst, and E. Martinoia. 1996. The human multidrug resistance-associated protein
functionally complements the yeast cadmium resistance factor 1. Proc. Natl. Acad. Sci.
USA 93:6743–6748.
141. Tong, A. H., G. Lesage, G. D. Bader, et al. 2004. Global mapping of the yeast genetic
interaction network. Science 303:808–813.
142. Ueda, K., A. M. Shimabuku, H. Konishi, Y. Fujii, S. Takebe, K. Nishi, M. Yoshida,
T. Beppu, and T. Komano. 1993. Functional expression of human P-glycoprotein in
Schizosaccharomyces pombe. FEBS Lett. 330:279–282.
143. Urban, M., T. Bhargava, and J. E. Hamer. 1999. An ATP-driven efflux pump is a
novel pathogenicity factor in rice blast disease. EMBO J. 18:512–521.
144. Urlinger, S., K. Kuchler, T. H. Meyer, S. Uebel, and R. Tampé. 1997. Intracellular
location, complex formation, and function of the transporter associated with antigen
processing in yeast. Eur. J. Biochem. 245:266–272.
145. Vik, A., and J. Rine. 2001. Upc2p and Ecm22p, dual regulators of sterol biosynthesis
in Saccharomyces cerevisiae. Mol. Cell. Biol. 21:6395–6405.
146. Volkman, S. K., A. F. Cowman, and D. F. Wirth. 1995. Functional complementation
of the ste6 gene of Saccharomyces cerevisiae with the pfmdr1 gene of Plasmodium
falciparum. Proc. Natl. Acad. Sci. USA 92:8921–8925.
147. Wang, D., F. Zheng, S. Holmberg, and G. B. Kohlhaw. 1999. Yeast transcriptional
regulator Leu3p. Self-masking, specificity of masking, and evidence for regulation by
the intracellular level of Leu3p. J. Biol. Chem. 274:19017–19024.
148. Wemmie, J. A., M. S. Szczypka, D. J. Thiele, and W. S. Moye-Rowley. 1994.
Cadmium tolerance mediated by the yeast AP-1 protein requires the presence of an
ATP-binding cassette transporter-encoding gene, YCF1. J. Biol. Chem. 269:32592–

32597.
149. Wendler, F., H. Bergler, K. Prutej, H. Jungwirth, G. Zisser, K. Kuchler, and
G. Högenauer. 1997. Diazaborine resistance in the yeast Saccharomyces cerevisiae
reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3.
J. Biol. Chem. 272:27091–27098.
150. Wilcox, L. J., D. A. Balderes, B. Wharton, A. H. Tinkelenberg, G. Rao, and S. L.
Sturley. 2002. Transcriptional profiling identifies two members of the ATP-binding
cassette transporter superfamily required for sterol uptake in yeast. J. Biol. Chem.
277:32466–32472.
151. Wolfger, H., Y. Mahé, A. Parle-McDermott, A. Delahodde, and K. Kuchler. 1997.
The yeast ATP-binding cassette (ABC) protein genes PDR10 and PDR15 are novel
targets for the Pdr1 and Pdr3 transcriptional regulators. FEBS Lett. 418:269–274.
152. Wolfger, H., Y. M. Mamnun, and K. Kuchler. 2001. Fungal ABC proteins:
pleiotropic drug resistance, stress response and cellular detoxification. Res. Microbiol.
152:375–389.
153. Wolfger, H., Y. M. Mamnun, and K. Kuchler. 2004. The yeast Pdr15p ATP-binding
cassette (ABC) protein is a general stress response factor implicated in cellular
detoxification. J. Biol. Chem. 279:11593–11599.
154. Yano, K., and T. Fukasawa. 1997. Galactose-dependent reversible interaction of Gal3p
with Gal80p in the induction pathway of Gal4p-activated genes of Saccharomyces
cerevisiae. Proc. Natl. Acad. Sci. USA 94:1721–1726.
155. Young, L., K. Leonhard, T. Tatsuta, J. Trowsdale, and T. Langer. 2001. Role of the
ABC transporter Mdl1 in peptide export from mitochondria. Science 291:2135–2138.
156. Zgurskaya, H. I., and H. Nikaido. 2002. Mechanistic parallels in bacterial and human
multidrug efflux transporters. Curr. Protein Pept. Sci. 3:531–540.
157. Zhang, X., Z. Cui, T. Miyakawa, and W. S. Moye-Rowley. 2001. Cross-talk between
transcriptional regulators of multidrug resistance in Saccharomyces cerevisiae. J. Biol.
Chem. 276:8812–8819.
158. Zhu, H., J. F. Klemic, S. Chang, et al. 2000. Analysis of yeast protein kinases using
protein chips. Nat. Genet. 26:283–289.
159. Zhu, H., and M. Snyder. 2003. Protein chip technology. Curr. Opin. Chem. Biol. 7:55–63.
Chapter 12
THE FHCRC/NCI YEAST ANTICANCER DRUG
SCREEN
Susan L. Holbeck1 and Julian Simon2

1
National Cancer Institute, Developmental Therapeutics Program, Information Technology
Brach, Rockville, MD; 2Clinical Research Division, Fred Hutchinson Cancer Research
Center, Seattle, WA
1 INTRODUCTION
Anticancer drug discovery has historically been done empirically,

screening for compounds that inhibit the growth of tumor cells in culture, or
that are effective against implanted tumors in mice. This approach is
analogous to the highly effective screens carried out in the 1950s and 1960s
for antimicrobial agents and has had its successes – indeed, many of the
anticancer drugs currently in clinical use were developed in this fashion.
However, as our understanding of the molecular mechanisms underlying the
development and growth of cancer cells has improved, new approaches to
drug discovery have begun to build on this knowledge. The recognition that
not all tumors arising in the same tissue are due to the same underlying
defects calls for treatments regimes that will be tailored to these molecular
alterations. The drug screen described in this chapter was initiated with the
hypothesis that single gene changes that are often associated with particular
hereditary and sporadic forms of cancer may serve as determinants of drug
sensitivity [27]. In principle, there are two possible mechanisms by which a
drug can be more toxic to a cell containing a particular genetic alteration.
First, by a mechanism in which damage caused by the drug is normally
repaired in a wild-type cell by a protein that has been deleted or altered in
the mutant. An example of this is the sensitivity of mutants defective in
recombinational repair of DNA double-strand breaks (DSBs) (e.g., yeast
rad50 mutants) to agents that cause DSBs (e.g., the topoisomerase poison
315
© 2007 Springer.
316 S. L. Holbeck and J. Simon
etoposide). The second mechanism is more interesting and potentially more

important as a anticancer therapy strategy. In this scenario, genes that are
normally nonessential are made essential by the deletion of the cancer-
related gene. Inhibition of the normally nonessential gene product by a small
molecule leads to cell death in the mutant background. This phenomenon is
called synthetic lethality and is an example of context-specific cytotoxicity.
By screening for agents that elicit synthetic lethality in cells harboring
cancer-related mutations, we may be able to identify a new armamentarium
of context-specific anticancer agents.
The drug discovery effort described here began in 1997 as a collaboration
between the Developmental Therapeutics Program (DTP) of the National
Cancer Institute (NCI) and the Fred Hutchinson Cancer Research Center
(FHCRC) and used the yeast Saccharomyces cerevisiae to screen a large
number of compounds for those that are selectively toxic to cells with
alterations in defined genes. This screen utilized a panel of yeast strains,
each with defined alterations in DNA repair genes or in cell cycle control
genes. Differential sensitivity of various DNA repair mutant strains to a
compound can identify pathways important for repair of damage caused by
that agent. DNA damage repair pathways represented in this screen included
DNA DSB repair (rad50 and rad52), ultraviolet (UV)-excision repair
(rad14), DNA mismatch repair (mlh1), removal of O6-methylguanine
(mgt1), and post-replication repair (rad18). Alterations in human homologs
of these genes have been identified in human tumors. The DNA mismatch
repair gene hMLH1 is mutated or silenced in hereditary and sporadic forms
of colon cancer, and is one source of the “Microsatellite Instability” (MIN)
phenotype. Defects in UV-excision repair lead to the cancer-prone syndrome
Xeroderma pigmentosum. The breast cancer predisposing gene BRCA1
associates with components of the DSB repair pathway. The screen also
included strains altered in cell cycle control – again, these reflect alterations
observed in human tumors. Bub3p is a component of the mitotic spindle
checkpoint, which ensures that chromosomes are attached to the mitotic
spindle before allowing passage through mitosis. Defects in a component of
this checkpoint have been seen in some human tumors, giving rise to the
“Chromosome Instability” (CIN) phenotype. The RAD53 gene is part of two
checkpoints, monitoring completeness of S-phase DNA synthesis, and also
arresting cell growth in response to DNA damage in G2. The screen also
includes a strain overexpressing the G1 cyclin, CLN2, which controls entry
into S-phase. Finally, the SGS1 gene is involved in an ever-growing list of
DNA transactions, including DNA replication, recombination, and telomere
function. There are several human homologs of SGS1, two of which, BLM
and WRN, lead to cancer-prone diseases, Bloom’s and Werner’s syndromes,
respectively.
Chapter 12: The FHCRC/NCI Yeast Anticancer Drug Screen 317
Nearly 100,000 compounds from the repository of the NCI-DTP were

screened for their ability to inhibit the growth of this panel of yeast strains.
The following sections describe the design of the screen, the compounds
used, more detailed information on the genes altered in the screen, general
results, and some specific examples. The data generated by this effort are
freely available to the public on a web site maintained by the NCI-DTP at
http://dtp.nci.nih.gov/yacds/index.html.
The results of the screen – the selective activity of agents in particular
genetic contexts – have been interpreted by comparing the sensitivity of a
panel of mutant yeast strains to Food and Drug Administration (FDA)-
approved anticancer agents [46] and other literature sources. Compounds
showing structural similarity to agents known to exhibit increased toxicity in
specific mutant backgrounds were segregated from structurally unique
compounds.
2 DESIGN OF THE SCREEN
The screen consisted of 3 stages. In the prescreen, Stage 0, compounds

were screened in duplicate at a single dose (50 µM) against a panel of 6
strains, each harboring 1 or 2 gene alterations of interest. In addition, all
strains were deleted for 3 additional genes. PDR1 and PDR3 encode
transcription factors that control the expression of drug efflux pumps. ERG6
alters the ergosterol composition of the plasma membrane, and increases
sensitivity to certain drugs. A complete list of the genotypes of the strains
used can be found at http://dtp.nci.nih.gov/yacds/index.html.
Compounds showing sufficient activity against at least one of the strains
in Stage 0 testing were selected for testing in the next stage (Stage 1). Here
compounds were tested in duplicate at 2 doses (5 and 50 µM) against the
same 6 strains used in Stage 0. In addition to confirmation of activity,
compounds were also examined for some degree of specificity. Compounds
that met a quantitative criterion of selectivity – greater than fivefold
difference in growth inhibition between the most and least sensitive strain –
were selected for Stage 2 testing.
Stage 2 used an expanded panel of yeast strains, with a single gene
alteration of interest in each strain. As in the earlier stages, all strains (except
one) were also deleted for the pdr1, pdr3, and erg6 genes. The single
exception, rad50, ERG6, PDR1, PDR3 was included to assess the effect of
drug uptake and efflux on growth inhibitory activity. Compounds were tested
in duplicate at 5 doses (1.2, 3.6, 11, 33, and 100 µM). Data for Stage 0,
Stage 1, and Stage 2 testing is available through the NCI-DTP web site at
http://dtp.nci.nih.gov/yacds/index.html.
3 GENES ALTERED IN THE SCREEN
3.1 DNA repair/DNA metabolism genes
3.1.1 MGT1
MGT1 encodes O6-methylguanine DNA methyltransferase, which

removes the methyl group from guanine residues in modified DNA by
covalently transferring the methyl group of either O6-methylguanine or O4-
methylthymine to a cysteine residue of Mgt1p [53, 56]. This residue also
reacts with free 06-benzyl guanine, which is used therapeutically to
inactivate the human homolog (MGMT) prior to chemotherapy with
alkylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)
[38]. O6-methylguanine lesions, if unrepaired, lead to insertion of T rather
than C opposite the altered base during DNA synthesis, which can then be
acted on by mismatch repair activities [41]. This is mutagenic, as seen by the
increased spontaneous mutation rate in mgt1 mutants [54]. Such lesions
occur following exposure to certain environmental agents (e.g., methyl
bromide) [39]. Mutations in the yeast gene can be complemented using the
human homolog [54]. mgt1 mutants show increased sensitivity to
streptozocin [46], MNNG [53] and other alkylating agents [55].
3.1.2 MLH1
MLH1 is involved in repair of DNA mismatches (both single base

mismatches and frameshifts) [19], in concert with MSH2 and PMS1. In
addition to repairing DNA mismatches, mismatch repair prevents
recombination between DNA sequences that have diverged [11]. Mlh1p
binds ATP and has a weak ATPase activity – ATP binding may facilitate its
interaction with other mismatch repair proteins [24]. MutLalpha, a
heterodimer of Mlh1p and Pms1p, has DNA-binding activity [25]. Mlh1p
was demonstrated to bind to the RecQ-like helicase Sgs1p [37] (which is
mutated in other strains in the NCI yeast anticancer drug screen). This
association is also seen with the human Mlh1 protein and the BLM gene, a
RecQ-like helicase mutated in the cancer-prone Bloom’s syndrome. Human
Mlh1 is mutated in many patients with the hereditary cancer-prone HNPCC,
characterized by microsatellite instability (the so-called MIN phenotype).
There are currently no compounds identified that specifically target mlh1
mutants. In fact, mismatch repair mutants show decreased sensitivity to
some DNA-damaging agents used in anticancer chemotherapy [14].
3.1.3 RAD14
RAD14 is one of a group of genes needed for excision repair of DNA

damaged by UV and other lesions that distort the helix. This process
involves recognition of damaged DNA (the step in which Rad14p
participates), followed by removal of a short stretch of the damaged DNA
strand and resynthesis, using the undamaged strand as a template [40].
RAD14 is the homolog of the human xeroderma pigmentosum comple-
mentation group A (XPA) gene [3]. Individuals with mutations in XPA
exhibit increased sensitivity to UV light and an increased incidence of skin
cancer. Rad14p and XPA each contain a zinc finger [22]. Rad14p is part of a
large protein complex, which also contains Rad1p, Rad7p, Rad10p, Rad16p,
Rad23p, RPA, RPB1, and TFIIH [43], although there is some controversy
over whether the complex is preformed in the absence of DNA damage [48],
or assembles in response to a lesion [23]. Strains defective in UV excision
repair have increased sensitivity to nitrogen mustard [33]. The null mutant
shows wild-type growth rates [3]. A rad14 null mutant shows increased
sensitivity to the alkylating agents cisplatin, thiotepa, mechlorethamine and
mitomycin C, to DNA synthesis inhibitors hydroxyurea, and actinomycin D,
to compounds that induce DNA breaks, such as bleomycin and doxorubicin,
and to pentostatin [46].
3.1.4 RAD18
RAD18 is needed for post-replication repair of DNA damage, and

mutants are hypersensitive to DNA alkylation. The Rad18 protein contains a
ring-finger motif, through which it interacts with Rad6p, a ubiquitin-
conjugating enzyme. Mutations in the ring-finger motif in the human Rad18
homolog cause sensitivity to UV light, methyl methanesulfonate, and
mitomycin C. In addition to ubiquitin-conjugating activity, the Rad18p-
Rad6p heterodimer binds DNA and hydrolyzes ATP [2].
3.1.5 RAD50
Rad50p, in concert with Mre11p and Xrs2p, forms a nuclease that is

needed for repair of double-stranded DNA breaks using either homologous
recombination [47] or nonhomologous end-joining (NHEJ) pathways [10,
15, 50]. The nuclease is manganese-dependent with both 3′–5′ exonuclease
and single-stranded DNA endonuclease activities [18, 50]. Rad50p is
required for activation of the Rad53 checkpoint in response to gamma-
irradiation, which induces DSBs [20]. The Rad50p/Mre11p/Xrs2p complex
is evolutionarily conserved – the analogous nuclease in humans is composed
of Rad50, Mre11, and NBS1. Patients with defects in NBS1 have the cancer-
prone Nijmegen breakage syndrome and are hypersensitive to ionizing
radiation. The breast cancer-associated gene BRCA1 associates with human
Rad50 [57]. Rad50p also has a role in telomere maintenance [8, 12].
Structural data on Rad50p suggest that Zn++ coordinated hooks on 2
Rad50p molecules may function to keep the DNA strands involved in repair
of a DSB connected with one another [28].
3.1.6 RAD52
Like Rad50p, Rad52p is needed for repair of double-stranded DNA

breaks by homologous recombination. However, Rad52p is not need for
NHEJ and does not have a role in telomere maintenance. Mutants lacking
RAD52 are extremely sensitive to DSBs, and are much more sensitive to
Mitomycin C than is the rad50 mutant. The role of Rad52p appears to be to
facilitate the interaction of the RecA homolog Rad51p with RPA and single-
stranded DNA, thereby promoting DNA strand exchange [35]. Rad52p is
needed for formation of Holliday junctions, an important recombination
intermediate [58].
3.1.7 SGS1
SGS1 is emerging as a player in many aspects of DNA metabolism,

including telomere maintenance, DNA recombination, and DNA replication.
It has recently been identified as a component of the intra-S checkpoint,
which senses DNA damage during S-phase, although it has no role in the G1
and G2 DNA damage checkpoints [17]. An sgs1 mutant has a several 100-
fold increase in genome rearrangements following mild DNA damage [34].
Sgs1p is a member of the RecQ-like helicase family, which includes two
human genes that cause cancer-prone syndromes when mutated. Defects in
the human RecQ-like BLM gene cause Bloom’s syndrome, characterized by
genome instability, sun-sensitivity, and increased incidence of cancer.
Defects in a second human RecQ-like gene, WRN, cause Werner’s
syndrome, characterized by premature aging, telomere shortening and
increased incidence of tumors. A third homolog, RECQ4, has been linked to
the premature aging and cancer-prone Rothmund–Thomson syndrome [31].
Two additional human homologs, of unknown function, have been
identified. RecQ-like helicases may be required to prevent improper
recombination at stalled DNA replication forks, in association with
topoisomerase III [4, 29]. While deletion mutants of sgs1 are viable on their
own, they are not able to tolerate deletion of certain other genes (synthetic
lethality), including DNA repair or metabolism genes rad50, sae2, hpr5,
mms4, top1, and pol32 [49]. Sgs1p interacts with the mismatch repair protein
Mlh1p – this interaction is also seen with the human BLM and MLH1
proteins [37]. Sgs1p can unwind G-quadraplexes, a structure that can be
formed by the G-rich single strand found at telomeres. This unwinding is
inhibited by compounds that bind to G-quadraplexes [26].
3.2 Cell cycle control genes
3.2.1 BUB3
Bub3p is a component of the mitotic spindle checkpoint, together with

Bub1p, Mad1p, Mad2p, Mad3p, and Mps1p. Bub3 mutants, lacking
checkpoint function, fail to arrest following treatment with spindle-
destabilizing drugs, such as the tubulin-binding benzimidazoles, leading to
chromosome mis-segregation and cell death. In the absence of spindle
damage, mutations in the mitotic spindle assembly checkpoint may con-
tribute to aneuploidy. The function of these checkpoint proteins is con-
served, with higher eukaryotic homologs binding to kinetochores. Bub3p has
WD40 repeats which mediate interactions with Mad2, Mad3, and Cdc20
(anaphase promoting complex activator) [16]. Bub3p is phosphorylated by
Bub1p kinase. Mutations in human Bub1 have been associated with the CIN
phenotype in colon carcinomas and other types of cancer [9].
3.2.2 CLN2
CLN2 encodes a G1/S-specific cyclin that regulates the activity of the

cyclin-dependent kinase (CDK) Cdc28p [42, 52]. Cln2p accumulates
through G1, becomes phosphorylated and ubiquitinated, then is rapidly
degraded by the proteasome, allowing cells to enter S phase. The strains
used in the screen overexpress this cyclin using the strong inducible GAL1
promoter. Overexpression of G1 cyclins or loss of CDK inhibitors is thought
to drive progression from the G1 to S phase in cancer cells, and high levels
of cyclin E have been associated with a poor prognosis in breast cancer [30].
3.2.3 RAD53 (mec2-1)
RAD53 is an essential gene in yeast, therefore the screen utilizes a point

mutant in this gene, rather than a gene disruption. RAD53 was identified
independently by several groups, giving rise to a host of synonyms (MEC2,
SPK1, SAD1). Rad53p is a component of two cell cycle checkpoints in yeast.
It is activated by the phosphatidylinositol-(3′) kinase-like protein kinases

Mec1p and Tel1p in response to DNA damage (which also activates Chk1p),
as well as by inhibition of DNA synthesis (which does not signal Chk1p)
[45]. The human homolog, CHEK2, is a serine/threonine kinase that
phosphorylates Cdc25C, preventing entry into mitosis [5]. CHEK2 is
autophosphorylated in response to DNA damage, which upregulates its
kinase activity.
4 COMPOUNDS
Compounds for the screen came from the repository of the NCI–DTP.
This collection contains compounds submitted for screening as possible
anticancer or antiviral compounds by a large number of suppliers. The
suppliers include academic groups, government scientists, small bio-
technology companies, and large pharmaceutical companies from all over
the globe. The acquistion of compounds began in 1956 and continues to this
day, with over 500,000 compounds currently registered. Interested parties
can find details about submitting compounds at http://dtp.nci.nih.gov/. About
half of the compounds are covered by a confidentiality agreement, which
precludes the NCI from disseminating data derived from these compounds.
Data derived from “open” compounds not covered by a confidentiality
agreement is publicly available through the DTP web site, and includes
compound structures, data from the NCI 60 human tumor cell line screen,
the NCI AIDS screen, and the NCI yeast anticancer drug screen. In addition,
if sufficient inventory is available, the NCI may be able to supply qualified
researchers with small quantities of “open” compounds from the DTP
repository for nonhuman use.
The NCI does not routinely verify the structures of submitted
compounds. In addition, the stability of different chemotypes varies widely.
Thus, researchers are encouraged to verify the structure of any compounds
before investing significant resources in lead development. Indeed, one of
the compounds identified as a specific inhibitor of the DSB repair mutants
rad50 and rad52 [13] had a structure different than that indicated by the
supplier.
Compounds submitted to the NCI–DTP may or may not have an
identified mechanism of action. Many compounds were synthesized as
chemically interesting structures, or were submitted as purified natural
products, with cellular targets yet to be determined. Other submitted
compounds have been rationally designed with specific targets in mind.
Finally, many compounds are structural analogs of anticancer agents with
known mechanisms of action. The end result is that the compounds in the
NCI–DTP repository represent a broad range of chemotypes with a broad

range of biological activities. To facilitate drug discovery in novel
molecularly targeted screens, the DTP has put together several plated sets of
compounds for distribution to qualified researchers. The diversity set is a
group of 1,990 compounds selected to represent a broad range of chemical
structures. All of the compounds in the set have been tested in both the NCI
60 human tumor cell line screen, as well as the NCI yeast anticancer drug
screen. The mechanistic diversity set is somewhat smaller, with 879
compounds, and was selected for diversity of behavior in the NCI 60 human
tumor cell line screen. Most of these compounds [662] have also been tested
in the NCI yeast anticancer drug screen.
Many of the compounds have been tested in the standard NCI–DTP
anticancer drug screen, which employs a panel of 60 human tumor cell lines.
Compounds are screened across a 5-log dose range for their ability to inhibit
the growth of, or to kill, these human tumor cell lines. The pattern of which
tumor cell lines are more sensitive, and which lines less so, provides a
fingerprint for each compound. The comparison of these fingerprints
between different compounds led to the development of the COMPARE
algorithm [36], which allows users to search for compounds with similar
patterns to a compound of interest. Compounds with similar mechanisms of
action tend to have similar fingerprints. The COMPARE algorithm has led to
the identification of numerous tubulin-binding agents, as well as a novel
structural class of cyclin-dependent kinase inhibitors. NCI–DTP databases
also include measurement of numerous “molecular targets” in the 60 cell line
panel. The pattern of molecular target expression can be compared to
compound sensitivity data, as a means of generating hypotheses about how a
compound of unknown mechanism might function. The 60 cell line screen
began in it’s current form in 1992. Compounds submitted in prior years may
have been tested against P388 or L1210 mouse tumor models [51]. This
data, structures of the compounds and access to the COMPARE algorithm, is
freely available through the NCI–DTP web site at http://dtp.nci.nih.gov/.
Some of the compounds showing selective activity in Stage 2 of the yeast
screen had not been through the 60 cell line screen. Many of these were sent
for 60 cell line testing, to allow users of the yeast screen data to link into
other NCI–DTP data on these compounds.
5 RESULTS
5.1 Stage 0 results
Compounds were screened in duplicate against a panel of 6 strains, each

harboring 1 or 2 gene alterations of interest, plus mutations in pdr1, pdr3,
and erg6. Stage 0 screening began in 1997 and was completed in 2001, with
a total of 97,261 compounds screened (some multiple times). Data for
87,264 “open” compounds tested in Stage 0 are available on the DTP web
site (http://dtp.nci.nih.gov/yacds/index.html). The remaining 10,003 com-
pounds are covered by a confidentiality agreement with the NCI, and were
screened while the Seattle Project was an NCI field station.
Stage 0 was designed as a prescreen, to eliminate inactive compounds
and determine whether compounds merited further multidose testing.
Inhibition of the growth of at least one of the strains by at least 70% was
required to trigger further testing in Stage 1. Nearly (14,466) compounds
(15% of those tested) met this criterion. While the cutoff of 70% growth
inhibition was selected for the purposes of this assay, this is somewhat
arbitrary, and does not mean that compounds below the cut-off are inactive.
Figure 1 displays the fraction of compounds with at least one strain meeting
various growth inhibition cutoffs.
Figure 1. A total of 97,261 compounds were tested in Stage 0. The figure indicates the
percentage of compounds where at least one of the strains exhibited growth inhibition equal to
or greater than the indicated cutoff. 70% growth inhibition in one of more strains was chosen
as the criterion for further testing.
Stage 0 was done in duplicate. The median difference between

measurements was 0.096 (based on fraction growth inhibition, with 1.0
denoting 100% growth inhibition), with the median of the average of
duplicates 0.05. For measurements where the average of the duplicates was
>0.7 growth inhibition (i.e., the criterion for further testing), the median
difference between duplicate measurements was 0.014, with most (90%) of
the duplicates having a difference of less than 0.11. There were instances
where there were large differences between measurements. Compounds were
chosen for further testing if any single measurement exceeded 70% growth
inhibition.
5.2 Stage 1 results
Compounds meeting the selection criterion in Stage 0 screening were

selected for rescreening in Stage 1. The same 6 strains as in Stage 0 were
used, but compounds were tested in duplicate at 2 doses: 50 µ (the
concentration used in Stage 0) and 5 µM. A total of 16,885 compounds were
screened. Data for 14,837 “open” compounds are publicly available through
the DTP web site.
Stage 1 was designed to confirm activity of compounds, and to weed out
generally cytotoxic compounds. Almost 5,270 compounds (31%) failed to
reconfirm activity, with no strain at either dose showing growth inhibition of
greater than 60%. Of these, 3,137 (19% of Stage 1 tested compounds) failed
to inhibit any strain at any dose by greater than 30%. Compounds with low
activity were not tested further.
About 1,283 compounds (7.6%) inhibited the growth of all strains at both
doses by at least 60%, and 773 compounds (4.6%) inhibited all strains at
both doses by at least 90%. A subset of approximately 100 of these
compounds were retested at sufficiently lower concentrations to allow IC50
determination. Most were found to be generally toxic, inhibiting the growth
of all strains at approximately the same concentration. Because of this
finding, the remaining potent compounds were excluded from further study,
however these could also be candidate antifungal agents. Several are natural
products that target essential processes such as protein synthesis. Those
compounds that exhibited at least a fivefold differential between the most
and least sensitive strains were selected for testing in Stage 2.
The majority of the Stage 0 and Stage 1 (50 µM) measurements agreed
fairly well with one another. There were 583,614 measurements in common
(same compound tested on the same strain at the same dose). Of these
524,307 (90%) differed from one another by less than 10% growth inhibition
(i.e., if one measurement was 60%, the other was between 50% and 70%).
Nearly 541,823 (93%) differed by less than 20% growth inhibition, 15,447
(2.6%) had values that differed by more than 50% growth inhibition, and
5,574 (1%) had values that differed by more than 80% growth inhibition.
5.2.1 Selective Stage 1 compounds
Three of the Stage 1 strains contained single mutations of interest. For

these, a brief summary of compounds selective for each follows.
5.2.1.1 Compounds specific for the bub3 strain in Stage 1

The bub3 mutant is deficient in the mitotic spindle checkpoint. It has
been shown to be sensitive to compounds that interact with tubulin (such as
benzimidazoles) as well as other components of the mitotic spindle.
Approximately two hundred eighty-four compounds selectively inhibited the
growth of the bub3 mutant (selectivity defined here as the bub3 strain
exhibiting at least 40% growth inhibition, that the bub3 inhibition be at least
5 times greater than that of the least sensitive strain, and that all other strains
were at least one-third less sensitive than the bub3 strain). Somewhat
surprisingly, the majority fall into structural classes without an identified
mechanism of action. Only 4% of the bub3-selective compounds were
structural analogs of compounds known to interfere with tubulin (including
steroid analogs), 4% contained heavy metals, 3% were analogs of
compounds that cause DNA damage, and 2% were analogs of compounds
that affect cell cycle or signal transduction. bub3-selective compounds will
be discussed in more detail in the Stage 2 results (section 5.3.1). We believe
that there are two reasons why more spindle poisons, which are abundant in
the NCI collection [36], were not identified as bub3-selective agents in the
screen. First, although yeast tubulin is highly homologous to human tubulin,
subtle differences in ligand-binding sites confer resistance to several
antimitotic agents (e.g., Paclitaxel) [21] in yeast. Second, the liquid growth
assay used in the drug screen relies on growth differences, as measured by
changes in optical density of the culture, over a small number of cell
divisions, typically 7–8 divisions, whereas colony formation assays require
up to 20 divisions. Cells arrested by spindle poisons in mitosis continue to
increase in size and therefore optical density leading to an underestimation
of growth inhibition in checkpoint proficient (BUB3+) strains in the liquid
assay. This is actually an advantage, allowing us to identify agents with
other mechanisms of action.
5.2.1.2 Compounds specific for the rad50 strain in Stage 1

The rad50 mutant is defective in DSB repair, so is expected to be
sensitive to compounds that introduce DSBs into DNA. This class would
include topoisomerase I (Topo I) and topoisomerase II (Topo II) poisons, as

well as bleomycins. In addition, agents that cause DNA damage (e.g.,
cisplatin) are more toxic to the rad50 mutant than to wild type. For these
agents, however, the rad18 mutant is the most sensitive. 311 compounds
showed selectivity for the rad50 mutant in Stage 1 testing (analogous to that
defined for the bub3 strain in section 5.2.1.1). Many of these compounds
(58%) were known Topo I or Topo II poisons, structural analogs of
topoisomerase poisons, or compounds with planar ring structures that might
plausibly intercalate into DNA. While 3% of the rad50-selective compounds
contained heavy metals. The remainder of the rad50-selective compounds
did not fall into structural classes expected to cause DSBs. rad50-selective
compounds will be discussed in more detail in the Stage 2 results (section
5.3.7).
5.2.1.3 Compounds specific for the rad53 (mec2-1) strain in Stage 1

The RAD53 gene is needed for both the S-phase DNA synthesis
checkpoint and the G2 DNA damage checkpoint. Fifty-six compounds
exhibited selectivity for the mec2-1 strain (analogous to that defined for the
bub3 strain in section 5.2.1.1.). And 13% of these compounds were
structural analogs of compounds known to interfere with DNA synthesis
(e.g., antifolates). Eleven percent of the mec2-1 selective compounds
contained heavy metals, a further 9% have structures suggesting they may be
DNA intercalators. The majority of the Stage 1 mec2-1 selective compounds
do not fall into structural classes expected to trigger either of the RAD53-
dependent checkpoints. rad53-selective compounds will be discussed in
more detail in the Stage 2 results (section 5.3.8).
5.3 Stage 2 results
Compounds that showed selective activity in Stage 1, with the strain with
the maximum growth inhibition at least five times greater than the strain
with the least growth inhibition, were selected for testing in Stage 2. A total
of 3,498 compounds were tested in duplicate at 5 doses (1.2, 3.7, 11, 33, and
100 µM). Compounds were screened against a panel of 13 strains, 2 of
which serve as wild-type controls. The remainder contained a single gene
alteration of interest. All but one of the strains was also deleted for the pdr1,
pdr3, and erg6 genes. The rad50 EPP+ strain, wild-type for ERG6, PDR1
and PDR3 genes, was included to determine whether further studies with
these compounds would be affected by these drug sensitivity mutations.
Data are publicly available for the 3,137 compounds that are not covered by
a confidentiality agreement. Stage 2 data, including bar graphs, dose-response
curves, links to compound structures and other screening data on these

compounds may be accessed at http://dtp.nci.nih.gov/yacds/index.html.
Nearly 703 of the Stage 2 tested compounds (20%) failed to inhibit the
growth of any of the strains by 70%. While active, 1,091 compounds (31%)
did not show much selectivity between the strains. The remaining 49% of the
tested compounds showed some degree of selectivity for one or more strains.
These compounds are discussed in more detail below.
5.3.1 Compounds selective for the bub3 strain in Stage 2
Using the criteria of a minimum 70% growth inhibition, 29 compounds

were found to be highly specific for bub3, inhibiting only the bub3 strain at 2
or more of the tested doses. A further 14 compounds inhibited the growth of
the bub3 mutant, plus one additional strain at 2 or more doses. A total of 286
compounds inhibited bub3, either alone or in combination with several other
strains, for at least one dose.
Figure 2. Structures are shown of representative compounds selective for the spindle
checkpoint mutant bub3 in Stage 2 testing. NSC 1331 was selective for bub3 at all 5 doses.
Two compounds with related structures were also bub3-selective. NSC 4000206 and two
related compounds were bub3-specific at multiple doses.
Figure 2 contains structures for selected compounds highly specific for

bub3. As stated previously, the bub3 strain is expected to be sensitive to
microtubule-targeting compounds. None of the major spindle poison
compound classes (i.e., vinca alkaloids, taxanes, benzimidazoles, or
colchicine analogs) scored as bub3-selective hits. The 28 bub3-selective
agents are a structurally diverse set of compounds, although there are some
scaffolds represented multiple times in this group. The 3 most highly
specific compounds (NSCs 1331, 16706, and 8513) may be structurally
related to Pedicin (NSC 255993), which is reported to inhibit microtubule
assembly [1]. Three bub3-selective compounds (NSCs 16461, 400206, and
30548) share a planar tricyclic anthrone structure, which could plausibly
intercalate into DNA. A dose-response curve for one bub3-specific
compound, NSC 1331, is shown in Figure 3. Very few of the bub3-selective

compounds were related to known tubulin-interacting agents, which are
highly represented in the DTP compound database. However, many of the
compounds that bind mammalian tubulin do not interact with yeast tubulin
[6].
Figure 3. Growth inhibition as a function of dose is plotted for cells treated with NSC 1331.
This compound specifically inhibited the growth of the bub3 spindle checkpoint mutant at all
doses tested in Stage 2, without significant inhibition of any of the other strains.
A number of the bub3-selective compounds were tested further in Rat1a

fibroblasts and human tumor cell lines. The following compounds had
effects in mammalian cells consistent with triggering of the spindle
checkpoint. NSC 5062, while having no effect on tubulin polymerization,
did cause an accumulation of mammalian fibroblasts in G2/M, with a defect
in chromosome segregation. NSC 142496-treated mammalian cells accumu-
late in G2, with the formation of tubulin rings. NSC 319447 treatment led to
mono-attached chromosomes in mammalian cells. NSC 672053 disrupted
microtubules in mammalian cells.
5.3.2 Compounds selective for the CLN2 overexpressing

strain in Stage 2
Two compounds were moderately specific for the strain overexpressing

the G1 cyclin CLN2, inhibiting only CLN2oe at 2 doses. Structures of these
compounds are displayed in Figure 4. NSC 180198 is related to the
carboxaldehyde thiosemicarbazone (e.g., triapine) class of ribonucleotide
reductase inhibitors. NSC 659206 is a bis-trihalocarbinol derivative of
cyclohexanone, unrelated to known biologically active agents. A total of 64
compounds inhibit CLN2oe +/– several other strains at least 1 dose.
Figure 4. These two compounds were selectively toxic to the G1 cyclin CLN2-over-
expressing strain at two of the doses tested in Stage 2. NSC 180198 is related to
ribonucleotide reductase inhibitors.
5.3.3 Compounds selective for the mgt1 strain in Stage 2
There were no compounds that inhibited only the mgt1 strain at more
than one dose. A total of 139 compounds inhibited mgt1 +/– 1 or more
strains at least 1 dose. The mgt1 mutant strain is known to be sensitive to
alkylative DNA damage in general and methylation at guanine residues at
the O6 position in particular. Even methylating agents, however, are more
toxic to the rad6 and rad18 post-replication repair mutant strains than to the
mgt1 strain. It is therefore expected that the mgt1 mutant strain would be
most sensitive among the panel of strains to few, if any, compounds.
5.3.4 V.3.4. Compounds selective for the mlh1 strain in Stage 2
There were no compounds that inhibited the growth of only the mlh1
mutant at 2 or more doses. A total of 164 inhibited the mlh1 strain +/– 1 or
more other strains at least 1 dose. Mlh1p is a component of mismatch repair
in yeast. There are no known agents that are selectively toxic to the mlh1
deletion strain.
5.3.5 Compounds selective for the rad14 strain in Stage 2
One compound, haloperidol (NSC 615296), inhibited the growth of just

the rad14 mutant at 3 doses. Haloperidol is used clinically as an
antipsychotic and is reported to bind to dopamine receptors [44]. Rad14p is
part of the nucleotide excision pathway for repair of bulky DNA adducts and
cross-links. Haloperidol does not contain reactive chemical groups that
would suggest DNA damage as the mechanism of action. Among the FDA-
approved agents, cross-linking agents mechlorethamine (nitrogen mustard)
and cisplatin are among the most active agents against the rad14 deletion
mutant [46]. As with the mgt1 mutant however, even with these agents, the
rad14 strain is not the most sensitive strain in the panel: the most sensitive
strains to mechlorethamine and cisplatin are rad6 and rad18 post-replication
repair mutants. Structures of two rad14-selective agents are shown in Figure 5.
Figure 5. Structures are shown for two compounds that selectively inhibited the growth of the
rad14 excision repair mutant.
5.3.6 Compounds selective for the rad18 strain in Stage 2
A large proportion of the Stage 2 tested compounds were highly specific

for the rad18 mutant. About 94 compounds (3% of all Stage 2 compounds)
inhibited only the rad18 mutant at 2 or more doses. Figure 6 presents
structures for 4 representative compounds.
Figure 6. Structures are given for representative compounds selective for the post-replication
repair mutant rad18 in Stage 2 testing. NSC 86324 is a nitrogen mustard, as were a number
of other rad18-seletive compounds. NSC 142226, an ---halocarbonyl, and NSC 3750, a
nitroaromatic, are representative of compound classes found multiple times among the rad18-
selective agents.
As already mentioned, the rad18 strain represents a loss-of-function in

post-replication, or daughter-strand gap repair. Loss of either rad6 and rad18
confers sensitivity toward a broad range of DNA damaging agents.
Compounds such as cisplatin, thiotepa, carmustine, hydroxyurea, bleomycin
as well as ionizing radiation are more toxic to rad18 mutant strain than to
wild type yeast. It is not surprising then that the vast majority of compounds
showing rad18 selectivity are electrophiles capable of damaging DNA. The
most highly represented classes of electrophiles include: nitrogen mustards
(e.g., NSC 86324) (i.e., bis-[2-chloroethyl]-amines) related to the FDA-
approved agents mechlorethamine, melphalan, chlorambucil, and cyclophos-
phamide; α-halocarbonyl compounds (e.g., NSC 142226); nitroaromatics
(e.g., NSC 3750) and aza-aromatic N-oxides (e.g., NSC 5094). Interestingly,
several compounds that appear to have low potential for causing DNA
damage (e.g., NSC 136325, a bromo-fluorene derivative) also score as rad18
selectives.
5.3.7 Compounds selective for the rad50 and rad52 strains in Stage 2
A large number of Stage 2 tested compounds exhibited specificity for the

DSB repair mutants rad50 and rad52. Almost 121 compounds (3% of Stage
2 tested compounds) were highly specific for these strains, inhibiting only
the DSB repair mutants at 2 or more doses. Representative data is shown in
Table 3. Three of the compounds were exceedingly specific, inhibiting only
the DSB repair mutants at all 5 doses. This included NSCs 295501 and
603071, analogs of the Topo I poison camptothecin; and NSC 669380, a
synthetic epi-podophylotoxin (i.e., related to etoposide) derivative.
Figure 7. Representative structures of rad50/rad52-specific compounds are shown. NSC

295501 is an analog of the topoisomerase I poison Camptothecin – this class is highly
represented among the agents selective for the DSB repair mutants. The remaining three
structures represent classes of known topoisomerase II poisons – NSC 669380 is an
epipodophyllotoxin, NSC 5159 is Chartreusin, and NSC 637992 in a pyrazoloacridine. All of
these compounds have planar ring structures that intercalate into DNA.
An additional 10 compounds inhibited the growth of only DSB repair

mutants at 4 of the 5 doses. Half of these have structures that might be
expected to interfere with topoisomerases. Chartreusin (NSC 5159) is
reported to bind DNA and inhibit the catalytic activity of Topo II [32]. NSC
302991 is a camptothecin analog, NSC 343501 is an analog of Amsacrine,
a Topo II poison. NSCs 129364, 637992 (pyrazoloacridone), 669965

(pyrimidoacridone) have planar ring structure with the potential to
intercalate into DNA. Of the remaining 108 rad50 rad52 specific
compounds, 92 are DNA intercalaters or Topo II poisons (or analogs
thereof ), and 13 are analogs of camptothecin. The remaining 16 compounds
are structurally unrelated to known topoisomerase poisons. A total of 953
compounds inhibit rad50 or rad52, either alone or in combination with a few
more strains, in at least one of doses.
5.3.8 V.3.8. compounds selective for the rad53 (mec2-1) strain

in Stage 2
Six compounds were highly specific for the rad53 mutant strain,
inhibiting only this strain in 2 of the doses. Of the 5 not covered by a
confidentiality agreement, 2 are structurally related to the antifolate
methotrexate, which is expected to trigger the S-phase DNA synthesis
checkpoint. The remaining 3 contain heavy metals (Au, Ru, Os). A total of
195 compounds inhibit the rad53 strain, either alone or in combination with
other strains, in at least 1 of the tested doses. 18 compounds (9%) (including
the 2 already mentioned) are analogs of nucleosides, antifolates, or other
compounds expected to interfere with DNA synthesis. 21 compounds (11%)
are related to topoisomerase poisons, alkylating agents, or other compounds
that introduce DNA damage.
5.3.9 Compounds selective for the sgs1 strain in Stage 2
There were no compounds highly selective for the sgs1 mutant. A total of
436 compounds inhibited sgs1, alone or in combination with a few more
strains in at least one dose. Of these 12 (3%) are analogs of compounds that
interfere with DNA synthesis, 14 (3%) are DNA damaging agents
(alkylating, topoisomerase poisons, etc.).
5.3.10 Compounds selective for several strains in Stage 2
A number of compounds were highly selective for multiple strains,

inhibiting the growth of only those strains by at least 70% at 2 or more
doses. For the purposes of this section, rad50 and rad52 will be counted
together, as both are needed for DSB repair, although there are a few
compounds that show differential activity between these two DSB repair
mutant strains. The predominant pattern observed for compounds that target
more than one strain is the activation of DNA damage responses. This is
perhaps not surprising given the high number of DNA damage response
mutants in the yeast strain panel and the abundance of DNA damaging
agents in the NCI compound collection.
Figure 8. Growth inhibition as a function of dose is plotted for cells treated with NSC 16258.
This compound was selectively toxic to the DSB repair mutants rad50 and rad52. It is
structurally unrelated to known topoisomerase poisons, which make up the majority of the
rad50/rad52 selective agents, and thus represents a novel structure selective for the DSB
repair mutants.
Figure 9. The structure of NSC 368252, a compound with modest selectivity for the rad53
checkpoint mutant is shown. In contrast to the more highly selective rad53 compounds, this
agent is not structurally related to known antimetabolites of DNA damaging agents.
In general, the rad18 mutant is the single most sensitive strain to DNA
damage and any compound that elicits a multistrain sensitivity profile that
includes rad18 has to be viewed as a potential DNA alkylator, cross-linker,
or cutter. Table 1 lists the NSC numbers and strain selectivity for compounds
that target multiple strains.
Table 1. Compounds listed in this table exhibited selectivity for multiple strains at two or
more doses. The strains inhibited, and the number of doses with this profile are indicated. For
this table, the rad50EP+ strain was not included.
NSC Strains inhibited Number of Structural characteristics

doses with
this pattern
613609 bub3 rad50 3 Planar ring
607088 bub3 sgs1 3 Mn++
400655 mec2 mlh1 3 Planar ring
21276 mec2 rad50 rad52 2 2-Chloro-1,3-dinitrobenzene
117619 rad14 rad50 rad52 2 1,2-Dibromo-1,2-diphenyl-
cyclopropane
134981 rad14 rad50 rad52 2 1,2-Diphenyl-cyclopropene
645457 rad14 sgs1 2
665606 rad14 sgs1 2
1510 rad18 rad50 rad52 2 Proflavine (Planar ring)
6208 rad18 rad50 rad52 2 Alkylator
21662 rad18 rad50 rad52 2 Phenazine bis-N-oxide (Planar
ring)
81052 rad18 rad50 rad52 2 Streptonigrin related (Planar
ring)
141586 rad18 rad50 rad52 2 Planar ring
400244 rad18 rad50 rad52 3 Planar ring
ring)
405789 rad18 rad50 rad52 2 (Planar ring)
ring)
507478 rad18 rad50 3
622143 rad18 rad52 2
24047 rad18 sgs1 4 bis-aromatic (Planar ring)
669264 rad50 sgs1 2 Guanine diphosphate ether
403546 bub3 rad18 rad50 3
683910 bub3 rad18 rad50 2 Planar ring
609959 mec2 rad50 rad52 sgs1 3 Camptothecin analog (Planar
ring)
50076 rad14 rad18 rad50 rad52 2 Nitrogen mustard
668523 rad14 rad18 rad50 rad52 2 Nitrogen mustard
NSC Strains inhibited Number of Structural characteristics

doses with
this pattern
632 rad18 rad50 rad52 sgs1 2 Aromatic propylene oxide ether
(Planar ring)
664867 rad18 rad50 rad52 sgs1 2
612071 wt1 mgt1 mlh1 sgs1 3 Weak uracil analog
658900 CLN2oe bub3 rad18 rad50 2
665773 CLN2oe rad14 mgt1 sgs1 2
108708 mec2 rad18 rad50 rad52 sgs1 2
154894 rad14 rad18 rad50 rad52 sgs1 2
329226 rad14 rad18 rad50 rad52 sgs1 4 Planar ring
373980 rad14 rad18 rad50 rad52 sgs1 2 Planar ring
652888 rad14 rad18 rad50 rad52 sgs1 2 Nitro-acridine (Planar ring)
270346 CLN2oe bub3 mec2 mgt1 mlh1 2
665727 CLN2oe rad14 rad18 rad52 mlh1 sgs1 2
621482 mec2 rad14 rad18 rad50 rad52 mgt1 mlh1 3
sgs1 wt2
78572 mec2 rad14 rad18 rad50 rad52 sgs1 3 Planar ring
373981 mec2 rad14 rad18 rad50 rad52 sgs1 2 Planar ring
382770 mec2 rad14 rad18 rad50 rad52 sgs1 2
610190 mec2 rad14 rad18 rad50 rad52 sgs1 3 Ellipticine analog (Planar ring)
667644 mec2 rad14 rad18 rad50 rad52 sgs1 2 Nitro-acridine (Planar ring)
404027 rad14 rad18 rad50 rad52 mgt1 mlh1 sgs1 wt2 2 (Planar ring)
5.3.10.1 Compounds selective for 2 strains

Ten compounds inhibited the rad18, rad50, and rad52 strains, but none
of the other strains, in at least 2 of the doses. Three of these, NSCs 21662,
402910, and 409304 are phenazine bis-N-oxides. These compounds undergo

bioreductive activation to generate radical species. A fourth compound,
proflavine (NSC 1510), has a somewhat related acridine structure. NSC
81052, a quinoline quinone that is related to the left - hand portion of
Streptonigrin, exhibits specificity for these three strains. Streptonigrin is
reported to cause multiple types of DNA damage, including DNA breaks and
DNA adducts [7]. The remaining rad18, rad50, rad52 selective compounds
(NSCs 6208, 141586, 400244, and 405789) are structurally unrelated. While
NSC 6208 is an alkylator, the remaining compounds appear to be of low
reactivity.
A single compound, NSC 507478, was selective for the rad18 and rad50
strains, inhibiting just these two at three of the tested doses. This is one of
the few compounds that showed inhibition of the rad50 mutant without
affecting the growth of the other DSB repair mutant, rad52. There is no
information available on mechanism of this compound.
A single compound, NSC 622143, was selective for the rad18 and rad52
mutants, while not inhibiting the rad50 mutant or any of the other strains.
Nothing is known regarding the mechanism of action of this compound.
Given the high concordance in sensitivity of rad50 and rad52 strains to both
topoisomerase poisons and DNA alkylating agents, compounds that affect
these strains differentially are of potential interest in dissecting the functions
of Rad50p and Rad52p.
Two compounds were selective for the rad50 and sgs1 mutants. One of
these is covered by a confidentiality agreement. The other compound (NSC
669264) is a guanine diphosphate ether containing a long aliphatic tail.
Unlike most compounds that inhibit the rad50 strain, these compounds were
much less active against the other DSB repair mutant, rad52. Both rad50
and sgs1 have been implicated in telomere function.
Two compounds (NSCs 645457 and 665606) were selective for rad14
and sgs1 at two of the doses. These compounds are unrelated to one another.
Neither contains electrophilic groups and both were relatively inactive in the
60 cell line screen. Since Rad14p is normally involved in nucleotide excision
repair of DNA damage, these compounds may highlight an additional
function of this protein.
Two compounds, NSCs 117619 and 134981, inhibited the growth of the
rad14, rad50, and rad52 strains, but no others, at two doses. These
compounds, a 1,2-dibromo-1,2-diphenyl-cyclopropane and a 1,2-diphenyl-
cyclopropene, share somewhat related structures and may behave as
electrophiles.
A single compound (NSC 21276) was selective for rad53 and rad50
rad52 at two doses. NSC 24047 was highly selective for the rad18 and sgs1
mutants, inhibiting the growth of just these strains in four of the tested doses.
This bis-aromatic appears to have little potential to cause DNA damage.

NSC 400655 exhibited specificity for both the rad53 and mlh1 mutants at
three of the doses. Very few compounds showed this degree of selectivity for
the mismatch repair mutant. One compound, NSC 607088, was selective for
the bub3 and sgs1 strains at three of the doses. A single compound (NSC
613609) selectively inhibited the growth of the bub3 and rad50 strains at 3
doses. This compound is one of the few seen that distinguishes between the
2 DSB repair mutants.
Since, with the exception of the DNA damage profile of some or all of
the rad18, rad14, rad50, rad52, rad53, and sgs1 strains, there is little
literature precedent for overlapping function of the mutants in our panel, it is
difficult to make definitive statements about the mechanism of action of
compounds that target multiple strains. It is impressive, however, to note the
seemingly large number of cellular responses demonstrating subtle
differences in the compounds’ mechanisms of action. Subtle differences
such as these may be the deciding factor between compounds that make it to
the clinic and those that do not.
5.3.10.2 Compounds selective for 3 strains

Two compounds, NSCs 50076 and 668523, were selective for the rad14,
rad18, rad50, and rad52 strains at 2 doses. These compounds share a related
structure related to the nitrogen mustard mechlorethamine (bis-[2-
chloroethyl]-amine) and are likely DNA alkylating and cross-linking agents.
Four compounds exhibited selectivity for the rad18, rad50 rad52, and
sgs1 strains. Three of these (NSCs 632, 634, and 338423) are structurally
related, highly reactive aromatic propylene oxide ethers and are also likely
DNA damaging agents. The fourth compound (NSC 664867) is structurally
unrelated but contains several electrophilic functionalities and may damage
DNA as well.
Two compounds (NSCs 403546 and 683910) were selective for bub3,
rad18, and rad50. While not close structural analogs, these two do share a
carbon double-bonded to a nitrogen.
NSC 180198 inhibited the growth of only the bub3, CLN2oe, and rad50
strains at two doses. A single compound, NSC 609959 was selective for the
rad53, sgs1, and rad50 rad52 mutants at three doses.
5.3.10.3 Compounds selective for more than 3 strains

Nine compounds were selective for the rad53, rad14, rad18, rad50
rad52, and sgs1 strains. This can be interpreted as a broad DNA damage
response. Five of these (NSCs 667644, 673793, 673795, 673799, and
673805) share similar structures, and demonstrated quite potent activity in
the NCI 60 human tumor cell line screen, with GI50 values of 10–7 to 10–8 M.
These compounds are called nitracrines (nitro-acridines) and are DNA-

intercalating alkylating agents. Although the exact mechanism of action is
undefined, these compounds have received attention in Europe, where
several have undergone clinical trials. The remaining 4 compounds (NSCs
78572, 373981, 382770, and 610190) are structurally unrelated, and were
about 2 logs less active in the 60 cell line screen. Interestingly, NSC 610190
is a synthetic ellipticine derivative. Several of these DNA-interactive agents
have been evaluated in pre-clinical studies.
Four compounds were selective for rad14, rad18, rad50, rad52, and sgs1
in at least two of the doses. Two of these compounds (NSCs 329226 and
373980) have structures similar to one another, while the other two (NSCs
154894 and 652888) are structurally unrelated. NSC 652888 is related to the
nitracrines mentioned in the previous section.
One compound, NSC 665773 showed specificity for the CLN2oe, rad14,
mgt1, and sgs1 strains at 2 doses. A single compound (NSC 658900) was
selective for bub3, rad18, CLN2oe, and rad50 rad52 at 2 doses.
5.4 Correlation of Stage 2 and Stage 1 results
For the 3 strains with a single mutation of interest in Stage 1 testing, we

can ask whether the Stage 2 data are consistent with that from Stage 1
testing. The discussion here pertains to those compounds that were highly
specific (i.e., that inhibited only the given strain in at least two of the Stage 2
doses) for the bub3 and rad53 strains. In addition, those compounds highly
selective for rad50 (with or without rad52) are compared to the Stage 1
rad50 results. For the remaining strains, the use of multiple alterations in the
Stage 1 strains would confound this type of analysis.
Most of the 29 compounds highly specific for bub3 in Stage 2 testing
were also specific for the bub3 mutant in Stage 1 tests. For 21 of these
compounds, the bub3 mutant was the most sensitive strain in Stage 1 testing.
One of the Stage 2 bub3-selective compounds was not selective for bub3 in
Stage 1. The remainder of these Stage 2 bub3-selective compounds inhibited
the bub3 mutant in Stage 1 testing, but inhibited the growth of some of the
other strains to the same extent. Five compounds were highly selective for
the rad53 mutant in the Stage 2 assay. For four of these, the rad53 mutant
was the most sensitive strain in Stage 1. The fifth compound inhibited the
growth of the rad53 stain in Stage 1, but inhibited other strains to the same
degree. 121 compounds were highly selective for the rad50 strain (and
rad52) in Stage 2 testing. For 112 of these, the rad50 mutant strain was the
most sensitive in Stage 1. The other nine compounds inhibited the rad50
mutant, as well as other strains in Stage 1.
Thus for the 155 compounds for which the Stage 1 and Stage 2 results
can be readily compared, the vast majority (88%) were selective for the same
strain in both stages. A further 11% were somewhat less selective in Stage 1
assays, inhibiting other strains in addition to that seen in Stage 2. Only one
of the these had discordant Stage 1 and Stage 2 results.
6 EFFECT OF ERG6, PDR1, AND PDR3

MUTATIONS ON DRUG SENSITIVITY
The erg6, pdr1, pdr3 (epp) mutations were included in all of the strains
used in Stage 0 and Stage 1 and in all but one strain in Stage 2 to increase
drug uptake (erg6) and decrease drug efflux (pdr1 and pdr3). By including
both rad50, erg6, pdr1, pdr3 (rad50epp-) and rad50, ERG6, PDR1, PDR3
(rad50EPP+) strains in the Stage 2 panel, we were able to quantify the effect
of the “drug-sensitizing” mutations. We stratified rad50-selective com-
pounds based on the following criterion. For each compound, if there were at
least two doses at which the rad50epp- strain but not rad50EPP+ had a 70%
or greater growth inhibition, then the compound was counted as having
increased activity in the mutant “drug uptake and efflux” background. Of the
116 rad50-selective compounds, 66 compounds, or 57% had increased
toxicity due to the epp mutations. For all Stage 2-tested compounds, about
one-third were aided by the drug-sensitizing mutations, with the rad50epp-
strain half a log more sensitive than the rad50EPP+ strain.
7 PDR AND MDR
Pleiotropic drug resistance (Pdr), genes in yeast encode a number of

ATP-binding cassette (ABC) transporter proteins that function as
detoxifying efflux pumps in a manner analogous to the mammalian multiple
drug resistance, or MDR proteins. Since multidrug resistance is a major
problem in cancer chemotherapy, we wondered whether sensitivity to PDR1
and PDR3 in yeast status correlated with MDR sensitivity in human cells. Of
the compounds where rad50epp- was at least tenfold more sensitive than the
rad50EPP+ strain, 161 have been tested in the 60 human tumor cell line
screen. Only 6% of these showed a significant negative correlation with
MDR activity in the 60 cell line panel.
8 SUMMARY
A major goal of anticancer drug discovery and development in the new

century is the identification of compounds that kill cancer cells selectively
without causing the catastrophic damage to normal tissues that is the
hallmark of current cytotoxic chemotherapy agents. Such selective agents
will arise in a number of ways. First, through rational selection of drug
targets. For example, the BCR-ABL translocation that is typical of chronic
myelogenous leukemia (CML) creates a hyper-active form of the ABL
tyrosine kinase. Inhibition of ABL, and BCR-ABL by analogy, is a
reasonable strategy to inhibit the growth and possibly kill BCR-ABL
expressing cells. Since normal cells have low levels of ABL activity,
inhibition of ABL in normal tissues is not expected to be a problem. This
approach led to the identification of Gleevec (imatinib mesylate), which has
proven to be exceptionally effective and free of side effects, validating the
rational target selection strategy. Other tumors, however, lack obvious
targets. While many breast cancers overexpress cyclin E, leading to
increased CDK activity, inhibition of that activity has, to date, failed to result
in impressive clinical responses. Another approach for the development of
selective anticancer agents is through phenotypic, cell-based screening for
compounds that selectively target specific cancer-related contexts and take
advantage of synthetic lethality. This is the approach outlined in this chapter.
We have assembled a panel of isogenic yeast strains containing
alterations in DNA damage response pathways that are homologous to those
often mutated in human cancers. This panel of strains was screened against
nearly 100,000 compounds from the NCI–DTP repository for differential
growth inhibition. The ultimate aim of this project is to identify agents that
will target tumors harboring the DNA damage mutations represented in the
yeast strain panel. If the major hurdle of the rational approach is the
selection of “valid” anticancer drug targets, then the major hurdle of our
unbiased approach is the identification of the drug targets. Many of the
compounds identified in these screens have very promising selectivity for a
specific mutation but it will take more time until we know the molecular
targets and mechanisms of action for these compounds. The yeast data
provides a very different metric of biological activity, a metric that
complements the NCI’s human cancer cell line screen.
The panel of strains was assembled based on a vast literature on the drug
sensitivity of various DNA response mutant yeast strains as well as our
preliminary evaluation of FDA-approved, small molecule anticancer agents.
We were able to evaluate agents and make rational choices as to whether a
particular agent was expected to target a mutant strain and, therefore,
represented a known mechanism of action, or whether the agent was
structurally unrelated to known compounds and potentially represented a

new mechanism of action and a new target. It is the latter class of
compounds that are the most interesting and will be studied in more detail in
the future.
The screen yielded over 1,500 compounds that showed selective growth
inhibition for one or more of the strains in the panel. Overall, the screen
design yielded highly reproducible results with 99% of the Stage 2 selective
compounds showing similar activity in Stage 1 screens. Selectivity for
individual strains was assessed using both strict numerical criteria as well
as a less quantitative, experience-based criteria. For example, several
compounds selective for the rad18, rad50, or bub3 strains showed selective
growth inhibition over the entire concentration range, with IC50s for mutant
and wild type strains differing by more than 100-fold. On the other hand, the
mlh1 and CLN2 overexpression strains yielded only mildly selective
compounds having at best a modest twofold difference in sensitivity. Thus
for each strain in the panel we applied slightly different criteria for the
selection of compounds.
The results presented in this chapter highlight the value of anticancer
drug screening using cell-based assays in genetically- defined model
organisms. This represents a unique resource, with data for nearly 90,000
compounds freely available through a public web site at http://dtp.nci.nih.
gov/yacds/index.html.
ACKNOWLEDGMENTS
The authors wish to thank Richard Klausner, Leland Hartwell, and

Stephen Friend for initiating this project; Heather Dunstan, John Lamb,
Philippe Szankasi, David Evans, and Michele Cronk for implementing the
screen; and Edward Sausville, Jill Johnson, Tim Myers, Daniel Zaharevitz
and David Segal for database and web site development, and for project
oversight.
REFERENCES
1. Alias, Y., K. Awang, A. H. Hadi, O. Thoison, T. Sevenet, and M. Pais. 1995. An
antimitotic and cytotoxic chalcone from Fissistigma lanuginosum. J. Nat. Prod.
58:1160–1166.
2. Bailly, V., S. Lauder, S. Prakash, and L. Prakash. 1997. Yeast DNA repair proteins
Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA binding, and
ATP hydrolytic activities. J. Biol. Chem. 272:23360–23365.
3. Bankmann, M., L. Prakash, and S. Prakash. 1992. Yeast RAD14 and human
xeroderma pigmentosum group A DNA-repair genes encode homologous proteins.
Nature 355:555–558.
4. Bennett, R. J., and J. C. Wang. 2001. Association of yeast DNA topoisomerase III and
Sgs1 DNA helicase: studies of fusion proteins. Proc. Natl. Acad. Sci. USA 98:11108–
11113.
5. Blasina, A., I. V. de Weyer, M. C. Laus, W. H. Luyten, A. E. Parker, and C. H.
McGowan. 1999. A human homologue of the checkpoint kinase Cds1 directly inhibits
Cdc25 phosphatase. Curr. Biol. 9:1–10.
6. Bode, C. J., M. L. Gupta, Jr., E. A. Reiff, K. A. Suprenant, G. I. Georg, and R. H.
Himes. 2002. Epothilone and paclitaxel: unexpected differences in promoting the
assembly and stabilization of yeast microtubules. Biochemistry 41:3870–3874.
7. Bolzan, A. D., and M. S. Bianchi. 2001. Genotoxicity of streptonigrin: a review. Mutat.
Res. 488:25–37.
8. Boulton, S. J., and S. P. Jackson. 1998. Components of the Ku-dependent non-
homologous end-joining pathway are involved in telomeric length maintenance and
telomeric silencing. EMBO J. 17:1819–1828.
9. Cahill, D. P., C. Lengauer, J. Yu, G. J. Riggins, J. K. Willson, S. D. Markowitz,
K. W. Kinzler, and B. Vogelstein. 1998. Mutations of mitotic checkpoint genes in
human cancers. Nature 392:300–303.
10. Critchlow, S. E., and S. P. Jackson. 1998. DNA-end-joining: from yeast to man.
Trends Biochem. Sci. 23:394–398.
11. Datta, A., M. Hendrix, M. Lipsitch, and S. Jinks-Robertson. 1997. Dual roles for
DNA sequence identity and the mismatch repair system in the regulation of mitotic
crossing-over in yeast. Proc. Natl. Acad. Sci. USA 94:9757–9762.
12. Diede, S. J., and D. E. Gottschling. 2001. Exonuclease activity is required for sequence
addition and Cdc13p loading at a de novo telomere. Curr. Biol. 11:1336–1340.
13. Dunstan, H. M., C. Ludlow, S. Goehle, M. Cronk, P. Szankasi, D. R. Evans, J. A.
Simon, and J. R. Lamb. 2002. Cell-based assays for identification of novel double-
strand break-inducing agents. J. Natl. Cancer Inst. 94:88–94.
14. Durant, S. T., M. M. Morris, M. Illand, H. J. McKay, C. McCormick, G. L. Hirst,
R. H. Borts, and R. Brown. 1999. Dependence on RAD52 and RAD1 for anticancer
drug resistance mediated by inactivation of mismatch repair genes. Curr. Biol. 9:51–54.
15. Evans, E., N. Sugawara, J. E. Haber, and E. Alani. 2000. The Saccharomyces
cerevisiae Msh2 mismatch repair protein localizes to recombination intermediates in
vivo. Mol. Cell. 5:789–799.
16. Fraschini, R., A. Beretta, L. Sironi, A. Musacchio, G. Lucchini, and S. Piatti. 2001.
Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does
not require intact kinetochores. EMBO J. 20:6648–6659.
17. Frei, C., and S. M. Gasser. 2000. The yeast Sgs1p helicase acts upstream of Rad53p in
the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci.
Genes Dev. 14:81–96.
18. Furuse, M., Y. Nagase, H. Tsubouchi, K. Murakami-Murofushi, T. Shibata, and
K. Ohta. 1998. Distinct roles of two separable in vitro activities of yeast Mre11 in
mitotic and meiotic recombination. EMBO J. 17:6412–6425.
19. Greene, C. N., and S. Jinks-Robertson. 1997. Frameshift intermediates in

homopolymer runs are removed efficiently by yeast mismatch repair proteins. Mol. Cell
Biol. 17:2844–2850.
20. Grenon, M., Gilbert, C., and N. F. Lowndes. 2001. Checkpoint activation in response
to double-strand breaks requires the Mre11/Rad50/Xrs2 complex. Nat. Cell Biol. 3:844–
847.
21. Gupta, M. L., Jr., C. J. Bode, G. I. Georg, and R. H. Himes. 2003 Understanding
tubulin-Taxol interactions: mutations that impart Taxol binding to yeast tubulin. Proc.
22. Guzder, S. N., P. Sung, L. Prakash, and S. Prakash. 1993. Yeast DNA-repair gene
RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA. Proc.
23. Guzder, S. N., P. Sung, L. Prakash, and S. Prakash. 1996. Nucleotide excision repair
in yeast is mediated by sequential assembly of repair factors and not by a pre-assembled
repairosome. J. Biol. Chem. 271:8903–8910.
24. Hall, M. C., P. V. Shcherbakova, and T. A. Kunkel. 2002. Differential ATP binding
and intrinsic ATP hydrolysis by amino-terminal domains of the yeast Mlh1 and Pms1
proteins. J. Biol. Chem. 277:3673–3679.
25. Hall, M. C., H. Wang, D. A. Erie, T. A. Kunkel. 2001. High affinity cooperative DNA
binding by the yeast Mlh1-Pms1 heterodimer. J. Mol. Biol. 312:637–647.
26. Han, H., D. R. Langley, A. Rangan, and L. H. Hurley. 2001. Selective interactions of
cationic porphyrins with G-quadruplex structures. J. Am. Chem. Soc. 123:8902–8913.
27. Hartwell, L. H., P. Szankasi, C. J. Roberts, A. W. Murray, and S. H. Friend. 1997.
Integrating genetic approaches into the discovery of anticancer drugs. Science.
278:1064–1068.
28. Hopfner, K. P., L. Craig, G. Moncalian, R. A. Zinkel, T. Usui, B. A. Owen,
A. Karcher, B. Henderson, J. L. Bodmer, C. T. McMurray, J. P. Carney, J. H.
Petrini, and J. A. Tainer. 2002. The Rad50 zinc-hook is a structure joining Mre11
complexes in DNA recombination and repair. Nature 418:562–566.
29. Kaliraman, V., J. R. Mullen, W. M. Fricke, S. A. Bastin-Shanower, and S. J. Brill.
2001. Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease.
Genes Dev. 15:2730–2740.
30. Keyomarsi, K., S. L. Tucker, T. A. Buchholz, M. Callister, Y. Ding, G. N.
Hortobagyi, I. Bedrosian, C. Knickerbocker, W. Toyofuku, M. Lowe, T. W.
Herliczek, and S. S. Bacus. 2002. Cyclin E and survival in patients with breast cancer.
N. Engl. J. Med. 347:1566–1575.
31. Kitao, S., A. Shimamoto, M. Goto, R. W. Miller, W. A. Smithson, N. M. Lindor,
and Y. Furuichi. 1999. Mutations in RECQL4 cause a subset of cases of Rothmund–
Thomson syndrome. Nat. Genet. 22:82–84.
32. Lorico, A., and B. H. Long. 1993. Biochemical characterisation of elsamicin and other
coumarin-related antitumour agents as potent inhibitors of human topoisomerase II. Eur.
J. Cancer. 29A:1985–1991.
33. McHugh, P. J., R. D. Gill, R. Waters, and J. A. Hartley. 1999. Excision repair of
nitrogen mustard-DNA adducts in Saccharomyces cerevisiae. Nucleic Acids Res.
27:3259–3266.
34. Myung, K., and R. D. Kolodner. 2002. Suppression of genome instability by redundant
S-phase checkpoint pathways in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA
99:4500–4507.
35. New, J. H., T. Sugiyama, E. Zaitseva, and S. C. Kowalczykowski. 1998. Rad52

protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature.
391:407–410.
36. Paull, K. D., R. H. Shoemaker, L. Hodes, A. Monks, D. A. Scudiero, L. Rubinstein,
J. Plowman, and M. R. Boyd. 1989. Display and analysis of patterns of differential
activity of drugs against human tumor cell lines: development of mean graph and
COMPARE algorithm. J. Natl. Cancer Inst. 81:1088–1092.
37. Pedrazzi, G., C. Perrera, H. Blaser, P. Kuster, G. Marra, S. L. Davies, G. H. Ryu,
R. Freire, I. D. Hickson, J. Jiricny, and I. Stagljar. 2001. Direct association of
Bloom’s syndrome gene product with the human mismatch repair protein MLH1.
Nucleic Acids Res. 29:4378–4386.
38. Pegg, A. E., M. Boosalis, L. Samson, R. C. Moschel, T. L. Byers, K. Swenn, and
M. E. Dolan. 1993. Mechanism of inactivation of human O6-alkylguanine-DNA
alkyltransferase by O6-benzylguanine. Biochemistry. 32:11998–12006.
39. Pletsa, V., M. J. Steenwinkel, M. Stoikidou, J. H. van Delft, R. A. Baan,
K. Katsouyanni, and S. A. Kyrtopoulos. 2002. Monitoring for DNA damage of
humans occupationally exposed to methyl bromide. Anticancer Res. 22:997–1000.
40. Prakash, S., and L. Prakash. 2000. Nucleotide excision repair in yeast. Mutat. Res.
451:13–24.
41. Rasmussen, L. J., and L. Samson. 1996. The Escherichia coli MutS DNA mismatch
binding protein specifically binds O(6)-methylguanine DNA lesions. Carcinogenesis
17:2085–2088.
42. Richardson, H. E., C. Wittenberg, F. Cross, and S. I. Reed. 1989. An essential G1
function for cyclin-like proteins in yeast. Cell. 59:1127–1133.
43. Rodriguez, K., J. Talamantez, W. Huang, S. H. Reed, Z. Wang, L. Chen, W. J.
Feaver, E. C. Friedberg, A. E. Tomkinson. 1998. Affinity purification and partial
characterization of a yeast multiprotein complex for nucleotide excision repair using
histidine-tagged Rad14 protein. J. Biol. Chem. 273:34180–34189.
44. Seeman, P. 2002. Atypical antipsychotics: mechanism of action. Can. J. Psychiatry
47:27–38.
45. Shimada, K., P. Pasero, and S. M. Gasser. 2002. ORC and the intra-S-phase
checkpoint: a threshold regulates Rad53p activation in S phase. Genes Dev. 16:3236–
3252.
46. Simon, J. A., P. Szankasi, D. K. Nguyen, C. Ludlow, H. M. Dunstan, C. J. Roberts,
E. L. Jensen, L. H. Hartwell, and S. H. Friend. 2000. Differential toxicities of
anticancer agents among DNA repair and checkpoint mutants of Saccharomyces
cerevisiae. Cancer Res. 60:328–333.
47. Sugawara, N., and J. E. Haber. 1992. Characterization of double-strand break-induced
recombination: homology requirements and single-stranded DNA formation. Mol. Cell
Biol. 12:563–575.
48. Svejstrup, J. Q., Z. Wang, W. J. Feaver, X. Wu, D. A. Bushnell, T. F. Donahue,
E. C. Friedberg, and R. D. Kornberg. 1995. Different forms of TFIIH for transcription
and DNA repair: holo-TFIIH and a nucleotide excision repairosome. Cell 80:21–28.
49. Tong, A. H., M. Evangelista, A. B. Parsons, H. Xu, G. D. Bader, N. Page,
M. Robinson, S. Raghibizadeh, C. W. Hogue, H. Bussey, B. Andrews, M. Tyers,
and C. Boone. 2001. Systematic genetic analysis with ordered arrays of yeast deletion
mutants. Science 294:2364–2368.
50. Usui, T., T. Ohta, H. Oshiumi, J. Tomizawa, H. Ogawa, and T. Ogawa. 1998.
Complex formation and functional versatility of Mre11 of budding yeast in
recombination. Cell 95:705–716.
51. Venditti, J. M., R. A. Wesley, and J. Plowman. 1984. Current NCI preclinical
antitumor screening in vivo: results of tumor panel screening, 1976–1982, and future
directions. Adv. Pharmacol. Chemother. 20:1–20.
52. Wittenberg, C., K. Sugimoto, and S. I. Reed. 1990. G1-specific cyclins of S.
cerevisiae: cell cycle periodicity, regulation by mating pheromone, and association with
the p34CDC28 protein kinase. Cell 62:225–237.
53. Xiao, W., B. Derfler, J. Chen, and L. Samson. 1991. Primary sequence and biological
functions of a Saccharomyces cerevisiae O6-methylguanine/O4-methylthymine DNA
repair methyltransferase gene. EMBO J. 10:2179–2186.
54. Xiao, W., and T. Fontanie. 1995. Expression of the human MGMT O6-methylguanine
DNA methyltransferase gene in a yeast alkylation-sensitive mutant: its effects on both
exogenous and endogenous DNA alkylation damage. Mutat. Res. 336:133–142.
55. Xiao, W., and L. Samson. 1992. The Saccharomyces cerevisiae MGT1 DNA repair
methyltransferase gene: its promoter and entire coding sequence, regulation and in vivo
biological functions. Nucleic Acids Res. 20:3599–3606.
56. Xiao, W., and L. Samson. 1993. In vivo evidence for endogenous DNA alkylation
damage as a source of spontaneous mutation in eukaryotic cells. Proc. Natl. Acad. Sci.
USA 90:2117–2121.
57. Zhong, Q., C. F. Chen, S. Li, Y. Chen, C. C. Wang, J. Xiao, P. L. Chen, Z. D.
Sharp, and W. H. Lee. 1999. Association of BRCA1 with the hRad50-hMre11-p95
complex and the DNA damage response. Science 285:747–750.
58. Zou, H., and R. Rothstein. 1997. Holliday junctions accumulate in replication mutants
via a RecA homolog-independent mechanism. Cell 90:87–96.
Chapter 13
YEAST AS A MODEL TO STUDY
THE IMMUNOSUPPRESSIVE AND
CHEMOTHERAPEUTIC DRUG RAPAMYCIN
John R. Rohde, Sara A. Zurita-Martinez and Maria E. Cardenas

Department of Molecular Genetics and Microbiology, Duke University Medical Center,
Durham, NC 27710, USA
1 INTRODUCTION
Rapamycin is a natural product of a soil bacterium and has potent

immunosuppressive, and antiproliferative actions. First identified in 1975 as
an antifungal drug, rapamycin languished in obscurity after it was found to
cause bone marrow suppression [112]. Interest in rapamycin was later
rekindled when it was discovered to be structurally related to the potent
T-cell inhibitor FK506 [65]. Subsequent studies, conducted first in yeast and
then in mammalian cells, revealed the molecular basis of therapeutic action.
Rapamycin diffuses into the cell and binds to a small cellular protein,
FKBP12, forming an FKBP12–rapamycin protein–drug complex that is the
active intracellular agent. This complex then binds to and inhibits the Tor
kinases, which function in nutrient sensing pathways that control cell growth
and differentiation (Figure 1) (reviewed in [101]). The Tor kinases and
FKBP12 are conserved from yeast to worms, flies, plants, and humans.
Rapamycin has received FDA approval as an immunosuppressant, and more
recently as an antiproliferative agent to inhibit restenosis following cardiac
stenting [84]. Recent studies indicate that rapamycin and its analogs will
find additional clinical applications as chemotherapeutic agents, topical
347
© 2007 Springer.
348 J. R. Rohde, S. A. Zurita-Martinez and M. E. Cardenas
immunosuppressive therapies in dermatology, and novel antifungal agents

[33, 45, 83, 88, 116]. We review here the use of yeast as a model to elucidate
the molecular basis of therapeutics for this exciting natural product.
The history of the budding yeast Saccharomyces cerevisiae as a model to
identify the targets and molecular basis of therapeutic action of rapamycin
and other immunosuppressive drugs began with the discovery of rapamycin
(sirolimus), cyclosporine A (CsA), and FK506 (tracrolimus) themselves.
These immunosuppressive drugs were identified in three independent
screens conducted at three different pharmaceutical companies. CsA was
discovered at Sandoz Pharmaceuticals in a screen of soil samples for
inhibitors of a mixed lymphocyte response (MLR) assay. CsA is produced
by a fungus, Tolypocladium inflatum, which was isolated from a soil sample
Figure 1. TOR responds to diverse signals to regulate cell growth. TOR is activated by
compounds associated with a favorable energy status including ATP, amino acids, and
phosphatidic acid. TOR is inactivated in response to nutrient depletion, mitochondrial
dysfunction as well as rapamycin–FKBP12. Active TOR promotes cell growth while
inactivation of TOR results in growth arrest and autophagy in yeast and growth arrest and
apoptosis in higher eukaryotes.
Chapter 13: Rapamycin in yeast 349
from northern Norway [13]. CsA received FDA approval in 1983 as an

immunosuppressant to prevent and treat graft rejection in organ transplant
recipients, revolutionized organ transplant therapy, and became the gold
standard for immunosuppressive therapies. FK506 was subsequently
discovered at Fujisawa Pharmaceuticals in 1987 in a soil sample taken from
the Tsukuba region of northern Japan and found to be a macrolide produced
by the soil bacterium S. tsukubaensis [65]. FK506 (tacrolimus) received
FDA approval in 1994 and has gone on to have considerable impact in
transplant medicine. Rapamycin was discovered from a screen for novel
natural products in 1975 at Wyeth–Ayerst Pharmaceuticals [112, 125].
In this case, the screen was for antifungal activity, and the rapamycin
producing bacterium, S. hygroscopicus, was discovered in an isolate from
the beaches of Easter Island (Rapa nui). Rapamycin remains one of the most
potent anti-Candida drugs ever discovered [5], however the early finding
that it caused bone marrow suppression halted development as an
antimicrobial agent. It was only following the discovery of FK506 in 1987,
and the appreciation that FK506 and rapamycin are structurally related, that
rapamycin was resurrected from the shelf and studies began again in earnest
to understand its molecular targets and possible therapeutic applications.
Studies in yeast that began in the late 1980s defined the molecular
targets of rapamycin, contributed to elucidate the mechanism of action of
FK506 and CsA, and fueled comparable studies in mammalian T-cells
that led to considerable insights into the molecular basis of therapeutic
action (reviewed in [20]). Early studies from Merck demonstrated that
FK506 and rapamycin inhibit T-cell proliferation by blocking different
signaling pathways [38, 39]. FK506, like CsA, blocked the T-cell antigen
response pathway necessary for signaling cascades to drive expression of
hundreds of genes required for T-cell activation. In contrast, rapamycin had
no effect on the T-cell antigen response pathway, but instead blocked T-cell
proliferation in response to interleukin-2 (IL-2). FK506 and rapamycin were
found to act as reciprocal antagonists [38, 39], suggesting that the two
exerted their actions via a common target. Concurrently, biochemical studies
led to the identification of a small abundant cellular binding protein, FK506
binding protein of 12 kDa (FKBP12), which is bound with high affinity to
either FK506 or to rapamycin [49, 117]. However, given that FKBP12 is an
abundant, ubiquitous protein expressed in all cells in the human body, a
general view was that its drug-binding activity might not be involved in a
very specific action in lymphocytes. Studies in yeast resolved this dilemma,
and established unequivocally the central role of the FKBP12 protein in
rapamycin action.
Around this time, an FKBP12 homolog that shared 54% amino acid
sequence identity with the human FKBP12 protein was identified in the
yeast S. cerevisiae [52, 132]. Subsequently the crystal structures were solved
for both the yeast and the human protein and found to be essentially
superimposable [104, 123]. The gene encoding the yeast FKBP12 protein
was cloned and disrupted. Whereas wild-type yeast cells are exquisitely
sensitive to growth inhibition by rapamycin, with a minimum inhibitory
concentration (MIC) of 25 ng/ml, yeast cells lacking FKBP12 were
completely viable and only slightly reduced in growth rate [52, 67, 132].
Thus inhibition of an essential function of FKBP12 could not explain the
potent toxic action of FKBP12. Taken together, these findings support a
model in which both the FKBP12 protein and its ligand rapamycin are both
required to exert a toxic effect in yeast. These findings also established
unequivocally that FKBP12 plays a central role in the action of rapamycin,
and the next challenge then was to identify the molecular target of the
FKBP12–rapamycin complex.
The targets of the FKBP12–rapamycin complex, the products of the
TOR1 and TOR2 genes (target of rapamycin), were discovered in a genetic
screen in yeast searching for rapamycin resistant mutants [51]. Mutations in
three different genes were identified. First, mutations in the FPR1 gene
encoding FKBP12 were found to be recessive, and resulted from amino acid
substitutions that based on structural studies of the FKBP12–rapamycin
complex were predicted to be critical for rapamycin binding. Importantly,
mutations in two other genes identified were genetically distinct from FPR1,
mapped to two different genomic locations and conferred dominant, or
semidominant, drug resistance in genetic crosses. Based on an unusual
genetic behavior between alleles of these three genes, known as nonallelic
noncomplementation, it was proposed that the three might form a physical
complex.
The TOR1 and TOR2 genes were subsequently cloned by the Hall and
Livi laboratories, revealing that they encode extremely large proteins, ~280
kDa, that Tor1 and Tor2 are homologs of each other, and that both share a
C-terminal domain with homology to lipid and protein kinases [18, 54, 71].
Further studies demonstrated that FKBP12–rapamycin forms a physical
complex with the yeast Tor1 and Tor2 proteins [22, 76, 118]. Later, work
from five different groups converged to identify the mammalian Tor
homolog (mTor) via its ability to bind the FKBP12–rapamycin complex [15,
27, 105, 106]. In addition, it was demonstrated that yeast Tor–mTor hybrid
genes are capable of providing Tor function in yeast cells [2]. Tor homologs
were later identified in other organisms; similar to S. cerevisiae two Tor
proteins have been characterized in S. pombe, and a single Tor homolog has
been identified in C. albicans, C. neoformans, D. melanogaster, A. thaliana,
and H. sapiens [15, 32, 51, 71, 82, 92, 105, 131, 136].
2 STRUCTURE OF THE TOR KINASES
The TOR proteins belong to a family of phosphatidylinositol kinase

(PIK)-related kinases which include the mammalian phosphatidylinositol
3-kinase itself, ataxia telangiectasia mutated (ATM), ataxia telangiectasia
related (ATR), DNA-dependent protein kinase, and the yeast MEC1, Rad53,
and TEL1 proteins, all of which are involved in regulating cell cycle
progression in response to exogenous or endogenous signals [62]. A
hallmark of the PIK-related family is a C-terminal kinase domain with
identity to both protein and lipid kinases. In fact, a specific protein kinase
activity has been demonstrated for both the mammalian and the yeast Tor
homologs [3, 16]. In addition to the kinase domain, the TOR proteins
contain an FKBP12–rapamycin binding (FRB) domain and a toxic effector
domain. Certain mutations in the FRB domain render Tor unable to bind
FKBP12–rapamycin and, as a consequence, confer dominant rapamycin
resistance [25, 76, 118]. Moreover, microinjection of the FRB domain into
human cells arrests cell growth in the G1 phase of the cell cycle [126].
Although, rapamycin blocks most Tor functions in living cells, the Tor
kinase activity is only partially inhibited by this drug in vitro, suggesting that
the FRB domain plays an important role in intact cells [3, 16]. Interestingly,
it has been suggested that phosphatidic acid interacts with the FRB domain,
blocking FRB and activating Tor [41]. However these studies await
further confirmation or a demonstration that they are conserved in other
organisms.
Extensive gene deletion analysis revealed that both the integrity of the
Tor protein as well as the kinase activity are required to provide Tor function
in yeast cells. Importantly, these studies led to the characterization of a 500
amino acid toxic domain in the central portion of the Tor protein that inhibits
cell growth when overexpressed [3]. BLAST searches with the yeast Tor
toxic domain revealed limited identity over a 240 amino acid region with the
PIK family members Atr, Rad3, Mei-41, and Atm [3]. In accord with these
results, recent sequence alignment analysis with multiple PIK family
members identified a shared domain named FAT (for Frap, Atm, Trrap) that
completely overlaps with the Tor toxic domain [14]. The dominant negative
effects of the FRB and toxic effector domains suggest that these domains
interact with important upstream regulators or downstream effectors of the
Tor signaling cascade; however, their exact in vivo functions remain to be
elucidated. The N-terminal region of the Tor proteins, amino acids 71–1147
in Tor1, contains multiple HEAT motifs and mediates association of Tor
with downstream targets and with cellular membranes [4, 11, 72]. Interest-
ingly, in mTor deletion of a C-terminal 30 amino acid region, which is
absent in the yeast Tor proteins, enhances kinase activity and signaling
[115]. This region includes Ser-2448, which was shown to be a target for
phosphorylation by protein kinase B, raising the possibility that in
mammalian cells Tor is subject to signaling by the PI3K/AKT pathway.
3 INITIATION OF TRANSLATION IS GOVERNED

BY TOR SIGNALING
The Tor pathway plays a central role in promoting translation in both

yeast and mammalian cells (Figure 2) [6, 9].
In mammalian cells, the Tor kinase has been implicated in
phosphorylating two proteins that function in translational control: PHAS-I
Figure 2. TOR controls translation in eukaryotes. In mammals and drosophila, mitogens and
nutrients inactivate Tsc1/Tsc2 to favor the GTP-bound form of Rheb. Rheb activates TOR
which in turn favors translation initiation by activating p70 S6 kinase as well as inactivating
the translational repressor PHAS-I. In yeast cells, TOR responds to nutrients and acts on
Eap1, a homolog of PHAS-I. Inactivation of TOR by nutrient limitation or rapamycin
treatment activates the Gcn2 kinase, resulting in eIF2α phosphorylation and inhibition of
overall translation. At this point it is not known if the yeast homolog of Rheb functions in the
TOR pathway or if the TOR–Gcn2 cross talk exists in mammals and drosophila.
and p70-S6 kinase [16, 19, 73, 98]. The PHAS-I protein forms a physical
complex with eIF4E, thereby blocking the ability of eIF4E to mediate
recognition of the 5′ CAP on mRNA and initiate recruitment of ribosomes to
the mRNA. Thus, PHAS-I is an inhibitor of translation initiation, and the
Tor pathway antagonizes the function of PHAS-I during culture conditions
that favor cell growth and division (reviewed in [44]). When nutrients are
limited, or rapamycin is present, the Tor signaling pathway is inactive or less
active, and PHAS-I forms a stable complex with eIF4E to prevent CAP
recognition and translation of the message. A central question in the field
has been whether or not the Tor kinase directly phosphorylates PHAS-I or
p70-S6 kinase in vivo [17]. This issue has been in part resolved by the
discovery of Tor interacting proteins that facilitate interaction with and
modification of these key substrate molecules (see below).
In yeast cells, rapamycin also leads to an inhibition of translation
initiation. Early studies implicated a different molecular target in yeast cells
as the translation initiation factor eIF4G, which physically interacts with the
5′ CAP-binding factor eIF4E and assists in the recruitment of additional
factors to the initiation complex. Rapamycin leads to degradation of eIF4G
in yeast cells and a profound block to translational initiation [10]. Two
possible distant homologs of the mammalian PHAS-I regulatory protein
have been identified in yeast and are called p20 and Eap1. Both proteins
physically interact with yeast eIF4E and thereby inhibit CAP dependent
translation initiation in a fashion analogous to the PHAS-I protein in
mammalian cells. While neither has been implicated as a direct substrate or
regulatory target of Tor in yeast cells, deletion of the EAP1 gene does confer
a modest level of resistance to rapamycin, indicating that liberating eIF4E
from this negative regulatory factor can enhance cell survival in the presence
of rapamycin [31]. It is thus quite remarkable that the role of the Tor
signaling pathway in translational control has been in large part conserved
from yeast to humans, even given that the direct molecular targets may be
distinct in these divergent organisms.
Recent studies have revealed a new and intriguing role for the Tor
pathway in regulating translation during amino acid limitation in yeast cells.
Amino acid limitation is normally sensed by the general control response, in
which the Gcn2 protein senses uncharged tRNAs and controls translation of
the mRNA encoding the global transcription factor Gcn4 [55]. The Gcn2
protein has two functional domains; a protein kinase and a domain that
shares identity with tRNA synthetases. Amino acid limitation leads to an
excess of uncharged tRNA molecules in the cell, which is sensed by the
Gcn2 kinase, leading to its dephosphorylation and activation. Gcn2 in turn
phosphorylates eIF2α, altering its activity in the cell to limit translation of
the vast majority of mRNA molecules but leads to enhanced translation of
the Gcn4 mRNA. The mRNA encoding Gcn4 is unique in that it contains
four upstream open reading frames, and during normal nutrient conditions,
ribosomes initiate translation at these upstream ORFs and rarely reach the
start codon for Gcn4 itself. In cells limited for amino acids, the action of
Gcn2 on eIF2α slows overall translation initiation allowing an increased
scanning rate and initiation to produce Gcn4. In turn, Gcn4 then activates the
expression of genes involved in amino acid biosynthesis, and a plethora of
other genes.
The first hints that the Tor pathway might impinge on the general control
response were studies demonstrating that rapamycin can stimulate
expression of a GCN4-lacZ reporter gene [122]. However, given previous
studies that implicate the Tor pathway in the control of amino acid
transporter stability in yeast, an alternative explanation was that rapamycin
was inducing the general control response via an indirect effect on amino
acid uptake. More recently, two studies have converged to implicate the Tor
pathway in a more direct action on the general control response [26, 70]. The
Gcn2 kinase is regulated by both uncharged tRNAs, which stimulate its
activity, and by a kinase that phosphorylates and inactivates Gcn2 in rich
medium. Inhibition of Tor by rapamycin leads to Ser 577 dephosphorylation
in Gcn2, activation of Gcn2, and increased eIF2α phosphorylation. The net
effect of rapamycin is then an increased level of Gcn4 expression and
activation of the general control response. The power of rapamycin to
activate Gcn2 also requires the ability of Gcn2 to bind uncharged tRNAs,
and thus provides an additional regulatory input to Gcn2 that allows the Tor
signaling cascade and limiting amino acids to be coordinately detected.
The physiological significance of this expanded regulatory network is at
present not clear, but it may allow enhanced induction of the general control
response when cells are more severely starved than simple amino acid
limitation affords. Alternatively, under conditions of amino acid starvation
Tor also regulates the general control response [87], especially since Tor
responds to amino acids in mammalian cells. These mechanisms allow
coordination between two global regulatory networks, the Tor and Gcn
pathways, which together function as both translational and transcriptional
regulatory cascades.
Whether or not the cross talk between these signaling pathways occurs in
mammalian cells is not yet known. It is intriguing however that the
mammalian Gcn2 protein has been identified as a key sensor to many
signaling inputs and controls the integrated stress response. Ron and
coworkers have proposed that mammalian cells utilize this ancient pathway
to integrate diverse stress signals to regulate gene expression of the targets of
the transcription factor ATF4, which is regulated in a manner analogous to
the yeast Gcn4 factor [48].
4 TOR REGULATES A COMPLEX

TRANSCRIPTIONAL PROGRAM
The recent development of genome wide arrays and the high specificity
of rapamycin to inhibit Tor function have aided the unraveling of a highly
complex transcriptional program regulated by the TOR pathway in yeast
cells (Figure 3). Earlier work had established a role for TOR signaling in the
synthesis of rRNA and tRNA [80, 135]. Although the targets of this
regulation are currently unknown, mutations that affect protein phosphatase
2A (PP2A) function result in alteration of both rRNA and tRNA gene
expression [119, 124]. Thus, an intriguing possibility is that regulation of
these genes by the TOR pathway is accomplished via PP2A.
Studies employing genome wide arrays revealed that ribosomal protein
(RP) genes expressed by PolII are repressed by the addition of rapamycin in
a manner that closely resembles nutritional limitation [21, 50, 68, 97]. The
Figure 3. TOR controls transcription of genes involved in growth and adaptation to starvation
and other stresses. In response to nutrients TOR activates the transcription of genes required
for ribosome biogenesis. At the same time TOR negatively regulates genes required for
adaptation to stress and nutrient scavenging. A common strategy for the repression of this
latter class of genes is through nuclear exclusion of the transcription factors. TOR inactivates
protein phosphatase 2A, which is required for nuclear localization of Msn2, Msn4, and Gln3
as well as translation of the message for the transactivator Gcn4. TOR also excludes the bZIP
transcription factors Rtg1 and Rtg3.
regulation of the RP genes by Tor signaling appears to be achieved via an

still unknown effector as two independent studies have demonstrated that
PP2A have little effect on regulating RP gene expression [40]. Most RP
gene promoters feature binding sites for the activator/repressor protein,
Rap1, and also for Abf1. Other transactivators, including Reb1, Gcr1, and
Gcr2 are proposed to cooperate with Rap1 and Abf1 in the regulation of
these genes (reviewed in [129]). In addition, the forkhead like transcription
factor Fhl1 and its activator Ifh1 as well as Yap5 have recently been shown
to bind to the majority of RP gene promoters and are likely to contribute to
RP gene expression [74]. Earlier work linked Rap1 mediated regulation of
RP genes to the cAMP and cell integrity pathways, however the precise
events in this regulation have not been elucidated [66, 89]. Recent studies
have identified components of the chromatin remodeling machinery that,
presumably in connection with Rap1 and Abf1, contribute to RP gene
expression in response to stress and nutrient signals and offer an opportunity
to fine tune expression of these genes by signal transduction cascades [100].
The histone acetylase Esa1 has been recently shown to be recruited to
RP gene promoters under conditions of active transcription [100]. We
demonstrated that rapamycin, like nutrient limitation, results in loss of Esa1
from the RP gene promoters, rapid histone deacetylation by the Rpd3–Sin3
complex, and decreased transcription [102]. In addition, our studies reveal
that rapamycin does not alter the occupancy of the Rap1 and Abf1 factors at
RP gene promoters [102] (see Figure 4). Evidently, the elucidation of the
detailed mechanisms by which Tor acts to promote occupancy of Esa1 and
associated proteins to the RP genes will require extensive analysis. Further
dissection of the regulatory events governing the RP genes is a challenging
problem and undoubtedly will unravel the molecular mechanism by which
nutrient and stress signals sensed by three signaling pathways converge at
RP promoters to regulate gene expression.
Recently, the Tor pathway was shown to control the expression of the
nitrogen catabolite repressed (NCR) genes, underscoring the central role of
Tor in nitrogen sensing [7, 11, 21, 50, 68]. The NCR genes are repressed by
the availability of preferred nitrogen sources, such as glutamine or ammonia,
and derepressed in the presence of limiting or poor nitrogen sources, such as
proline or urea (for review see [79]). Many NCR genes are regulated by a set
of GATA transcription factors, including the transactivators Gln3 and Nil1,
and by their inhibitor Ure2 [30, 79]. Under rich growth conditions both Gln3
and Ure2 are phosphoproteins and upon shift to poor nitrogen sources or
treatment with rapamycin, these factors are rapidly dephosphorylated [7, 21,
50]. More recent evidence suggests that Tor1, Gln3, and Ure2 form a
complex. Accordingly, it was shown that Gln3 binds to the HEAT repeats on
Figure 4. TOR signaling links nutrient sensing to histone acetylation to regulate gene
expression.
Tor1, and that Ure2 is recruited to the complex indirectly by its interaction
with Gln3 [11]. In the same study evidence was presented that Tor1 is
capable of directly phosphorylating Gln3 in vitro [11]. However, these
studies await independent confirmation.
In the phosphorylated state, Ure2 and Gln3 form a complex and
dephosphorylation of this complex by Tor inhibition promotes complex
dissociation and nuclear translocation of Gln3 [7, 11]. Indirect evidence
indicates that the rapid dephosphorylation of Ure2 and Gln3 is regulated by
Tor via protein phosphatase type 2A. Thus, rapamycin treatment or entry
into stationary growth phase induces the dissociation of the PP2A catalytic
subunit Sit4 from its regulatory subunit Tap42 [36]. Although earlier studies
suggested that Tap42 ejected an inhibitory role on Sit4 for the expression of
the NCR genes, two independent studies have concluded that both of these
factors are required for expression of these genes [7, 22, 41]. Finally it was
shown that nuclear import of Gln3 requires the importin Srp1 while
Crm1/Xpo is necessary for the nuclear export [23].
A prominent set of genes that responds to nitrogen catabolite repression

encodes enzymes that operate the glyoxalate and TCA cycles. Expression of
these genes is directed by the Rtg1–Rtg3 heterodimeric transcription factor
and their regulators Rtg2 and Mks1. When yeast cells are grown in poor
nitrogen sources, such as urea, these cycles are upregulated to afford
production of α-ketoglutarate that in turn is utilized in the de novo synthesis
of glutamate and glutamine. The TOR signaling pathway controls the
activity of the Rtg proteins in a manner strikingly similar to that of the Gln3
protein [68]. It has been shown that Rtg2 and Mks1 form a complex in the
cytosol which negatively regulates the activity of the Rtg1–Rtg3 dimer
[37, 113, 114]. Tor inhibition by glutamate or glutamine depletion, or by
rapamycin, results in dephosphorylation of Mks1 and Rtg3 and nuclear
translocation of Rtg1 and Rtg3 [37, 113]. The importin Msn5 was shown to
be required for the export of Rtg1 and Rtg3 as msn5 mutations result in
permanent nuclear localization of these factors. Interestingly, an msn5
mutation does not cause activation of the target genes for Rtg1 and Rtg3;
instead, addition of rapamycin is still required for Rtg directed gene
expression. These studies illustrate that TOR signaling functions at two
levels to regulate Rtg directed transcription: first, to trigger the nuclear
localization of the transactivators, and second at a later step to achieve
proper gene expression.
A growing body of data supports the notion that the control of nuclear
localization may be a general mechanism by which the TOR kinases regulate
transcription. Tor signaling also influences the mechanisms by which the
transcription factors Msn2 and Msn4 are sequestered in the cytoplasm
through interaction with the negative regulators Bmh1 and Bmh2 [7].
Treatment with rapamycin results in expression of the STRE genes by 2 h
postexposure, although in this case the activation of the target genes is not
precisely correlated with the rapid nuclear localization of Msn2 and Msn4
that was reported by Beck and Hall, suggesting additional levels of control
for these genes (as is the case with Rtg controlled genes) [21, 50]. In a more
recent study, rapamycin was found to have no effect on nuclear import of
Msn2, but instead delayed nuclear export [40]. In addition, loss of Tap42
function in temperature sensitive mutant strains shifted to the nonpermissive
temperature led to an induction of the stress response genes, implicating a
role for Tap42 in the pathway [40]. This finding is again in contrast to an
earlier report based on studies with the tap42-11 mutant allele that were
interpreted as excluding a role for Tap42 in the Tor pathway leading to
control of the stress response genes [8]. Taken together, the current view is
that the Tor–Tap42–Sit4 pathway controls the nuclear export of the Msn2
and Msn4 transcription factors that regulate expression of the stress response
genes. These results may help explain the marked delay in induction of the
STRE genes by rapamycin. Taken together these studies reveal that the Tor
pathway controls both nuclear import and nuclear export of key regulatory
transcription factors.
The Tor pathway has also been shown to control gene silencing at
subtelomeric regions. These studies capitalized on the Saccharomyces
toolbox-ordered sets of gene deletions. Zheng and coworkers screened
~4000 viable gene deletions for altered sensitivity to rapamycin [24]. One of
these mutants, sir3, displayed an increased resistance to rapamycin. Studies
of this phenomenon revealed that Tor negatively regulates MPK1, the MAP
kinase of the cell integrity pathway. MPK1 in turn phosphorylates Sir3 to
result in increased expression of subtelomeric genes involved in the repair of
cell wall damage [1]. Once again the powerful tools available in yeast were
able to quickly identify an important mode of cross talk between two
different signal transduction pathways.
Finally, observations in mammalian cells suggest that mTOR and PP2A
control the activity of the STAT3 protein in a fashion similar to the control
of Gln3 activity in yeast. STAT3 is retained in the cytoplasm until activation
by the JAK kinases that act by phosphorylating tyrosine residues in response
to cytokines. Phosphorylation of serine and threonine residues (most notably
Ser727) also occurs but it is not yet clear if phosphorylation positively or
negatively regulates the ability of STAT3 to activate transcription. Inhibition
of PP2A activity increases STAT3 phosphorylation on serine and threonine
residues and results in a concominant exclusion of STAT3 from the nucleus
[133]. Reportedly STAT3 directed transcription is blocked by rapamycin
treatment and in vitro phosphorylation studies indicate that mTOR may
phosphorylate this protein directly [134]. While these studies convincingly
show the involvement of mTOR and PP2A in regulating the Ser727
phosphorylation status of STAT3, the physiological consequences of this
phosphorylation remain unknown. Importantly, constitutive activation of
STAT3 results in malignant transformation and cancer.
5 TOR SIGNALS VIA A CONSERVED PROTEIN

PHOSPHATASE COMPLEX
The Tor signaling cascade functions to control the activity of the

PP2A-related catalytic subunits Sit4, Pph21, and Pph22 and their associated
regulatory factor Tap42. Studies by Arndt and colleagues first demonstrated
that the Sit4 catalytic subunit and related proteins physically interact with
the essential Tap42 protein [36, 77]. Most interestingly, the Sit4–Tap42
complex disassociates when cells are limited for nutrients or exposed to
rapamycin, and certain TAP42 mutant alleles, such as tap42-11, confer
partial resistance to rapamycin in yeast cells. Subsequent studies have

demonstrated that Sit4 plays a key role in controlling the Ure2–Gln3
regulatory complex that governs expression of the NCR genes [7], and also
more recently in the stress response pathway governed by Msn2/4 [40].
Although Tap42 was originally thought to only function as a negative factor,
studies implicate Tap42 as playing both positive and negative signaling roles
downstream of the Tor kinase. Earlier studies demonstrated that shift of the
tap42-11 conditional allele to the nonpermissive temperature arrested cell
growth but did not lead to an induction of the NCR gene response, indicating
that Tap42 must play a role in addition to simply inhibiting Sit4 in the
signaling cascade [21]. More recently, this finding was confirmed employing
additional temperature sensitive Tap42 conditional mutants [40]. Tap42 may
play a role in targeting Sit4 to different locations in the cell, or to different
substrates. One report has suggested that Tap42 might be a direct substrate
of the Tor kinases [60], but this finding has not yet been independently
confirmed. An additional layer of regulation on the Tap42–Sit4 complex was
introduced by the identification of a Tap42 interacting protein, named Tip41
[59]. Tor signaling promotes the phosphorylation of Tip41 and stabilization
of the Tap42–Sit4 complex. Rapamycin treatment results in Tip41
dephosphorylation, mediated at least in part by Sit4, which favors Tip41
association with Tap42 and presumably leads to the release of an activated
form of Sit4.
In mammalian cells, the Tap42 homolog is the α4 protein, which forms
physical complexes with protein phosphatase catalytic subunits related to the
yeast Sit4 protein. The role of the Tor pathway in controlling PP2A activity
in mammalian cells is at present controversial. Moreover, it is not clear that
the stability of the PP2A–α4 complex is regulated by Tor signaling in the
same fashion as the yeast Sit4–Tap42 complex and some findings consistent
with a direct effect of mTor on PP2A itself have been reported [85, 86, 95].
A tempting model then is that the Tor proteins which contain a C-
terminal protein kinase domain signals in conjunction with the associated
phosphatases Sit4–Tap42, Pph21–Tap42, and Pph22–Tap42. This regulatory
network may therefore function to more rapidly and dynamically control the
phosphorylation state of target substrates than either the kinase or the phos-
phatase alone could achieve. By coupling the activity of the two, the Tor
cascade could rapidly mediate the dephosphorylation of proteins that are
also substrates of the Tor kinase activity. This would lead to very rapid
alterations in cell physiology in response to changing nutrient conditions,
much more rapid than could be achieved by inhibiting only the activity of
the kinase alone since protein turnover and new synthesis would then be
required to lead to the production of dephosphorylated target molecules.
6 REGULATION OF PROTEIN DEGRADATION

AND AUTOPHAGY
Autophagy is a response to nitrogen, carbon, or sulfur starvation in which

indiscriminate portions of the cytosol are engulfed by a membrane to form
autophagic bodies. These autophagic bodies then fuse with lysosomes or, in
yeast, with the vacuole, and proteins are degraded, resulting in the release of
free amino acids which are in turn recycled by the cell. Tor inactivation by
rapamycin treatment induces autophagy and the evidence indicates that Tor
regulation is exerted at the transcriptional as well as a posttranscriptional
level [90]. The pathway that mediates autophagy is operated by the APG
genes [42, 90]. Among the genes activated by Tor inhibition and regulated
via activation of Gln3 are the APG genes: APG1, AGP3, AGP5, APG7,
APG8, APG12, and APG13 (reviewed in [99]). In addition, Tor regulates the
interaction of Apg13 with Apg1 to result in activation of the Apg1 protein
kinase required for autophagy [61]. Thus, nutrient starvation or rapamycin
treatment results in dephosphorylation of Apg13, leading to Apg13–Apg1
complex formation, Apg1 activation, and autophagy [61]. In addition to the
Tor pathway two other nutrient signaling pathways operated by the Snf1 and
Pho85 kinases have recently been shown to regulate autophagy [128]. It will
be of considerable interest to establish if these regulatory cascades converge
at the Apg1 activation step.
7 TOR REGULATES POLARIZATION

OF THE ACTIN CYTOSKELETON
In yeast, Tor1 and Tor2 have overlapping rapamycin-sensitive functions

in the control of cell growth, translation, and transcription. In addition, Tor2
has a unique and essential rapamycin-insensitive function involving
polarization of the actin cytoskeleton [110, 111, 138]. Establishment and
regulation of the actin cytoskeleton is controlled in part by small GTP-
binding proteins encoded by the RHO1, RHO2, and CDC42 genes which are
highly conserved throughout evolution. Activity of these proteins is
governed by a switch between the GDP-bound inactive, and GTP-bound
active states. Genetic analysis of the Tor2-unique function has led to a
model whereby Tor2 controls this switch. Lethal tor2 mutations can be
suppressed by mutations in the SAC7 gene, which encodes a GTPase
activating protein (GAP) for Rho1 and Rho2, by overexpression of Rho1 or
Rho2 themselves, or by Rom2, a GTP exchange factor (GEF) that promotes
the GTP (active) state of the Rho proteins [110]. Furthermore, alleles of
TOR2 that are defective for the Tor2-unique function have been
characterized and were found to cause a G2/M cell cycle arrest consisting of
cells with unpolarized actin [53]. Importantly, Rho1 is known to activate the
PKC signaling pathway in yeast and overexpression of Pkc1 rescues alleles
of tor2 defective in the Tor2-unique function. In mammalian cells, mTOR
regulates the activity of PKC but it is as yet unclear how similar this
connection will be to that of yeast. A link between the actin cytoskeleton
and mTOR has not yet been established in mammalian cells and PKC
activation by mTOR is rapamycin-sensitive in mammals, whereas the Tor2-
unique function is rapamycin-insensitive in yeast [138]. Recently, careful
studies have revealed a rapamycin-sensitive and cell cycle dependent
polarization of the yeast actin cytoskeleton [127]. These studies suggest
that Tor1 may have a greater role in actin polarization than previously
appreciated.
8 TOR REGULATES MICROTUBULE FUNCTION
A second function for the Tor kinases at the G2/M phase of the cell cycle
is beginning to emerge. Recent reports suggest that the Tor kinases play a
role in the assembly or function of microtubules [12, 29]. This appears to be
a rapamycin-sensitive function that is shared between yeast Tor1 and Tor2
and that is conserved from yeast to mammals [12]. Genetic and biochemical
evidence in yeast suggests that this process involves the kinesin-related
proteins (KRPs), which are important for spindle–pole separation at
anaphase [29]. Consistent with this, rapamycin treatment causes defects in
chromosome and nuclear segregation in both yeast and mammalian cells
[12, 29].
9 TOR INTERACTING PROTEINS AND NUTRIENT

SENSING
The downstream elements of the Tor signaling pathway likely involve

the recently identified Tor partner proteins, Raptor/Kog1, Avo1/2/3, and
Lst8/GβL [47, 63, 64, 75, 130]. Kog1/Raptor is conserved from yeast to
humans and plays an essential function in yeast as part of a complex of
proteins (called the Torc1 complex) involving the Tor1 [130] or Tor2
kinases [75] and Lst8. Interestingly, Lst8 contains 7 WD-40 repeats and is
also conserved in mammals, where it is known as G • L. Some evidence
suggests that nutrient limitation promotes the formation of the mTor–raptor
complex in mammalian cells resulting on mTOR inhibition. Furthermore, it
has been proposed that raptor facilitates Tor association with substrates
[63]; Hara, 2002 #3227]. In particular, the mammalian raptor protein can
bind to the Tor substrates p70 S6 kinase and PHAS-I via a previously
defined domain known as the TOR signaling motif (TOS) [91, 108, 109].
The TOS domain allows PHAS-I to dock onto the mTor/Raptor protein
complex, and PHAS-I is then phosphorylated to promote release and
activation of eIF4E and cell growth. In yeast cells, a second complex, called
the Torc2 complex, can be detected containing Tor2, Lst8, and the Avo1,
Avo2, and Avo3 proteins [75, 130]. FKBP12–rapamycin can physically
associate with the Torc1 complex but not with the Torc2 complex,
suggesting that this second complex mediates the rapamycin-insensitive role
of Tor2 in controlling polarization of the actin cytoskeleton. If and how any
of these newly discovered Tor associated proteins communicate nutrient
sensing via Tor to PP 2A or any of its associated subunits is as yet unknown.
10 TOR, THE TUBEROUS SCLEROSIS PROTEINS,

RHEB, AND NUTRIENT SENSING
A new regulatory cascade that is conserved from budding and fission

yeast to humans involving the tuberous sclerosis gene products and the small
G protein Rheb has emerged that may link activation of the Tor pathway to
nutrients. The mammalian TSC1 and TSC2 genes encode the proteins
hamartin and tuberin, which physically interact to form a protein complex
that functions as a tumor suppressor [69]. Mutations in the TSC1 or TSC2
genes are associated with both renal cell carcinomas and the formation of
benign hamartomas. Studies conducted concurrently in drosophila and
mammalian cells first linked the functions of both the mammalian and the
fly Tsc1/2 homologs to the activation of the p70 S6 kinase cascade that is
controlled by Tor [43, 58]. The Tsc1 and Tsc2 proteins were found to
negatively regulate Tor signaling, and to form a physical complex with the
drosophila Tor homolog known as dTOR. Mutants lacking either Tsc1/2
protein exhibited a Tor-dependent increase in p70 S6 kinase activity, and the
Tsc1/2 proteins also control the activity of the Tor regulated protein PHAS-I.
A series of recent studies converged to identify the small G protein Rheb as
the target of the Tsc2 GTPase activating protein (GAP) function. These
findings suggests that Rheb is an intermediary in the control of Tor activity
by the Tsc1/2 protein complex [57, 107, 120, 137].
Earlier studies in budding and fission yeast and pathogenic fungi have
implicated the Rheb signaling in nitrogen sensing and the control of amino
acid transport. Budding yeast mutants lacking Rheb were found to be viable
but hypersensitive to amino acid analogs, such as canavanine, and also
exhibited an increase in amino acid uptake [121]. Fission yeast mutants
lacking the Rheb homolog Rhb1 were also viable but exhibited defects in
nutrient sensing and arrested in G1 more rapidly than wild-type cells in
response to nitrogen limitation. Furthermore, in the rhb1 mutant cells several
nitrogen starvation induced genes were found to be expressed under rich
nitrogen conditions [78]. Homologs of the mammalian Tsc1 and Tsc2
proteins in fission yeast are known to regulate the proper targeting and
function of amino acid permeases, a role consistent with related studies on
the Rheb homolog [81]. The Aspergillus fumigatus Rheb homolog, rhbA, is
induced in response to nitrogen starvation. The rhbA mutant cells are viable
but their growth was compromised in poor nitrogen sources, nitrogen rich
conditions failed to repress their asexual development, and they were
hypersensitive to rapamycin [93, 94]. Taken together, these findings are
consistent with a role for the small G protein Rheb in nutrient sensing in
budding yeast, fission yeast, and pathogenic fungi and suggest Rheb might
play a globally conserved role in activating the Tor kinases in response to
certain nutritional cues. Rheb is not essential for growth in any of these
fungi, in contrast to Tor, and thus Rheb likely represents one of several
redundant regulatory inputs.
The key questions that remain are how the Tor kinases sense nutrients
and how they communicate this information to their downstream effectors.
Two recent studies suggest that the mTOR kinase is activated in response to
two small molecules: ATP and PA [35, 41]. Phosphatidic acid is produced
by phospholipase D and has been reported to bind mTOR and thereby
stimulate its kinase activity, in vitro and in vivo [41]. Phospholipase D is
conserved in yeasts, where it plays a known role in promoting morphological
changes to pheromone and in meiosis [46, 103]. As yet, no role has been
adduced for phospholipase D in controlling Tor signaling in yeasts. Recent
studies reveal that Pld1 is required for filamentous growth in Candida
albicans [56], and our studies have shown that the Tor pathway is necessary
for filamentous growth in S. cerevisiae, C. albicans, and C. neoformans [34].
Finally, Thomas and colleagues have suggested that the low intrinsic Km of
mTOR for ATP poises the enzyme to serve as a general ATP sensor for the
cell [35]. Thus PA and ATP may contribute to the activation of mTOR via or
in addition to nutrient sensing mechanisms.
11 SUMMARY REMARKS AND PERSPECTIVES
From its original discovery in 1975 as an antifungal natural product with

an undesired immunosuppressive effect, rapamycin emerged as a powerful
immunosuppressive drug that received FDA approval in 1999. Rapamycin
also inhibits growth and proliferation of a variety of other cells types,
including smooth muscle cells and several neoplastic cell lines. The recent
finding that cardiac stents impregnated with rapamycin can significantly
reduce restenosis, and that rapamycin can inhibit growth of a variety of
different tumors, will lead to new clinical indications for this novel natural
product. In light of these significant clinical advances, a more detailed
understanding of the targets of rapamycin and their function in growth
promoting pathways should provide considerable insight into the molecular
basis of therapeutic action and support the development of additional and
novel targets for therapeutic intervention.
Of particular interest and importance is the clinical utility of rapamycin
as a novel chemotherapy agent. A flurry of recent reports have identified
unique tumor cell lines that are exquisitely sensitive to growth inhibition by
the mTOR inhibitor rapamycin and its analogs. Particularly, in cells and
mice lacking the tumour suppressor PTEN, a lipid phosphatase that controls
signaling via the PI3 kinase pathway, rapamycin inhibits p70 S6 kinase
activity and oncogenesis [28, 45, 83, 88, 96, 116]. These findings apply to a
broad range of tumor types, including tumors of the immune system
(multiple myeloma), common solid organ tumors (breast, prostate), and less
common but devastating solid organ tumors (glioblastoma). There are a
variety of phase II and phase III trials that are ongoing now, and the years
ahead hold excitement and promise of a significant impact for rapamycin in
a multitude of diverse clinical applications.
Much of our current understanding of rapamycin targets and mechanism
of action stems directly from genetic and molecular studies conducted in the
model yeast S. cerevisiae. In summary, these contributions include:
1. Identification of the rapamycin binding protein FKBP12

2. Demonstration that FKBP12 is not essential for growth but is required
for rapamycin action
3. Identification of Tor1 and Tor2 as the targets of the FKBP12–rapamycin
complex
4. Definition of functional domains of the Tor kinases
5. Elucidation of the role of the Tor pathway in translation
6. Finding that Tor controls PP 2A
7. Illumination of the Tor pathway role in nutrient sensing
8. Discovery that the Tor pathway controls transcriptional programs for
cell growth
9. Application of a “chemical genomics” approach to identify novel Tor
functions
10. Identification of novel Tor binding proteins conserved from yeast to
humans (Kog1/raptor and Lst8/GβL)
These findings highlight the vast potential of simple, genetically

tractable model systems to inform our understanding of drug targets and
mechanisms of action in more complex systems such as mammalian cells.
These advances also highlight the power of yeast genetics to define the
direct targets of rapamycin as the conserved FKBP12 and Tor proteins. The
challenges that lie ahead are: (1) to define the upstream regulatory pathways
that allow Tor to sense nutrients, (2) to elucidate the detailed mechanisms by
which Tor controls the translational and transcriptional machineries to effect
developmental and transcriptional programs required to respond to altered
growth conditions to promote cell growth and proliferation; and (3) to bring
these molecular insights to bear on clinical applications that will improve
health and prolong life.
REFERENCES
1. Ai, W., P. G. Bertram, C. K. Tsang, T. F. Chan, and X. F. Zeng. 2002. Regulation of
subtelomeric silencing during stress response. Mol. Cell. 10:1295–1305.
2. Alarcon, C. M., M. E. Cardenas, and J. Heitman. 1996. Mammalian RAFT1 kinase
domain provides rapamycin-sensitive TOR function in yeast. Genes Dev. 10:279–288.
3. Alarcon, C. M., J. Heitman, and M. E. Cardenas. 1999. Protein kinase activity and
identification of a toxic effector domain of the target of rapamycin TOR proteins in
yeast. Mol. Biol. Cell 10:2531–2546.
4. Andrade, M. A., and P. Bork. 1995. HEAT repeats in the Huntington’s disease
protein. Nat. Genet. 11:115–116.
5. Baker, H., A. Sidorowicz, S. N. Sehgal, and C. Venzina. 1978. Rapamycin (AY-
22,989), a new antifungal antibiotic. III. In vitro and in vivo evaluation. J. Antibiot.
31:539–545.
6. Barbet, N. C., U. Schneider, S. B. Helliwell, I. Stansfield, M. F. Tuite, and M. N.
Hall. 1996. TOR controls translation initiation and early G1 progression in yeast. Mol.
Biol. Cell 7:25–42.
7. Beck, T., and M. N. Hall. 1999. The TOR signalling pathway controls nuclear
localization of nutrient-regulated transcription factors. Nature 402:689–692.
8. Beck, T., A. Schmidt, and M. N. Hall. 1999. Starvation induces vacuolar targeting and
degradation of the tryptophan permease in yeast. J. Cell Biol. 146:1227–1238.
9. Beretta, L., A.-C. Gingras, Y. V. Svitkin, M. N. Hall, and N. Sonenberg. 1996.
Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation
of translation. EMBO J. 15:658–664.
10. Berset, C., H. Trachsel, and M. Altmann. 1998. The TOR (target of rapamycin) signal
transduction pathway regulates the stability of translation initiation factor eIF4G in the
yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 95:4264–4269.
11. Bertram, P. G., J. H. Choi, J. Carvalho, W. Ai, C. Zeng, T. F. Chan, and X. F.
Zheng. 2000. Tripartite regulation of Gln3p by TOR, Ure2p, and phosphatases. J. Biol.
Chem. 275:35727–35733.
12. Bonatti, S., M. Simmili, A. Galli, P. Bagnato, S. Pigullo, R. H. Schiestl, and A.
Abbondandolo. 1998. Inhibition of the Mr 70,000 S6 kinase pathway by rapamycin
results in chromosome malsegregation in yeast and mammalian cells. Chromosoma

107:498–506.
13. Borel, J. F. 1976. Comparative study of in vitro and in vivo drug effects on cell-
mediated cytotoxicity. Immunology 31:631–641.
14. Bosotti, R., A. Isacchi, and E. L. Sonnhammer. 2000. FAT: a novel domain in PIK-
related kinases. Trends Biochem. Sci. 25:225–227.
15. Brown, E. J., M. W. Albers, T. B. Shin, K. Ichikawa, C. T. Keith, W. S. Lane, and
S. L. Schreiber. 1994. A mammalian protein targeted by G1-arresting rapamycin-
receptor complex. Nature 369:756–759.
16. Brunn, G. J., C. C. Hudson, A. Sekulic, J. M. Williams, J. Hosoi, P. J. Houghton,
J. J. C. Lawrence, and R. T. Abraham. 1997. Phosphorylation of the translational
repressor PHAS-I by the mammalian target of rapamycin. Science 277:99–101.
17. Burnett, P. E., R. K. Barrow, N. A. Cohen, S. H. Snyder, and D. M. Sabatini. 1998.
RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc.
18. Cafferkey, R., P. R. Young, M. M. McLaughlin, D. J. Bergsma, Y. Koltin, G. M.
Sathe, L. Faucette, W.-K. Eng, R. K. Johnson, and G. P. Livi. 1993. Dominant
missense mutations in a novel yeast protein related to mammalian phosphatidylinositol
3-kinase and VPS34 abrogate rapamycin cytotoxicity. Mol. Cell Biol. 13:6012–6023.
19. Calvo, V., C. M. Crews, T. A. Vik, and B. E. Bierer. 1992. Interleukin 2 stimulation
of p70 S6 kinase activity is inhibited by the immunosuppressant rapamycin. Proc. Natl.
Acad. Sci. USA 89:7571–7575.
20. Cardenas, M. E., M. C. Cruz, M. D. Poeta, N. Chung, J. R. Perfect, and
J. Heitman. 1999. Antifungal activities of antineoplastic agents: Saccharomyces
cerevisiae as a model system to study drug action. Clin. Microbiol. Rev. 12:583–611.
21. Cardenas, M. E., N. S. Cutler, M. C. Lorenz, C. J. D. Como, and J. Heitman. 1999.
The TOR signaling cascade regulates gene expression in response to nutrients. Genes
Dev. 13:3271–3279.
22. Cardenas, M. E., and J. Heitman. 1995. FKBP12-rapamycin target TOR2 is a
vacuolar protein with an associated phosphatidylinositol-4 kinase activity. EMBO J.
14:5892–5907.
23. Carvalho, J., P. G. Bertram, S. R. Wente, and X. F. Zheng. 2001. Phosphorylation
regulates the interaction between Gln3p and the nuclear import factor Srp1p. J. Biol.
Chem. 276:25359–25365.
24. Chan, T. F., J. Carvalho, L. Riles, and X. F. Zheng. 2000. A chemical genomics
approach toward understanding the global functions of the target of rapamycin protein
(TOR). Proc. Natl. Acad. Sci. USA 97:13227–13232.
25. Chen, J., X.-F. Zheng, E. J. Brown, and S. L. Schreiber. 1995. Identification of an
11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-
associated protein and characterization of a critical serine residue. Proc. Natl. Acad. Sci.
USA 92:4947–4951.
26. Cherkasova, V. A., and A. G. Hinnebusch. 2003. Translational control by TOR and
TAP42 through dephosphorylation of eIF2alpha kinase GCN2. Genes Dev. 17:859–872.
27. Chiu, M. I., H. Katz, and V. Berlin. 1994. RAPT1, a mammalian homolog of yeast
Tor, interacts with the FKBP12/rapamycin complex. Proc. Natl. Acad. Sci. USA
91:12574–12578.
28. Choe, G., S. Horvath, T. F. Cloughesy, K. Crosby, D. Seligson, A. Palotie, L. Inge,
B. L. Smith, C. L. Sawyers, and P. S. Mischel. 2003. Analysis of the
phosphatidylinositol 3′-kinase signaling pathway in glioblastoma patients in vivo.
Cancer Res. 63:2742–2746.
29. Choi, J. H., N. R. Adames, T. F. Chan, C. Zeng, J. A. Cooper, and X. F. Zeng. 2000.
TOR signaling regulates microtubule structure and function. Curr. Biol. 10:861–864.
30. Cooper, T. G. 2002. Transmitting the signal of excess nitrogen in Saccharomyces
cerevisiae from the Tor proteins to the GATA factors: connecting the dots. FEMS
Microbio. Rev. 26:223–238.
31. Cosentino, G. P., T. Schmelzle, A. Haghighat, S. B. Helliwell, M. N. Hall, and
N. Sonenberg. 2000. Eap1p, a novel eukaryotic translation initiation factor 4E-
associated protein in Saccharomyces cerevisiae. Mol. Cell Biol. 20:4604–4613.
32. Cruz, M. C., L. M. Cavallo, J. M. Görlach, G. Cox, J. R. Perfect, M. E. Cardenas,
and J. Heitman. 1999. Rapamycin antifungal action is mediated via conserved
complexes with FKBP12 and TOR kinase homologs in Cryptococcus neoformans. Mol.
Cell. Biol. 19:4101–4112.
33. Cruz, M. C., A. L. Goldstein, J. Blankenship, M. Del Poeta, J. R. Perfect, J. H.
McCusker, Y. L. Bennani, M. E. Cardenas, and J. Heitman. 2001. Rapamycin and
less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus
neoformans via FKBP12-dependent inhibition of TOR. Antimicrob. Agents Chemother.
45:3162–3170.
34. Cutler, N. S., X. Pan, J. Heitman, and M. E. Cardenas. 2001. The TOR signal
transduction cascade controls cellular differentiation in response to nutrients. Mol. Biol.
Cell 12:4103–3113.
35. Dennis, P. B., A. Jaeschke, M. Saitoh, B. Fowler, S. C. Kozma, and G. Thomas.
2001. Mammalian TOR: a homeostatic ATP sensor. Science 294:1102–1105.
36. Di Como, C. J., and K. T. Arndt. 1996. Nutrients, via the Tor proteins, stimulate the
association of Tap42 with type 2A phosphatases. Genes Dev. 10:1904–1916.
37. Dilova, I., C. Y. Chen, and T. Powers. 2002. Mks1 in concert with TOR signaling
negatively regulates RTG target gene expression in S. cerevisiae. Curr. Biol. 12:389–
395.
38. Dumont, F. J., M. R. Melino, M. J. Staruch, S. L. Koprak, P. A. Fischer, and N. H.
Sigal. 1990. The immunosuppressive macrolides FK-506 and rapamycin act as
reciprocol antagonists in murine T-cells. J. Immunol. 144:1418–1424.
39. Dumont, F. L., M. J. Staruch, S. L. Koprak, M. R. Melino, and N. H. Sigal. 1990.
Distinct mechanisms of suppression of murine T cell activation by the related
macrolides FK506 and rapamycin. J. Immunol. 144:251–258.
40. Duvel, K., A. Santhanam, S. Garrett, L. Schneper, and J. R. Broach. 2003. Multiple
roles of Tap42 in mediating rapamycin-induced transcriptional changes in yeast. Mol.
Cell 11:1467–1478.
41. Fang, Y., M. Vilella-Bach, R. Bachmann, A. Flanigan, and J. Chen. 2001.
Phosphatidic acid-mediated mitogenic activation of mTOR signaling. Science
294:1942–1945.
42. Funakoshi, T., A. Matsuura, T. Noda, and Y. Ohsumi. 1997. Analyses of APG13
gene involved in autophagy in yeast, Saccharomyces cerevisiae. Gene 192:207–213.
43. Gao, X., Y. Zhang, P. Arrazola, O. Hino, T. Kobayashi, R. S. Yeung, B. Ru, and D.
Pan. 2002. Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling. Nat.
Cell. Biol. 4:699–704.
44. Gingras, A. C., B. Raught, and N. Sonenberg. 2001. Regulation of translation
initiation by FRAP/mTOR. Genes Dev. 15:807–826.
45. Grunwald, V., L. DeGraffenried, D. Russel, W. E. Friedrichs, R. B. Ray, and
M. Hidalgo. 2002. Inhibitors of mTOR reverse doxorubicin resistance conferred by
PTEN status in prostate cancer cells. Cancer Res. 62:6141–6145.
46. Hairfield, M. L., A. B. Ayers, and J. W. Dolan. 2001. Phospholipase D1 is required

for efficient mating projection formation in Saccharomyces cerevisiae. FEM Yeast Res.
1:225–232.
47. Hara, K., Y. Maruki, X. Long, K. Yoshino, N. Oshiro, S. Hidayat, C. Tokunaga,
J. Avruch, and K. Yonezawa. 2002. Raptor, a binding partner of target of rapamycin
(TOR), mediates TOR action. Cell 110:177–189.
48. Harding, H. P., I. Novoa, Y. Zhang, H. Zeng, R. Wek, M. Schapira, and D. Ron.
2000. Regulated translation initiation controls stress-induced gene expression in
mammalian cells. Mol. Cell 6:1099–1108.
49. Harding, M. W., A. Galat, D. E. Uehling, and S. L. Schreiber. 1989. A receptor for
the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature 341:758–
760.
50. Hardwick, J. S., F. G. Kuruvilla, J. K. Tong, A. F. Shamji, and S. L. Schreiber.
1999. Rapamycin-modulated transcription defines the subset of nutrient-sensitive
signaling pathways directly controlled by the Tor proteins. Proc. Natl. Acad. Sci. USA
96:14866–14870.
51. Heitman, J., N. R. Movva, and M. N. Hall. 1991. Targets for cell cycle arrest by the
immunosuppressant rapamycin in yeast. Science 253:905–909.
52. Heitman, J., N. R. Movva, P. C. Hiestand, and M. N. Hall. 1991. FK506-binding
protein proline rotamase is a target for the immunosuppressive agent FK506 in
53. Helliwell, S. B., I. Howald, N. Barbet, and M. N. Hall. 1998. TOR2 is part of two
related signaling pathways coordinating cell growth in Saccharomyces cerevisiae.
Genetics 148:99–112.
54. Helliwell, S. B., P. Wagner, J. Kunz, M. Deuter-Reinhard, R. Henriquez, and M. N.
Hall. 1994. TOR1 and TOR2 are structurally and functionally similar but not identical
phosphatidylinositol kinase homologues in yeast. Mol. Biol. Cell 5:105–118.
55. Hinnebusch, A. G., and K. Natarajan. 2002. Gcn4p, a master regulator of gene
expression, is controlled at multiple levels by diverse signals of starvation and stress.
Eukaryot. Cell 1:22–32.
56. Hube, B., D. Hess, C. A. Baker, M. Schaller, W. Schafer, and J. W. Dolan. 2001.
The role and relevance of phospholipase D1 during growth and dimorphism of Candida
albicans. Microbiology 147:879–889.
57. Inoki, K., Y. Li, T. Xu, and K. L. Guan. 2003. Rheb GTPase is a direct target of TSC2
GAP activity and regulates mTOR signaling. Genes Dev. 17:1829–1834.
58. Inoki, K., Y. Li, T. Zhu, J. Wu, and K. L. Guan. 2002. TSC2 is phosphorylated and
inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4:648–657.
59. Jacinto, E., B. Guo, K. T. Arndt, T. Schmelzle, and M. N. Hall. 2001. TIP41 interacts
with TAP42 and negatively regulates the TOR signaling pathway. Mol. Cell 8:1017–
10126.
60. Jiang, Y., and J. R. Broach. 1999. Tor proteins and protein phosphatase 2A
reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J. 18:2782–2792.
61. Kamada, Y., T. Funakoshi, T. Shintani, K. Nagano, M. Ohsumi, and Y. Ohsumi.
2000. Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J. Cell
Biol. 150:1507–1513.
62. Keith, C. T., and S. L. Schreiber. 1995. PIK-related kinases: DNA repair,
recombination, and cell cycle checkpoints. Science 270:50–51.
63. Kim, D. H., D. D. Sarbassov, S. M. Ali, J. E. King, R. R. Latek, H. Erdjument-
Bromage, P. Tempst, and D. M. Sabatini. 2002. mTOR interacts with raptor to form a
nutrient-sensitive complex that signals to the cell growth machinery. Cell 110:163–175.
64. Kim, D. H., D. Sarbassov dos, S. M. Ali, R. R. Latek, K. V. Guntur, H. Erdjument-

Bromage, P. Tempst, and D. M. Sabatini. 2003. GbetaL, a positive regulator of the
rapamycin-sensitive pathway required for the nutrient-sensitive interaction between
raptor and mTOR. Mol. Cell 11:895–904.
65. Kino, T., H. Hatanaka, S. Miyata, N. Inamura, H. Hishiyama, T. Yajima, T. Goto,
M. Okuhara, M. Kohsaka, H. Aoki, and T. Ochiai. 1987. FK-506, a novel
immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-
506 in vitro. J. Antibiot. 40:1256–1265.
66. Klein, C., and K. Struhl. 1994. Protein kinase A mediates growth-regulated expression
of yeast ribosomal protein genes by modulating RAP1 transcriptional activity. Mol. Cell
Biol. 14:1920–1928.
67. Koltin, Y., L. Faucette, D. J. Bergsma, M. A. Levy, R. Cafferkey, P. L. Koser, R. K.
Johnson, and G. P. Livi. 1991. Rapamycin sensitivity in Saccharomyces cerevisiae is
mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding
protein. Mol. Cell Biol. 11:1718–1723.
68. Komeili, A., K. P. Wedaman, E. K. O’Shea, and T. Powers. 2000. Mechanism of
metabolic control. Target of rapamycin signaling links nitrogen quality to the activity of
the Rtg1 and Rtg3 transcription factors. J. Cell Biol. 151:863–878.
69. Krymskaya, V. P. 2003. Tumour suppressors hamartin and tuberin: intracellular
signalling. Cell Signal. 15:729–739.
70. Kubota, H., T. Obata, K. Ota, T. Sasaki, and T. Ito. 2003. Rapamycin-induced
translational derepression of GCN4 mRNA involves a novel mechanism for activation
of the eIF2 alpha kinase GCN2. J. Biol. Chem. 278:20457–20460.
71. Kunz, J., R. Henriquez, U. Schneider, M. Deuter-Reinhard, N. R. Movva, and
M. N. Hall. 1993. Target of rapamycin in yeast, TOR2, is an essential
phosphatidylinositol kinase homolog required for G1 progression. Cell 73:585–596.
72. Kunz, J., U. Schneider, I. Howald, A. Schmidt, and M. N. Hall. 2000. HEAT repeats
mediate plasma membrane localization of Tor2p in yeast. J. Biol. Chem. 275:37011–
37020.
73. Kuo, C. J., J. Chung, D. F. Fiorentino, W. M. Flanagan, J. Blenis, and G. R.
Crabtree. 1992. Rapamycin selectively inhibits interleukin-2 activation of p70 S6
kinase. Nature 358:70–73.
74. Lee, T. I., N. J. Rinaldi, F. Robert, D. T. Odom, Z. Bar-Joseph, G. K. Gerber,
N. M. Hannett, C. T. Harbison, C. M. Thompson, I. Simon, J. Zeitlinger, E. G.
Jennings, H. L. Murray, D. B. Gordon, B. Ren, J. J. Wyrick, J. B. Tagne, T. L.
volkert, E. Fraenkel, D. K. Gifford, and R. A. Young. 2002. Transcriptional
regulatory networks in Saccharomyces cerevisiae. Science 298:799–804.
75. Loewith, R., E. Jacinto, S. Wullschleger, A. Lorberg, J. L. Crespo, D. Bonenfant,
W. Oppliger, P. Jenoe, and M. N. Hall. 2002. Two TOR complexes, only one of
which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell
10:457–468.
76. Lorenz, M. C., and J. Heitman. 1995. TOR mutations confer rapamycin resistance by
preventing interaction with FKBP12-rapamycin. J. Biol. Chem. 270:27531–27537.
77. Luke, M. M., F. Della Seta, C. J. Di Como, H. Sugimoto, R. Kobayashi, and K. T.
Arndt. 1996. The SAP, a new family of proteins, associate and function positively with
the SIT4 phosphatase. Mol. Cell Biol. 16:2744–2755.
78. Mach, K. E., K. A. Furge, and C. F. Albright. 2000. Loss of Rhb1, a Rheb-related
GTPase in fission yeast, causes growth arrest with a terminal phenotype similar to that
caused by nitrogen starvation. Genetics 155:611–622.
79. Magasanik, B., and C. A. Kaiser. 2002. Nitrogen regulation in Saccharomyces

cerevisiae. Gene 290:1–18.
80. Mahajan, P. B. 1994. Modulation of transcription of rRNA genes by rapamycin. Int. J.
Immunopharmacol. 16:711–721.
81. Matsumoto, S., A. Bandyopadhyay, D. J. Kwiatkowski, U. Maitra, and
T. Matsumoto. 2002. Role of the Tsc1-Tsc2 complex in signaling and transport across
the cell membrane in the fission yeast Schizosaccharomyces pombe. Genetics 161:1053–
1063.
82. Menand, B., T. Desnos, L. Nussaume, F. Berger, D. Bouchez, C. Meyer, and
C. Robaglia. 2002. Expression and disruption of the Arabidopsis TOR (target of
rapamycin) gene. Proc. Natl. Acad. Sci. USA 99:6422–6427.
83. Mita, M. M., A. Mita, and E. K. Rowinsky. 2003. Mammalian target of rapamycin: a
new molecular target for breast cancer. Clin. Breast Cancer 4:126–137.
84. Morice, M. C., P. W. Serruys, J. E. Sousa, J. Fajadet, E. Ban Hayashi, M. Perin,
A. Colombo, G. Schuler, P. Barragan, G. Guagliumi, F. Molnar, and R. Falotico.
2002. A randomized comparison of a sirolimus-eluting stent with a standard stent for
coronary revascularization. N. Engl. J. Med. 346:1773–1780.
85. Murata, K., J. Wu, and D. L. Brautigan. 1997. B cell receptor-associated protein
alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein
phosphatase 2A. Proc. Natl. Acad. Sci. USA 94:10624–10629.
86. Nanahoshi, M., T. Nishiuma, Y. Tsujishita, K. Hara, S. Inui, N. Sakaguchi, and
K. Yonezawa. 1998. Regulation of protein phosphatase 2A catalytic activity by alpha4
protein and its yeast homolog Tap42. Biochem. Biophys. Res. Commun. 251:520–526.
87. Natarajan, K., M. R. Meyer, B. M. Jackson, D. Slade, C. Roberts, A. G.
Hinnebusch, and M. J. Marton. 2001. Transcriptional profiling shows that Gcn4p is a
master regulator of gene expression during amino acid starvation in yeast. Mol. Cell
Biol. 21:4347–4368.
88. Neshat, M. S., I. K. Mellinghoff, C. Tran, B. Stiles, G. Thomas, R. Petersen,
P. Frost, J. J. Gibbons, H. Wu, and C. L. Sawyers. 2001. Enhanced sensitivity of
PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc. Natl. Acad. Sci. USA
98:10314–10319.
89. Neuman-Silberberg, F. S., S. Bhattacharya, and J. R. Broach. 1995. Nutrient
availability and the RAS/cyclic AMP pathway both induce expression of ribosomal
protein genes in Saccharomyces cerevisiae but by different mechanisms. Mol. Cell Biol.
15:3187–3196.
90. Noda, T., and Y. Ohsumi. 1998. Tor, a phosphatidylinositol kinase homologue,
controls autophagy in yeast. J. Biol. Chem. 273:3963–3966.
91. Nojima, H., C. Tokunaga, S. Eguchi, N. Oshiro, S. Hidayat, K. Yoshino, K. Hara,
N. Tanaka, J. Avruch, and K. Yonezawa. 2003. The mammalian target of rapamycin
(mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through
their TOR signaling (TOS) motif. J. Biol. Chem. 278:15461–15464.
92. Oldham, S., J. Montagne, T. Radimerski, G. Thomas, and E. Hafen. 2000. Genetic
and biochemical characterization of dTOR, the Drosophila homolog of the target of
rapamycin. Genes Dev. 14:2689–2694.
93. Panepinto, J. C., B. G. Oliver, T. W. Amlung, D. S. Askew, and J. C. Rhodes. 2002.
Expression of the Aspergillus fumigatus rheb homologue, rhbA, is induced by nitrogen
starvation. Fungal Genet. Biol. 36:207–214.
94. Panepinto, J. C., B. G. Oliver, J. R. Fortwendel, D. L. Smith, D. S. Askew, and J. C.
Rhodes. 2003. Deletion of the Aspergillus fumigatus gene encoding the Ras-related
protein RhbA reduces virulence in a model of invasive pulmonary aspergillosis. Infect.

Immun. 71:2819–2826.
95. Peterson, R. T., B. N. Desai, J. S. Hardwick, and S. L. Schreiber. 1999. Protein
phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of
FKBP12-rapamycin-associated protein. PNAS 96:4438–4442.
96. Podsypanina, K., R. T. Lee, C. Politis, I. Hennessy, A. Crane, J. Puc, M. Neshat,
H. Wang, L. Yang, J. Gibbons, P. Frost, V. Dreisbach, J. Blenis, Z. Gaciong,
P. Fisher, C. Sawyers, L. Hedrick-Ellenson, and R. Parsons. 2001. An inhibitor of
mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/- mice. Proc.
Natl. Acad. Sci. USA 98:10320–10325.
97. Powers, T., and P. Walter. 1999. Regulation of ribosome biogenesis by the rapamycin-
sensitive TOR-signaling pathway in Saccharomyces cerevisiae. Mol. Biol. Cell 10:987–
1000.
98. Price, D. J., J. R. Grove, V. Calvo, J. Avruch, and B. E. Bierer. 1992. Rapamycin-
induced inhibition of the 70-kilodalton S6 protein kinase. Science 257:973–977.
99. Reggiori, F., and D. J. Klionsky. 2002. Autophagy in the eukaryotic Cell Eukaryot.
Cell 1:11–21.
100. Reid, J. L., V. R. Iyer, P. O. Brown, and K. Struhl. 2000. Coordinate regulation of
yeast ribosomal protein genes is associated with targeted recrutiment of Esa1 histone
acetylase. Mol. Cell 6:1297–1307.
101. Rohde, J., J. Heitman, and M. E. Cardenas. 2001. The TOR kinases link nutrient
sensing to cell growth. J. Biol. Chem. 276:9583–9586.
102. Rohde, J. R., and M. E. Cardenas. 2003. The Tor pathway regulates gene expression
by linking nutrient sensing to histone acetylation. Mol. Cell Biol. 23:629–635.
103. Rose, K., S. A. Rudge, M. A. Frohman, A. J. Morris, and J. Engebrecht. 1995.
Phospholipase D signaling is essential for meiosis. Proc. Natl. Acad. Sci. USA
92:12151–12155.
104. Rotonda, J., J. J. Burbaum, H. K. Chan, A. I. Marcy, and J. W. Becker. 1993.
Improved calcineurin inhibition by yeast FKBP12-drug complexes. J. Biol. Chem.
268:7607–7609.
105. Sabatini, D. M., H. Erdjument-Bromage, M. Lui, P. Tempst, and S. H. Snyder.
1994. RAFT1: A mammalian protein that binds to FKBP12 in a rapamycin-dependent
fashion and is homologous to yeast TORs. Cell 78:35–43.
106. Sabers, C. J., M. M. Martin, G. J. Brunn, J. M. Williams, F. J. Dumont, G.
Wiederrecht, and R. T. Abraham. 1995. Isolation of a protein target of the FKBP12-
rapamycin complex in mammalian cells. J. Biol. Chem. 270:815–822.
107. Saucedo, L. J., X. Gao, D. A. Chiarelli, L. Li, D. Pan, and B. A. Edgar. 2003. Rheb
promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell
Biol. 5:566–571.
108. Schalm, S., D. C. Fingar, D. M. Sabatini, and J. Blenis. 2003. TOS motif-mediated
raptor binding regulates 4E-BP1 multisite phosphorylation and function. Curr. Biol.
13:797–806.
109. Schalm, S. S., and J. Blenis. 2002. Identification of a conserved motif required for
mTOR signaling. Curr. Biol. 12:632–639.
110. Schmidt, A., M. Bickle, T. Beck, and M. N. Hall. 1997. The yeast
phosphatidylinositol kinase homolog TOR2 activates RHO1 and RHO2 via the
exchange factor ROM2. Cell 88:531–542.
111. Schmidt, A., J. Kunz, and M. N. Hall. 1996. TOR2 is required for organization of the
actin cytoskeleton in yeast. Proc Natl Acad Sci USA 93:13780–13785.
112. Sehgal, S. N., H. Baker, and C. Vezina. 1975. Rapamycin (AY-22,989), a new
antifungal antibiotic. II. Fermentation, isolation and characterization. J. Antibiot.
28:727–732.
113. Sekito, T., Z. Liu, J. Thronton, and R. A. Butow. 2002. RTG-dependent
mitochondria-to-nucleus signaling is regulated by MKS1 and is linked to formation of
yeast prion [URE3]. Mol. Biol. Cell 13:795–804.
114. Sekito, T., J. Thornton, and R. A. Butow. 2000. Mitochondria-to-nuclear signaling is
regulated by the subcellular localization of the transcription factors Rtg1p and Rtg3p.
Mol. Biol. Cell 11:2103–2115.
115. Sekulic, A., C. C. Hudson, J. L. Homme, P. Yin, D. M. Otterness, L. M. Karnitz,
and R. T. Abraham. 2000. A direct linkage between the phosphoinositide 3-kinase-
AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated
and transformed cells. Cancer Res. 60:3504–3513.
116. Shi, Y., J. Gera, L. Hu, J. H. Hsu, R. Bookstein, W. Li, and A. Lichtenstein. 2002.
Enhanced sensitivity of multiple myeloma cells containing PTEN mutations to CCI-779.
Cancer Res. 62:5027–5034.
117. Siekierka, J. J., S. H. Y. Hung, M. Poe, C. S. Lin, and N. H. Sigal. 1989. A cytosolic
binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase
activity but is distinct from cyclophilin. Nature 341:755–757.
118. Stan, R., M. M. McLaughlin, R. T. Cafferkey, R. K. Johnson, M. Rosenberg, and
G. P. Livi. 1994. Interaction between FKBP12-Rapamycin and TOR involves a
conserved serine residue. J. Biol. Chem. 269:32027–32030.
119. Stettler, S., N. Chiannilkulchai, S. H.-L. Denmat, D. Lalo, F. Lacroute, A. Sentenac,
and P. Thuriaux. 1993. A general suppressor of RNA polymerase I, II and III
mutations in Saccharomyces cerevisiae. Mol. Gen. Genet. 239:169–176.
120. Tabancay, A. P., Jr., C. L. Gau, I. M. Machado, E. J. Uhlmann, D. H. Gutmann,
L. Guo, and F. Tamanoi. 2003. Identification of dominant negative mutants of Rheb
GTPase and their use to implicate the involvement of human Rheb in the activation of
p70S6K. J. Biol. Chem. [Epub ahead of print].
121. Urano, J., A. P. Tabancay, W. Yang, and F. Tamanoi. 2000. The Saccharomyces
cerevisiae Rheb G-protein is involved in regulating canavanine resistance and arginine
uptake. J. Biol. Chem. 275:11198–11206.
122. Valenzuela, L., C. Aranda, and A. Gonzalez. 2001. TOR modulates GCN4-dependent
expression of genes turned on by nitrogen limitation. J. Bacteriol. 183:2331–2334.
123. van Duyne, G. D., R. F. Standaert, P. A. Karplus, S. L. Schreiber, and J. Clardy.
1991. Atomic structure of FKBP-FK506, an immunophilin-immunosuppressant
complex. Science 252:839–842.
124. van Zyl, W., W. Huang, A. A. Sneddon, M. Stark, S. Camier, M. Werner,
C. Marck, A. Sentenac, and J. R. Broach. 1992. Inactivation of the protein
phosphatase 2A regulatory subunit A results in morphological and transcriptional
defects in Saccharomyces cerevisiae. Mol. Cell Biol. 12:4946–4959.
125. Vezina, C., A. Kudelski, and S. N. Sehgal. 1975. Rapamycin (AY-22,989), a new
antifungal antibiotic. I. Taxonomy of the producing Streptomycete and isolation of the
active principle. J. Antibiot. 28:721–726.
126. Vilella-Bach, M., P. Nuzzi, Y. Fang, and J. Chen. 1999. The FKBP12-rapamycin-
binding domain is required for FKBP12-rapamycin-associated protein kinase activity
and G1 progression. J. Biol. Chem. 274:4266–4272.
127. Wang, H., and Y. Jiang. 2003. The Tap42-protein phosphatase type 2A catalytic
subunit complex is required for cell cycle-dependent distribution of actin in yeast. Mol.
Cell. Biol. 23:3116–3125.
128. Wang, Z., W. A. Wilson, M. A. Fujino, and P. J. Roach. 2001. Antagonistic controls
of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-
activated protein kinase, and the cyclin-dependent kinase Pho85p. Mol. Cell. Biol.
21:5742–5752.
129. Warner, J. R. 1999. The economics of ribosome biosynthesis in yeast. Trends
Biochem. Sci. 24:437–440.
130. Wedaman, K. P., A. Reinke, S. Anderson, J. Yates, J. M. McCaffery, and
T. Powers. 2003. Tor kinases are in distinct membrane-associated protein complexes in
Saccharomyces cerevisiae. Mol. Biol. Cell 14:1204–1220.
131. Weisman, R., and M. Choder. 2001. The fission yeast TOR homolog, tor1+, is
required for the response to starvation and other stresses via a conserved serine. J. Biol.
Chem. 276:7027–7032.
132. Wiederrecht, G., L. Brizuela, K. Elliston, N. H. Sigal, and J. J. Siekierka. 1991.
FKB1 encodes a nonessential FK506-binding protein in Saccharomyces cerevisiae and
contains regions suggesting homology to the cyclophilins. Proc. Natl. Acad. Sci. USA
88:1029–1033.
133. Woetmann, A., M. Nielsen, S. T. Christensen, J. Brockdorff, K. Kaltoft, A. M.
Engel, S. Skov, C. Brender, C. Geisler, A. Svejgaard, J. Rygaard, V. Leick, and
N. Odum. 1999. Inhibition of protein phosphatase 2A induces serine/threonine
phosphorylation, subcellular redistribution, and functional inhibition of STAT3. Proc.
Natl. Acad. Sci. USA 96:10620–10625.
134. Yokogami, K., S. Wakisaka, J. Avruch, and S. A. Reeves. 2000. Serine
phosphorylation and maximal activation of STAT3 during CNTF signaling is mediatd
by the rapamycin target mTOR. Curr. Biol. 10:47–50.
135. Zaragoza, D., A. Ghavidel, J. Heitman, and M. C. Schultz. 1998. Rapamycin induces
the G0 program of transcriptional repression in yeast by interfering with the TOR
signaling pathway. Mol. Cell Biol. 18:4463–4470.
136. Zhang, H., J. P. Stallock, J. C. Ng, C. Reinhard, and T. P. Neufeld. 2000. Regulation
of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 14:2712–
2724.
137. Zhang, Y., X. Gao, L. J. Saucedo, B. Ru, B. A. Edgar, and D. Pan. 2003. Rheb is a
direct target of the tuberous sclerosis tumour suppressor proteins. Nat. Cell Biol. 5:578–
581.
138. Zheng, X.-F., D. Fiorentino, J. Chen, G. R. Crabtree, and S. L. Schreiber. 1995.
TOR kinase domains are required for two distinct functions, only one of which is
inhibited by rapamycin. Cell 82:121–130.
Chapter 14
USE OF YEAST AS A MODEL SYSTEM
FOR IDENTIFYING AND STUDYING
ANTICANCER DRUGS
Jun O. Liu1 and Julian A. Simon2

1
Departments of Pharmacology and Neuroscience, Johns Hopkins School of Medicine,
Baltimore, MD 21205; 2Divisions of Clinical Research, Basic Sciences and Human Biology,
Fred Hutchinson Cancer Research Center, Seattle, WA 98109
1 INTRODUCTION
As signified in part by the publication of the current book, yeast is

becoming a more widely used system for studying small molecule–protein
interactions in general and anticancer agents in particular. There are a
number of reasons for this surging popularity. As a eukaryote, it contains
many, if not most, of the essential genes involved in the control of cell cycle,
cellular structure, metabolism, and stress responses that are conserved from
yeast to humans, making it possible to use yeast to study the function of
those genes. For this reason, what is learned in a yeast cell can often been
extrapolated into a mammalian counterpart. Unlike mammalian cells, yeast
is relatively easy to grow and manipulate, with its natural habitat being the
harsh environment of the wild. Moreover, decades of research on both the
biochemistry and genetics of the budding yeast has accumulated a wealth of
information about yeast and made available a number of tools that allows for
the manipulation of genes in yeast, including knockout and ectopic
expression of both endogenous and exogenous genes.
Several recent advances have greatly facilitated the use of yeast as a
powerful system to study small molecules. They include the advent of the
yeast two-hybrid system, the sequencing of the genome of the budding yeast
Saccharomyces cerevisiae and the development of DNA chip and
375
© 2007 Springer.
376 J. O. Liu and J. A. Simon
microarrays that allow for the monitoring of the expression of the complete
yeast genome. In this chapter, we will cover three different ways yeast has
been used to facilitate the identification, characterization, and mechanistic
studies of anticancer drugs. These include (1) the use of yeast as a surrogate
system to identify and study anticancer drugs; (2) the use of yeast as self-
contained microvessles to perform large-scale parallel analysis of interactions
between proteins and small ligands; and (3) the use of the yeast transcriptional
profiling for validating the molecular target for given drugs. For the most part,
we have selected examples from our own laboratories. We thus have
attempted to be more illustrative than comprehensive in this chapter.
2 USE OF YEAST AS A SURROGATE SYSTEM TO

IDENTIFY AND STUDY ANTICANCER DRUGS
Many of the essential genes, including those involved in the control of

cell cycle, cell structure, transcription and basic metabolism, are conserved
between yeast and humans. It is those essential genes involved in the
regulation of cell proliferation that are often mutated in human cancer cells,
leading to uncontrolled growth. For those genes, yeast represents an ideal
surrogate system to identify and study anticancer drugs. In some instances, a
conserved gene between yeast and human can be studied even if the
fundamental physiological functions differ, as long as there is a suitable
phenotypic readout upon inhibition of the gene function. We will use two
examples to illustrate this point, one being a family of anti-angiogenic
natural products, fumagillin and their common protein target, the type 2
methionine aminopeptidase (MetAP2) and the other being the identification
of the NAD+-dependent histone deacetylase Sir2p in yeast involved in
silencing, aging, and cell cycle control.
2.1 Studies of the fumagillin family of anti-angio-

genicinhibitors for the type 2 methionine
aminopeptidase (MetAP2)
Fumagillin and ovalicin (Figure 1) are a family of structurally related

natural products that were serendipitously discovered as potent angiogenesis
inhibitors [7, 25]. A structural analog of fumagillin, known as TNP-470 or
AGM-1470, has been undergoing testing for treatment of a variety of
cancers. The molecular mechanism of inhibition of angiogenesis by this
class of inhibitors was elucidated through the detection, isolation, and
identification of the molecular target for these inhibitors. A 67 kDa protein
was detected by photoaffinity labeling using photosensitive probes derived
Chapter 14: Use of Yeast as a Model System 377
from fumagillin. The target protein was isolated using affinity chro-
matography and identified by mass spectrometry as the MetAP2 [16, 41].
Figure 1. The chemical structures of fumagillin, TNP-470, and ovalicin.
Methionine aminopeptidases represent a family of highly conserved

proteins from bacterial to humans [26, 33]. Their primary function is to
remove the N-terminal methionine from newly synthesized proteins for
subsequent posttranslational modifications. In prokaryotes, there is only one
copy of methionine aminopeptidase gene. Knockout of the gene has a lethal
phenotype, indicating that methionine aminopeptidase is an essential gene in
bacteria [4]. In eukaryotes, however, there are two different genes for
methionine aminopeptidases, hence the names type 1 (MetAP1) and type 2
(MetAP2) [1, 28]. Despite their different N-terminal extension sequences,
the catalytic domains of MetAP1 and MetAP2, including the five amino acid
residues involved in the formation of a bimetallic active site, are highly
conserved (Figure 2). Genetic analyses in yeast revealed that the two
methionine aminopeptidase genes can be deleted individually without
affecting viability, although they lead to a slow growth phenotype [5, 28].
Double knockout of the two MetAP genes, however, is lethal, suggesting
that the two genes encoding methionine aminopeptidases are functionally
redundant and together they are essential for yeast growth [28].
Figure 2. Schematic comparison of the domain structures of eukaryotic MetAP1 and MetAP2.
The five amino acid residues in the catalytic domain are shown with single letter code.
Since the MetAP2 was isolated from mouse embryo extract via affinity
chromatography using the fumagillin or ovalicin as an affinity ligand, a
major question was the specificity of fumagillin for MetAP2, given the
extremely high sequence similarity between MetAP1 and MetAP2 [1, 28].
Using the wild type and map1 and map2 mutant strain of yeast, it was found
that only the map1 mutant strain of yeast is sensitive to inhibition by
fumagillin or ovalicin, providing strong evidence that the drugs are highly
specific for MetAP2 (Figure 3) [16, 41]. In addition to demonstrating the
extraordinary specificity of fumagillin for MetAP2, these studies also raised
the interesting question of how inhibition of MetAP2 alone led to cell cycle
inhibition of human endothelial cells. Although the map1 mutant has not
been further exploited, it is apparent that the mutant can be used for
screening of small molecule libraries to identify novel inhibitors of MetAP2.
Figure 3. Specific inhibition of MetAP2 by TNP-470 in yeast. The wild type, map1 (yeast
MetAP1) and map2 (yeast MetAP2) mutant strains were plated on YEPD plates containing
either dimethylsulfoxide as a control or 50 nM of either TNP-470 or ovalicin. The plates were
incubated at 30ºC for 4 days before they were photographed.
2.2 Identification and studies of Sir2p inhibitors in yeast
Sir2p (Sir stands for Silent Information Regulator) is a founding member

of a family of NAD+-dependent protein deacetylases that are conserved
from bacteria to humans [11]. Eukaryotic Sir2 homologs have been shown to
be involved in the regulation of transcription, cell cycle control, repair of
DNA damage, and aging [13, 18]. At the molecular level, Sir2p and its
family members regulate protein–protein interactions through removal of
acetyl groups from lysine residues. Regulation of chromatin structure is
accomplished by deacetylation of histone proteins within targeted chromatin
regions [18]. In yeast, Sir2p is known to be responsible for silencing
transcription of the ribosomal DNA array (rDNA), the silent mating-type
loci HMR and HML as well as regions near telomeres [24, 27, 42]. A human
homolog of the yeast Sir2p, hSIR2, was recently shown to deacetylate the
tumor suppressor gene p53, downregulating the transcriptional activity of
p53 and repressing p53-dependent apoptosis of tumor cells in response to
DNA damage and oxidative stress [34, 43]. Thus, inhibitors of the human
Sir2p homologs may be useful in potentiating cytotoxic chemotherapy by
enhancing the p53-depenent apoptosis and cell cycle arrest.
That Sir2p in yeast is responsible for silencing the chromatin in proximity
of telomeres was demonstrated by the repression of a reporter gene
integrated into the telomere proximal region. It was shown that the uracil
biosynthsesis gene URA3 is silenced when it is in close proximity of a
telomere [15]. The deletion of the yeast SIR2 gene leads to the derepression
of the transcription of the URA3 gene, as well as other genes in normally
silent loci.
To identify small molecule inhibitors of the yeast Sir2p as a first step
toward identifying inhibitors for its human counterpart, two similar
approaches were taken, both involving the exploitation of the URA3 gene as
a reporter placed within a Sir2p-dependent silent locus [2, 17]. It was
expected that inhibition of yeast Sir2p would lead to the derepression of the
silenced URA3. The two groups used slightly different readouts, one
exploiting the URA3 auxotroph [2] and the other utilizing the URA3-
dependent toxicity by a small molecule 5-fluoroorotic acid [17]. The
advantage of the use of the yeast URA3 auxotroph is that it is a positive
selection, eliminating potential false positive hits that may be toxic to yeast
by mechanisms other than inhibition of Sir2p. Upon screening a library of
6,000 chemical compounds for those that enabled the growth of the yeast
with a telomeric URA3 gene in the absence of uracil, 11 hits emerged. One
out of the 11 hits was confirmed with a second reporter yeast strain with the
TRP1 gene integrated at another silent locus growing in the absence of
tryptophan. The hit was named splitomicin (Figure 4).
Figure 4. Structure of splitomicin (A) and its effect on yeast responsiveness to α factor (B).
The helo of cells indicates that splitomicin induced loss of sensitivity of yeast to α factor-
induced silencing in the mating type loci.
The yeast system not only enabled the identification of hits, but also
facilitated the subsequent verification of splitomicin as a relatively selective
inhibitor for yeast Sir2p and identification of residues in Sir2p that are
critical for its binding to splitomicin [2]. Yeast contains four SIR2 homologs,
HST1-4 (HST stands for Homologue of Sir Two). To determine whether
splitomicin is specific for Sir2p, the transcriptional profiles of wild- type
yeast cells treated with splitomicin and the isogenic yeast strains with
deletion of SIR2 and each of its four homologs were obtained and compared
to each other. It was found that the majority of the changes (88%) in
transcription as a consequence of splitomicin treatment are seen in the sir2
mutant yeast strain. Interestingly, a small number of changes in transcription
(9%) were identical to those seen in the hst1 mutant strain. In contrast, there
was no overlap in transcriptional profile changes between splitomicin-treated
yeast cells and the hst2-4 mutant strains. These results clearly indicated that
splitomicin is selective toward Sir2p and Hst1p, but does not interact with
the other members of the Sir2p family of proteins. PCR-mediated mutagenesis
of the SIR2 open reading frame was used to identify three drug-resistant
mutants of Sir2p using similar reporter screens in yeast. The drug-resistant
mutations all occurred in a region of the protein that is part of the putative
active site for Sir2 and is highly conserved between yeast Sir2p and Hst1p,
suggesting that splitomicin is likely to interact with the active sites of both
Sir2p and Hst1p.
In an independent study, a similar approach was used to screen for the
yeast Sir2p inhibitors with the yeast strain containing a telomeric copy of
URA3 gene [17]. Three small molecule inhibitors of Sir2p were identified,
which also inhibited the human SIRT2 protein with similar potencies.
It is noteworthy that the screens employed in the studies would not have
been possible without the use of yeast. Even though the human SIRT2 is
known, its celluar function is largely unknown and it is much more difficult
to devise a similar reporter system that would allow for a phenotypic screen
for the human SIRT2 inhibitors directly.
3 YEAST CELLS AS MINIATURIZED

BIOCHEMICAL REACTION VESSELS
FOR ASSESSING DRUG–PROTEIN
INTERACTIONS IN LARGE NUMBERS
The ability of yeast cells to take up one or more plasmids and express the
specific proteins encoded by the plasmids makes it possible to use individual
yeast cells as independent “containers” for analyzing the interaction between
the proteins expressed in the yeast and the small molecules administered.
Given the ease of culturing yeast cells and the ease with which exogenous
plasmids can be introduced into yeast, large-scale parallel analyses are
feasible. Thus, each yeast cell can be turned into a self-contained reaction
vessel to assay the interaction between a ligand and its target.
One of the major challenges is to develop a suitable readout that allows
for quick and easy detection of the binding between a drug and its protein
target in yeast cells. One type of reporter system couples a ligand-binding
event with the transcription of a specific reporter gene introduced into the
yeast. Another reporter system links the ligand–protein binding event with
the reconstitution of an enzyme that can be easily detected. In this chapter,
we have chosen a few systems that have the potential of being adapted for
such an application or have been used to assess drug-protein interactions.
3.1 Yeast two-hybrid system and its variants
Since its development, the yeast two-hybrid system has become one of the
most powerful tools for uncovering new protein–protein interactions [6, 8].
The yeast two-hybrid system exploits an intrinsic property of eukaryotic
transcription factors to function with two separable and portable domains, a
DNA-binding domain and a transcriptional activation domain. By splitting a
transcription factor into two separate proteins and fusing into each two
interacting proteins, it is possible to reconstitute the transcription activity.
In theory, the yeast two-hybrid system is ideal for identifying inhibitors
for protein–protein interactions. Unfortunately, due to the relatively large
binding interface often associated with protein–protein interactions [32], it is
in general difficult to discover small molecules that directly compete for a
protein–protein interaction. Nevertheless, there do exist a few successful

examples of small molecule antagonists for protein–protein interactions
identified through the two-hybrid system [23, 44].
Some protein–protein interactions are mediated by defined protein
modules and a short, contigous oligopeptide. These include the binding of
SH2 and SH3 domains to phosphotyrosine-containing peptides or proline-
rich peptides, respectively [37]. The detection of an SH3 domain and its
target sequence can be easily established using the yeast two-hybrid system.
The binding of SH2 domains to their target requires a phosphorylation step,
which usually occurs in mammalian cells upon activation of specific
signaling pathways but is absent in yeast. To circumvent the problem, a new
version of the yeast two-hybrid system was developed in which a tyrosine
kinase is coexpressed in yeast, along with the fusion proteins containing
the SH2 domain and the phosphorylated peptide and the system is dubbed the
yeast tribrid system [36]. Thus, the expressed tyrosine-containing peptide
will be phosphorylated by the expressed kinase, enabling it to form a
complex with the SH2 domain fusion protein. This system seems to be
ideally suited for high-throughput screen to identify inhibitors of the SH2
domain–ligand interaction as well as inhibitors for the kinase. It remains to
be seen how successful such screening system is for identifying inhibitors
for specific SH2 domains.
3.2 Yeast three-hybrid system
The yeast three-hybrid system is adapted from the yeast two-hybrid

system, except that the DNA-binding domain and the transactivation domain
is bridged by two ligand–receptor pairs, instead of two interacting proteins in
the two-hybrid system (Figure 5). Thus, the receptor for one ligand is fused
to the DNA-binding domain and the receptor for a second ligand is fused to
the transcriptional activation domain. In addition to the two hybrid fusion
proteins, the system requires a third hybrid small molecule, a dimer of two
ligands. Thus, when the two hybrid fusion proteins are expressed in yeast,
the small hybrid ligand would enter the cells by diffusion and form a ternary
complex with the two fusion proteins, reconstituting a functional trans-
cription factor to drive reporter gene expression, similar to the yeast
two-hybrid system.
Figure 5. The basic components of yeast three-hybrid system. The triangle and semicircle
represent two different chemical ligands. UAS, upstreat activating sequences.
The three-hybrid system can be conceptually divided into two ligand–

receptor pairs. One ligand–receptor pair can be prefixed, consisting of a
known ligand and a known receptor. For the second ligand–receptor pair,
either the ligand or the receptor can be unknown, which can be identified
through screening with the three-hybrid system. In the case of a known
ligand with unknown receptor, the ligand can be covalently linked to the
prefixed known ligand and the resulting hybrid ligand can be used in screen
a cDNA library fused to the transcriptional activation domain to identify the
potential protein target for the ligand. By the same token, if the receptor is
known but a suitable ligand is needed, a library of ligands all tethered to the
prefixed known ligand can be screened to identify a lead that is capable of
binding to the receptor target. These two complementary applications should
significantly facilitate both ligand and receptor discovery processes.
The idea was first reduced to practice by employing the hormone-binding
domain of the rat glucocorticoid receptor and human FKBP, along with a
synthetic dimer of their respective ligands, dexamethasone and FK506 [29].
The hormone-binding domain of the glucocorticoid receptor was fused with
the LexA DNA-binding domain and the human FKBP was fused with the
B42 transcriptional activation domain [19]. When the two fusion proteins
were expressed in yeast, the addition of the dexamethasone-FK506 hybrid
ligand did not lead to activation of the LacZ reporter gene. It turned out that
the wild-type rat glucocorticoid recceptor, when expressed in yeast, suffers
from a significant decrease in binding affinity for dexamethasone. To
increase the affinity of the hormone-binding domain of the rat glucocorticoid

receptor for dexamethasone, several mutants were created which were
known to enhance the affinity of the glucocorticoid receptor expressed in
yeast for dexamethasone [3, 12]. One of the double mutants, a C656G and
F620S double mutant, when employed to the three-hybrid setting, yielded
strong reporter gene activity in the presence of the dexamethasone-FK506
dimer. The reporter gene activation was shown to be completely dependent
on the interaction between the two ligands and the receptors, as the three-
hybrid reporter gene activation is sensitive to inhibition by free FK506.
When the expression of the FKBP-B42 fusion protein was turned off by
switching the carbon source from galactose to glucose, the activation of the
reporter gene was also abrogated. These control experiments unambiguously
demonstrated that the activation of reporter gene was a result of the three-
hybrid ligand–protein interactions.
The three-hybrid system was used to screen a cDNA library to isolate
cDNAs that encode for FK506-binding proteins with the dexamethasone-
FK506 as a bait. Similar to the yeast two-hybrid screen, the three-hybrid
screen is rapid and convenient. Unlike the typical two-hybrid screen,
additional controls are available for the three-hybrid screen that ensures the
elimination of false positive clones (Figure 6). One control is the dependence
of the reporter gene activation on the presence of the hybrid ligand. In fact,
the majority of the clones identified in the initial three-hybrid screen were
eliminated by this control. Another critical control is the sensitivity of
the three-hybrid interaction to competition by free FK506, which enabled
the elimination of another six of the nine potential positive clones. All three
remaining clones were found to harbor plasmids encoding variants of
cDNAs for human FKBP12. The rapid isolation of cDNAs for human
FKBP12 by the three-hybrid system (in 2–3 weeks) clearly demonstrated the
power of this approach for identifying protein targets for small “orphan”
ligands.
Figure 6. Summary of the yeast three-hybrid screen for target proteins for FK506.
The yeast three-hybrid system does have its limitations, including the
requirement of the protein receptors to exist as a fusion protein with either
the DNA-binding domain or transcription activation domain of a trans-
cription factor. The ability of hybrid ligands to penetrate the yeast
membranes is another barrier to its general application. Solutions to these
potential problems are beginning to be addressed.
The three-hybrid system based on the glucocorticoid receptor–
dexamethasone pair has been shown to work when the other ligand–receptor
pair is wild-type FKBP and FK506. It has been found that when the affinity
between FKBP and FK506 is further decreased by replacing FKBP12 with
other members of the FKBP family, such as FKBP13 and FKB P25 or by
generating FKBP12 mutants that have decreased affinity for FK506, the
reporter gene activation can no longer be detected (E. Griffith and J. O. Liu,
unpublished results). This is understandable as the three-hybrid system
involves the formation of a ternary complex from three partners, the two
hybrid fusion proteins and the hybrid ligand, in contrast to the two individual
fusion proteins required for two-hybrid system.
In addition to the dexamethasone-FK506 as the hybrid ligand, two alter-
native hybrid ligands were recently developed and tested in a three-hybrid
setting, which further validated the concept of the three-hybrid system. Both
groups employed dihydrofolate reductase and its potent inhibitor metho-
trexate as one receptor–ligand pair to replace FKBP12-FK506 [22, 30].
Cornish and colleagues used the LexA DNA-binding domain and B42
transactivation domain [30] while Henthorn and coworkers employed
the Gal4 DNA-binding domain and Gal4 transactivation domain [22]. In
both cases, robust LacZ reporter gene activity was observed in yeast expres-
sing appropriate fusion proteins in the presence of the dexamethasone-
methotrexate hybrid ligands. Cornish and colleagues found that at the same
concentration of the hybrid ligand, the reporter gene activity was about
150- fold higher for the dexamethasone-methotrexate system than for the
dexamethasone-FK506-based system [30]. Assuming that the reporter gene
activation is proportional to the overall affinity of the hybrid ligand for
the two fusion receptors, this is expected as the affinity of methotrexate for
dihydrofolate reductase is in the picomolar range [38] while the affinity of
FK506 for FKBP12 is in the low nanomolar range [20, 40]. Using the Gal4-
based system, Henthorn and coworkers further demonstrated the utility of
the system to screen a cDNA library to identify clones encoding dihydro-
folate reductase [22]. Similar to the three-hybrid screen for FKBPs, no false
positive clones were found after their elimination using the competition
assay with excess amount of free methotrexate. It will be interesting to see
whether there is a significant improvement in sensitivity of the three-hybrid
system when the dihydrofolate reductase–methotrexate pair is used to
replace the glucocorticoid receptor–dexamethasone pair.
4 YEAST WHOLE GENOME GENE EXPRESSION

PROFILES AS FINGERPRINTS FOR TARGET
VALIDATION, TARGET IDENTIFICATION,
AND TOXICITY PROFILING
The expression of genes in yeast, like in any other organisms, is highly

coordinated. Perturbation of one pathway or interference with the function of
one protein often leads to the changes in the levels of expression of many
other genes in a defined manner. The completion of the sequencing of the
yeast genome allows for the comparison of expression profile of the entire
yeast genome by either DNA chip or microarrays [9, 39]. The change in the
expression of each gene, or lack of it, represents a point in a one-dimensional
space. With nearly 6,000 yeast genes, the expression profiles in yeast
represents a 6,000-dimentional space that allows for extremely precise
comparison of gene expression patterns. This, together with the collection of
the yeast knockout mutants, offers a great opportunity to validate drug

targets that are conserved between yeast and humans.
A pioneering set of experiments validating the concept with microarrays
was carried out with the immunosuppressant drugs cyclosporin A (CsA),
FK506 and yeast mutants in which the drug-binding proteins are deleted
[35]. CsA and FK506 represent a new class of drugs that act by forming
ternary complexes with their respective immunophilins and the ultimate
target, a calcium-dependent protein phosphatase known as calcineurin [10,
31]. Thus, CsA binds to cyclophilins while FK506 binds to FKBPs. It is the
binary CsA–cyclophilin complex and the FK506–FKBP complex that then
binds to calcineurin, inhibiting its protein phosphatase activity. As calci-
neurin and immunophilins are conserved from yeast to humans, yeast has
been exploited as a model system to gain insight into the molecular mecha-
nism of inhibition of calcineurin by the immunosuppressants [21]. Although
calcineurin has been shown to be a target for these immunosuppressants,
how specific they are remains unknown.
Using microarrays, the “signatures” in gene expression patterns associated
with the treatment of wild-type yeast with the immunosuppressants and
those associated with the deletion of calcineurin and immunophilins were
determined. It was found that treatment of wild-type yeast with FK506
caused a greater than twofold change in expression of 36 genes. A strikingly
similar signature was observed when an isogenic calcineurin mutant strain
was compared with the wild-type yeast or when the wild-type yeast was
treated with CsA with high correlation coefficients (Figure 7). These
signatures, however, showed no statistically significant correlation with 40
other perturbations in the form of either genetic mutations or drug treatment
that are known to be unrelated to calcineurin pathway. These results gave
indication that calcineurin is a likely target for both CsA and FK506.
Figure 7. Correlation coefficients for gene expression profiles as a result of the different types
of perturbation of yeast using microarray.
To validate that the convergence of the gene profile signatures of

calcineurin mutant and those upon treatment of yeast with CsA and FK506
did not result from coincidence, the signatures of the calcineurin mutant
upon treatment with both drugs were obtained. The profile of calcineurin
mutant treated with CsA or FK506 is completely different from those of
wild-type yeast treated with the drugs or calcineurin mutant, indicating that
calcineurin is necessary for the changes in gene expression in wild-type
yeast cells. Since the inhibition of calcineurin by FK506 required the
preformation of the FK506–FKBP complex, the FK506 treatment signature
was also obtained with fpr1 mutant strain of yeast. Similar to calcineurin
mutant, the gene expression profile for fpr1 mutant is also different from the
wild-type FK506 treatment signature, indicating that FKBP in yeast is also
required for FK506 activity in yeast. In contrast, treatment of a mutant strain
that is deficient in cyclophilin, the cph1 mutant, with FK506 yielded a
signature that is similar to the wild type, consistent with the fact that
cyclophilin does not mediate the action of FK506. These results confirm that
calcineurin is a common target for both CsA and FK506 while FKBP a
target that is specific for FK506, but not CsA.
Each of the more than 6,000 individual genes and putative open reading
frames has been deleted in yeast [14]. The time will come when a
compendium of signatures of gene expression will be obtained for all the
viable mutants of yeast and possibly the inviable mutants with conditional
knockouts. Once such a database exists and experimental conditions are
standardized to allow direct comparisons of newly obtained experimental
data and the archival database, it is conceivable that every drug whose target
has a yeast counterpart can be subjected to the same validation process,
which will not only help to validate the putative target, but will also provide
information about the specificity of the drug toward the intended target.
It is worth noting, however, this approach, while powerful, does have its
limitations. The major limitation lies at the limited size of the yeast genome
in comparison to that of humans. As a consequence, many genes present in
mammalian cells, including those involved in cell–cell communications and
intracelluar signaling such as protein tyrosine kinases, are absent in yeast.
The collection of the compendium of the yeast mutants is not applicable to
studying drugs that interact with those proteins. Another limitation will be
those genes that are essential to yeast growth. More sophisticated methods of
downregulating those genes such as reduction in the expression level without
complete knockout will be important for studying drugs that affect those
genes. Nevertheless, the existing mutants and their signatures should take us
a long way toward facilitating the study of many types of drugs already.
REFERENCES
1. Arfin, S. M., R. L. Kendall, L. Hall, L. H. Weaver, A. E. Stewart, B. W. Matthews,

and R. A. Bradshaw. 1995. Eukaryotic methionyl aminopeptidases: two classes of
cobalt-dependent enzymes. Proc. Natl. Acad. Sci. USA 92:7714–7718.
2. Bedalov, A., T. Gatbonton, W. P. Irvine, D. E. Gottschling, and J. A. Simon. 2001.
Identification of a small molecule inhibitor of Sir2p. Proc. Natl. Acad. Sci. USA
98:15113–15118.
3. Chakraborti, P. K., M. J. Garabedian, K. R. Yamamoto, and S. S. Simons, Jr.
1991. Creation of “super” glucocorticoid receptors by point mutations in the steroid
binding domain. J. Biol. Chem. 266:22075–22078.
4. Chang, S. Y., E. C. McGary, and S. Chang. 1989. Methionine aminopeptidase gene of
Escherichia coli is essential for cell growth. J. Bacteriol. 171:4071–4072.
5. Chang, Y.-H., U. Teichert, and J. A. Smith. 1992. Molecular cloning, sequencing,
deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces
6. Chien, C.-T., P. L. Bartel, R. Sternglanz, and S. Fields. 1991. The two-hybrid system:
a method to identify and clone genes for proteins that interact with a protein of interest.
7. Corey, E. J., A. Guzman-Perez, and M. C. Noe. 1994. Short enantioselective
synthesis of (-)-ovalicin, a potent inhibitor of angiogenesis, using substrate-enhanced
catalytic asymmetric dihydroxylation. J. Am. Chem. Soc. 116:12109–12110.
8. Fields, S., and O.-K. Song. 1989. A novel genetic system to detect protein–protein
interactions. Nature 340:245–246.
9. Fodor, S. P., R. P. Rava, X. C. Huang, A. C. Pease, C. P. Holmes, and C. L. Adams.
1993. Multiplexed biochemical assays with biological chips. Nature 364:555–556.
10. Friedman, J., and I. Weissman. 1991. Two cytoplasmic candidates for immunophilin
action are revealed by affinity for a new cyclophilin: One in the presence and one in the
abasence of CsA. Cell 66:799–806.
11. Frye, R. A. 2000. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like
proteins. Biochem. Biophys. Res. Commun. 273:793–798.
12. Garabedian, M. J., and K. R. Yamamoto. 1992. Genetic dissection of the signaling
domain of a mammalian steroid receptor in yeast. Mol. Biol. Cell 3:1245–1257.
13. Gartenberg, M. R. 2000. The Sir proteins of Saccharomyces cerevisiae: mediators of

transcriptional silencing and much more. Curr. Opin. Microbiol. 3:132–137.
14. Giaever, G., A. M. Chu, L. Ni, C. Connelly, L. Riles, S. Veronneau, S. Dow,
A. Lucau-Danila, K. Anderson, B. Andre, A. P. Arkin, A. Astromoff, M. El-
Bakkoury, R. Bangham, R. Benito, S. Brachat, S. Campanaro, M. Curtiss,
K. Davis, A. Deutschbauer, K. D. Entian, P. Flaherty, F. Foury, D. J. Garfinkel,
M. Gerstein, D. Gotte, U. Guldener, J. H. Hegemann, S. Hempel, Z. Herman, D. F.
Jaramillo, D. E. Kelly, S. L. Kelly, P. Kotter, D. LaBonte, D. C. Lamb, N. Lan,
H. Liang, H. Liao, L. Liu, C. Luo, M. Lussier, R. Mao, P. Menard, S. L. Ooi, J. L.
Revuelta, C. J. Roberts, M. Rose, P. Ross-Macdonald, B. Scherens, G. Schimmack,
B. Shafer, D. D. Shoemaker, S. Sookhai-Mahadeo, R. K. Storms, J. N. Strathern,
G. Valle, M. Voet, G. Volckaert, C. Y. Wang, T. R. Ward, J. Wilhelmy, E. A.
Winzeler, Y. Yang, G. Yen, E. Youngman, K. Yu, H. Bussey, J. D. Boeke,
M. Snyder, P. Philippsen, R. W. Davis, and M. Johnston. 2002. Functional profiling
of the Saccharomyces cerevisiae genome. Nature 418:387–391.
15. Gottschling, D. E., O. M. Aparicio, B. L. Billington, and V. A. Zakian. 1990.
Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription.
Cell 63:751–762.
16. Griffith, E. C., Z. Su, B. E. Turk, S. Chen, Y.-H. Chang, Z. Wu, K. Biemann, and
J. O. Liu. 1997. Methionine aminopeptidase (type 2) is the common target for
angiogenesis inhibitors AGM-1470 and ovalicin. Chem. Biol. 4:461–469.
17. Grozinger, C. M., E. D. Chao, H. E. Blackwell, D. Moazed, and S. L. Schreiber.
2001. Identification of a class of small molecule inhibitors of the sirtuin family of NAD-
dependent deacetylases by phenotypic screening. J. Biol. Chem. 276:38837–38843.
18. Guarente, L. 2000. Sir2 links chromatin silencing, metabolism, and aging. Genes Dev.
14:1021–1026.
19. Gyuris, J., E. A. Golemis, H. Chertkov, and R. Brent. 1993. Cdi1, a human G1 and S
phase protein phosphatase that associates with Ckd2. Cell 75:791–803.
20. Harding, M. W., A. Galat, D. E. Uehling, and S. L. Schreiber. 1989. A receptor for
the immuno-supressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature 341:758–
760.
21. Hemenway, C. S., and J. Heitman. 1999. Calcineurin. Structure, function, and
inhibition. Cell Biochem. Biophys. 30:115–151.
22. Henthorn, D. C., A. A. Jaxa-Chamiec, and E. Meldrum. 2002. A GAL4-based yeast
three-hybrid system for the identification of small molecule-target protein interactions.
Biochem. Pharmacol. 63:1619–1628.
23. Huang, J., and S. L. Schreiber. 1997. A yeast genetic system for selecting small
molecule inhibitors of protein–protein interactions in nanodroplets. Proc. Natl. Acad.
Sci. USA 94:13396–13401.
24. Imai, S., C. M. Armstrong, M. Kaeberlein, and L. Guarente. 2000. Transcriptional
silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature
403:795–800.
25. Ingber, D., T. Fujita, S. Kishmoto, K. Sudo, T. Kanamaru, H. Brem, and
J. Folkman. 1990. Synthetic analogues of Fumagillin that inhibit angiogenesis and
suppress tumour growth. Nature 348:555–557.
26. Keeling, P. J., and W. F. Doolittle. 1996. Methionine aminopeptidase-1: the MAP of
the mitochondrion? Trends Biochem. Sci. 21:285–286.
27. Landry, J., A. Sutton, S. T. Tafrov, R. C. Heller, J. Stebbins, L. Pillus, and
R. Sternglanz. 2000. The silencing protein SIR2 and its homologs are NAD-dependent
protein deacetylases. Proc. Natl. Acad. Sci. USA 97:5807–5811.
28. Li, X., and Y.-H. Chang. 1995. Amino-terminal protein processing in Saccharomyces
cerevisiae is an essential function that requires two distinct methionine aminopeptidases.
29. Licitra, E. J., and J. O. Liu. 1996. A three-hybrid system for detecting small ligand-
protein receptor interactions. Proc. Natl. Acad. Sci. USA 93:12817–12821.
30. Lin, H., W. M. Abida, R. T. Sauer, and V. W. Cornish. 2000. Dexamethasone-
Methotrexate: an efficient chemical inducer of protein dimerization in vivo. J. Am.
Chem. Soc. 122:4247–4248.
31. Liu, J., J. D. Farmer, W. S. Lane, J. Friedman, I. Weissman, and S. L. Schreiber.
1991. Calcineurin is a common target of cyclophilin–cyclosporin A and FKBP–FK506
complexes. Cell 66:807–815.
32. Liu, J. O. 1999. Recruitment of proteins to modulate protein-protein interactions. Chem.
Biol. 6:R213–215.
33. Lowther, W. T., and B. W. Matthews. 2000. Structure and function of the methionine
aminopeptidases. Biochim. Biophys. Acta 1477:157–167.
34. Luo, J., A. Y. Nikolaev, S. Imai, D. Chen, F. Su, A. Shiloh, L. Guarente, and
W. Gu. 2001. Negative control of p53 by Sir2alpha promotes cell survival under stress.
Cell 107:137–148.
35. Marton, M. J., J. L. DeRisi, H. A. Bennett, V. R. Iyer, M. R. Meyer, C. J. Roberts,
R. Stoughton, J. Burchard, D. Slade, H. Dai, D. E. Bassett, Jr., L. H. Hartwell,
P. O. Brown, and S. H. Friend. 1998. Drug target validation and identification of
secondary drug target effects using DNA microarrays. Nat. Med. 4:1293–1301.
36. Osborne, M. A., S. Dalton, and J. P. Kochan. 1995. The yeast tribrid system–genetic
detection of trans-phosphorylated ITAM-SH2-interactions. Biotechnology 13:1474–
1478.
37. Pawson, T., and G. D. Gish. 1992. SH2 and SH3 domains: from structure to function.
Cell 71:359–362.
38. Sasso, S. P., R. M. Gilli, J. C. Sari, O. S. Rimet, and C. M. Briand. 1994.
Thermodynamic study of dihydrofolate reductase inhibitor selectivity. Biochim.
39. Schena, M., D. Shalon, R. W. Davis, and P. O. Brown. 1995. Quantitative monitoring
of gene expression patterns with a complementary DNA microarray. Science 270:467–
470.
40. Siekierka, J. J., S. H. Y. Hung, M. Poe, C. S. Lin, and N. H. Sigal. 1989. A cytosolic
binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase
activity but is distinct from cyclophilin. Nature 341:755–757.
41. Sin, N., L. Meng, M. Q. W. Wang, J. J. Wen, W. G. Bornmann, and C. M. Crews.
1997. The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine
aminopeptidase, MetAP-2. Proc. Natl. Acad. Sci. USA 94:6099–6103.
42. Smith, J. S., C. B. Brachmann, I. Celic, M. A. Kenna, S. Muhammad, V. J. Starai,
J. L. Avalos, J. C. Escalante-Semerena, C. Grubmeyer, C. Wolberger, and J. D.
Boeke. 2000. A phylogenetically conserved NAD+-dependent protein deacetylase
activity in the Sir2 protein family. Proc. Natl. Acad. Sci. USA 97:6658–6663.
43. Vaziri, H., S. K. Dessain, E. Ng Eaton, S. I. Imai, R. A. Frye, T. K. Pandita,
L. Guarente, and R. A. Weinberg. 2001. hSIR2(SIRT1) functions as an NAD-
dependent p53 deacetylase. Cell 107:149–159.
44. Young, K., S. Lin, L. Sun, E. Lee, M. Modi, S. Hellings, M. Husbands,
B. Ozenberger, and R. Franco. 1998. Identification of a calcium channel modulator
using a high throughput yeast two-hybrid screen. Nat. Biotechnol. 16:946–950.
Chapter 15
GENETIC ANALYSIS OF CISPLATIN
RESISTANCE IN YEAST AND MAMMALS
Seiko Ishida and Ira Herskowitz *

Department of Biochemistry and Biophysics, University of California, San Francisco, CA
Cisplatin is one of the most widely prescribed anticancer drugs. Its

cytotoxic effect was discovered serendipitously in a study designed to test
the effects of electric fields on the growth of the bacterium Escherichia coli
[49]. In the presence of electric fields, bacterial growth continues but the
cells do not divide. This inhibition was found to derive from the presence of
a platinum complex, cis-diamminedichloroplatinum [II] (cisplatin), formed
by electrolysis at the platinum electrodes [50]. The observation that cisplatin
inhibits bacterial cell division raised the possibility that it might have
anticancer activity, which was first demonstrated against a mouse sarcoma
[51]. Since its approval by the FDA in 1979, cisplatin has been included in
chemotherapeutic regimens for many different types of cancers. Notably,
cisplatin-based chemotherapy is curative for the majority of patients with
advanced testicular cancer, even with metastases, which was fatal in the pre-
cisplatin era [43]. Cisplatin is also effective for ovarian, bladder, cervical,
head and neck, and small-cell lung cancers. Many patients with these
cancers, however, eventually relapse and become refractory to cisplatin. In
addition, cisplatin has minimal activity against some common cancers such
as colorectal cancer. Increasing dosage to overcome resistance can cause
serious side effects in the kidneys and the inner ear. Understanding the
mechanism of intrinsic and acquired resistance to cisplatin is critical in
developing a more effective cure for cancer. In this chapter, we review
mammalian genes that have been genetically shown to be involved in
cisplatin resistance and discuss use of bakers’ yeast, Saccharomyces
*
Deceased
393
© 2007 Springer.
394 S. Ishida and I. Herskowitz
cerevisiae, for identifying genes and understanding their functions in

cisplatin resistance.
1 CISPLATIN – ITS MODE OF ACTION
Intravenously administered cisplatin retains its neutral structure in

plasma, where the chloride concentration is ~100 mM [52]. The mechanism
by which cisplatin enters cells is not known [22]. Once cisplatin passes
through the plasma membrane into the cytoplasm of a cell, where the
chloride concentration drops to ~3 mM, its chlorides are displaced by water
molecules. This aquated product is the reactive form, a potent electrophile
that can react with any nucleophile, including the sulfhydryl groups on
proteins and nitrogen donor atoms on nucleic acids.
Figure 1. An overview of cisplatin uptake and drug action.
Work from many laboratories has implicated DNA as a critical target for
cisplatin cytotoxicity, the most revealing evidence being the hypersensitivity
to cisplatin of both prokaryotic and eukaryotic cells deficient in DNA repair
[3, 20, 45]. The most prevalent cisplatin-induced DNA adduct is the
intrastrand cross-link in which the platinum is covalently bound to the N7
positions of the imidazole ring of two adjacent guanines [15]. The
intrastrand cross-links are repaired by the nucleotide excision repair pathway
[4]. Unrepaired DNA damage triggers cell cycle arrest, and cells eventually
undergo programmed cell death (apoptosis) [13].
Chapter 15: Genetic Analysis of Cisplatin Resistance in Yeast and Mammals 395
2 GENES THAT GOVERN CISPLATIN

RESISTANCE IN MAMMALIAN CELLS
In principle, increased cisplatin resistance could result from decreasing

the number of cisplatin adducts or inhibiting apoptosis in response to
cisplatin-induced DNA damage. A reduction in the number of cisplatin
adducts can be achieved by reducing the cellular level of cisplatin by a
decrease in drug uptake or an increase in drug efflux, reducing the reactivity
of the drug, reducing the accessibility of DNA to the drug, or enhancing
removal of the adducts from DNA. Factors that antagonize activation of
apoptotic pathways in response to DNA damage could increase cell survival
to cisplatin treatment.
In mammalian systems, genes that affect cellular sensitivity to cisplatin
have been identified using a candidate-gene approaches described below (see
Table 1).
2.1 Mammalian genes that inhibit cisplatin accumulation

in cells – cMOAT/MRP2 and ATP7B
Canalicular multispecific organic anion transporter (cMOAT), also

known as apical mulitdrug resistant protein (MRP2), is predominantly
localized to the canalicular membrane of hepatocytes and to the apical
membrane of proximal tubules in the kidney. Because of its structural and
functional similarities with MRP1, an integral membrane protein associated
with multidrug resistance, a potential involvement of cMOAT/MRP2 in
cisplatin resistance was tested. Human hepatic cancer cell lines transfected
with antisense cMOAT/MRP2 cDNA exhibit an increase in cisplatin
sensitivity accompanied by an increase in cellular accumulation of cisplatin
[33]. In addition, overexpression of human cMOAT/MRP2 increases
cisplatin resistance in various mammalian cell lines [10, 31] and decreases
drug accumulation in Chinese hamster ovary cells [31]. These observations
demonstrate that cMOAT/MRP2 inhibits cisplatin accumulation in cells,
possibly by enhancing drug efflux.
ATP7B is a copper-transporting P-type ATPase that transports copper
into the lumen of the Golgi and into secretory vesicles. It belongs to a class
of heavy metal transporting P-type ATPases that pump cadmium, zinc,
silver, and lead in addition to copper. In a study designed to investigate a
role of ATP7B in resistance to various heavy metals including the platinum
compound cisplatin, it was noted that overexpression of human ATP7B in
human epidermoid carcinoma cells conferred increased cisplatin resistance,
accompanied by an increase in cisplatin efflux [34]. These findings suggest
that ATP7B protein may serve as a pump to assist cisplatin efflux.
2.2 Mammalian genes that inactivate cisplatin by

increasing thiols – γ -glutamylcysteine synthetase and
metallothionein
Cisplatin is a potent electrophile inside cells and reacts with any

nucleophile, including the sulfhydryl groups on proteins and nucleophilic
groups on nucleic acids. Glutathione (γ-glutamylcysteinyl glycine, GSH),
one of the most abundant thiols in cells, binds to cisplatin and the GSH–
cisplatin complex is transported across the plasma membrane [27]. The rate-
limiting step of GSH synthesis is catalyzed by γ-glutamylcysteine synthetase
(γ-GCS). Overexpression of human γ-GCS in a human small-cell lung
cancer cell line increases the cellular GSH content, accompanied by a
decrease in cellular cisplatin level and an increase in cisplatin resistance
[37]. Furthermore, human colon cancer cells transfected with a hammerhead
ribozyme against γ-GCS mRNA exhibit a reduction in the level of γ-GCS
transcripts, an increase in cisplatin sensitivity [48], and a decrease in
cisplatin efflux [25], suggesting that γ-GCS protein protects cells from
cisplatin toxicity by sequestering intracellular cisplatin and exporting it.
Metallothioneins (MTs) are cysteine-rich proteins that constitute a major
fraction of intracellular protein thiols. Cisplatin has been shown to
covalently bind MT [36]. Overexpression of human MT–IIA in mouse cells
results in increased cisplatin resistance [32]. Cells derived from MT-null
mice lacking both MTI and MTII exhibit enhanced sensitivity to cisplatin
[35]. The MT-null mice manifest more severe kidney and liver damage after
cisplatin treatment and exhibit reduced survival after cisplatin injection
compared to wild-type mice [42]. Taken together, these results demonstrate
that MTs play a role in detoxification of cisplatin.
2.3 Mammalian genes that mediate cytotoxic response

to cisplatin adducts – MSH2 and MLH1
Mismatch repair is a post-replication repair system that corrects unpaired

or mispaired nucleotides. Deficiency in this repair system predisposes cells
to genomic instability. A defect in mismatch repair plays an important role in
hereditary nonpolyposis colon cancer and may play a role in a variety of
sporadic tumors as well. In addition to its role in processing mismatched
bases, mismatch repair in E. coli has been implicated in cytotoxic effects of
DNA-damaging agents including cisplatin [19]. A mismatch recognition
complex in human consists of Msh2 and GTBP (hMsh6), whose binding
to mismatched bases is further stabilized by the association of Mlh1 and
Pms2 proteins. Purified human Msh2 binds to cisplatin G–G intrastrand
adducts [12, 47], and both hMsh2 and hMlh1 are detected on cisplatin-
treated DNA after incubation with nuclear extract [16]. Human cancer cell
lines deficient in mismatch repair due to mutations in hMSH2 or hMLH1 are
more resistant to cisplatin treatment than cells whose repair deficiency is
complemented by introduction of chromosome 3 (containing hMSH2) or
chromosome 2 (containing hMLH1) [1]. No difference is observed in the
level of cisplatin bound to DNA or in the rate of removal of cisplatin-DNA
adducts between these mismatch repair-deficient and -proficient cells [9].
Furthermore, cell lines deleted for the MSH2 gene demonstrate increased
resistance to cisplatin compared to wild-type cell lines in vitro and when
implanted in nude mice [17].
It is hypothesized that mismatch repair proteins sense the cisplatin-induced
damage and initiate a series of events that results in cell cycle arrest and death.
Two models have been proposed to explain the connection between the
mismatch repair pathway and sensitivity to DNA damage caused by anticancer
drugs such as cisplatin. In one model, DNA lesions are recognized and
processed by the mismatch repair pathway, but because mismatch repair
excises a tract from the newly incorporated strand, damage in the parental
strand is not removed. Repeated attempts at processing are proposed to create
persistent gaps that trigger cell cycle arrest or cell death. This model is
consistent with the observations that mismatch repair-proficient cells show a
reduced level of nascent DNA synthesis as measured by incorporation of
radiolabeled nucleotides into chromosomal DNA after treatment of cells with
cisplatin [56] or into cisplatinated plasmids after incubation in vitro with cell
extracts [14]. In addition, mismatch repair-proficient cells are less efficient
in reactivation of cisplatin-damaged plasmid DNA as measured by luciferase
activity from a cisplatin-damaged reporter plasmid [9]. It is possible that a
more direct signaling pathway links the mismatch repair pathway and
cisplatin cytotoxicity. Assembly of mismatch repair proteins on cisplatin-
DNA adducts, whose DNA structure is different from that of unpaired or
mispaired bases [55], could initiate a signaling cascade that leads to cell
death.
3 UNDERSTANDING MECHANISMS OF
CISPLATIN RESISTANCE IN THE BUDDING
YEAST SACCHAROMYCES CEREVISIAE
3.1 Yeast as an experimental system to understand

mechanisms of cisplatin resistance
Yeast provides excellent opportunities to identify genes and functions of

their products in cisplatin resistance for three reasons. First, yeast cells are
sensitive to cisplatin treatment. This enables isolation of mutants with
increased cisplatin resistance. Second, at least 30% of the yeast genome has
mammalian homologs [5]. Genes identified in yeast may thus have
mammalian counterparts that function in a similar manner. Third, genetic
manipulations are much easier and faster in yeast compared to mammalian
systems. The doubling time is ~90 min compared to >20 h in cultured
mammalian cell lines [23]. The fact that yeast can exist as a haploid with
only one copy of each chromosome makes it possible to carry out random
mutagenesis or targeted deletion of a gene and observe a loss-of-function
phenotype. Mutations in different genetic loci can be combined by crossing
haploid mutants and sporulating them.
Two genetic approaches can be taken to identify genes involved in
cisplatin resistance. One is to look for genes that confer increased cisplatin
resistance when overexpressed. The other is to mutagenize cells and look for
mutants that show increased cisplatin resistance and identify genes altered in
the mutants. The two methods are complementary. In overexpression
screens, one may miss genes whose products act in a complex with other
proteins. In this case, overexpressing one component of the complex may not
result in a phenotype. Such genes may be uncovered by mutagenesis screens,
since disruption of any one of the components may inactivate the entire
complex. In mutagenesis screens, genes that have redundant functions
will be missed. Such genes, however, may be identified in overexpression
screens.
3.2 Identification of yeast genes that govern cisplatin

resistance
Three methods have been undertaken to identify yeast genes which could
potentially govern cisplatin resistance – searching for genes whose products
bind to platinated DNA and genes whose overexpression or loss of function
allows cells to grow in the presence of a toxic level of cisplatin (Table 1).
Table 1. Genes that govern cisplatin resistance.

Genes that govern cisplatin Protein Suggested role in cisplatin
resistance resistance
Human cMOAT/MRP2 Canalicular multispecific Cisplatin efflux
anion transporter/apical
multidrug resistant protein
Human ATP7B Copper-transporting P-type Cisplatin efflux
ATPase on Golgi
Human γ-glutamylcysteine Enzyme involved in Inactivation of cisplatin
synthtase glutathione synthesis
Human MT Metallothionein Inactivation of cisplatin
Human MSH2 Mispatch repair protein Enhancing cell death
Human MLH1 Mispatch repair protein Enhancing cell death
Human SRPK1 Kinase Unknown
Mouse CTR1 Copper transporter Cisplatin uptake
Yeast 1XR1 HMG-domain protein Shielding adducts from
repair
Yeast PHR1 Photolyase Unknown
Yeast CIN5 Unknown Unknown
Yeast YDR259c Unknown Unknown
Yeast PDE2 cAMP phosphodiesterase Unknown
Yeast ZDS2 Unknown Unknown
Yeast SKY1 Kinase Unknown
Yeast CTR1 Copper transporter Cisplatin uptake
In an effort to identify genes whose products bind to platinated DNA, a

λ-gt11 yeast expression library was screened with radiolabeled platinated
DNA, leading to the identification of IXR1 [6]. IXR1 encodes a protein with
an high mobility group (HMG)-box motif known to bind irregular DNA
structures. Ixr1p specifically binds to platinated DNA but not to UV-
damaged DNA. Disruption of IXR1 results in a twofold increase in
resistance to cisplatin. In another study, Phr1p, a yeast photolyase that binds
to UV-induced cyclobutane dimers and repairs the damage was shown to
bind to DNA damage caused by cisplatin [18]. Cisplatin sensitivity
correlates with gene dosage of PHR1.
Two screens have been carried out to search for genes that confer
resistance to cisplatin when overexpressed in wild-type cells or in cells
sensitized to cisplatin by inactivation of nucleotide excision repair. Wild-type
cells transformed with multicopy plasmids carrying CIN5 or YDR259c, both
encoding proteins with bZIP motifs commonly present in AP-1 transcription
factors, exhibit increased resistance to cisplatin [21]. Cellular accumulation of
cisplatin in these cells is not affected. Deletion of either CIN5 or YDR259c,
however, does not make cells more sensitive to cisplatin. rad4 mutants, which
are defective in nucleotide excision repair, become more resistant to cisplatin
when transformed with multicopy plasmids carrying PDE2, a cAMP
phosphodiesterase, or ZDS2, which is implicated in cell cycle control and

resistance to multiple drugs [7]. The increase in cisplatin resistance is not
accompanied by a reduction in drug accumulation. The effect of over-
expression of these genes in wild-type cells has not been reported.
A transposon library was used by two groups to mutagenize cells and to
isolate mutants with increased cisplatin resistance. In this method, cells are
transformed with yeast genomic fragments whose sequence is interupted at
random places by a transposon [8]. Transposon-mediated mutagenesis
allows rapid identification of the sequence disrupted by the transposon in the
mutant and subsequent identification of the affected gene. Screening of
transposon-mutagenized rad4∆ mutants led to the identification of the SKY1
gene, whose deletion in a rad4∆ background and in wild-type cells causes an
increase in cisplatin resistance [53]. Overexpression of SKY1 in rad4∆
results in increased sensitivity to the drug. Sky1p is a kinase thought to
phosphorylate regulators of RNA processing. How SKY1 affects cisplatin
resistance is not known. rad4∆ sky1∆ mutants display similar levels of
cisplatin in cells and in DNA compared to rad4∆ mutants [54]. In another
study, transposon-mediated mutagenesis was performed in wild-type cells, in
which case inactivation of the MAC1 gene resulted in increased cisplatin
resistance [26]. MAC1 encodes a transcription factor [29, 57, 38, 44]. In
subsequent studies, it was shown that deletion of the high-affinity copper
transporter gene CTR1, one of the genes whose transcription is activated by
Mac1p, causes cisplatin resistance similar to that of mac1∆ mutants [26].
Both mac1∆ and ctr1∆ mutants show a decreased level of platinum in cells
and in DNA.
3.3 Understanding mechanisms of increased cisplatin

resistance in yeast mutants and implications in
mammals
Measurement of platinum levels in cells and in DNA is a powerful

method for understanding the basis for cisplatin resistance in mutant strains.
For many genes, increased resistance is not accompanied by changes in
cellular accumulation of the drug or the cisplatin adduct level.
Overexpression of CIN5 or YDR259c in wild-type cells or overexpression of
PDE2 or ZDS2 in rad4∆ mutants confers an increase in cisplatin resistance,
but the level of cellular cisplatin is unchanged [21, 7]. Deletion of SKY1 in
rad4∆ mutants causes an increase in resistance, but no change is observed in
the levels of cisplatin in cells or in DNA [54]. Among the yeast genes
implicated in cisplatin resistance, IXR1 and CTR1 are the most characterized
for their roles in governing cisplatin sensitivity, which appear to be con-
served in mammals as well.
3.3.1 Ixr1p shields cisplatin adducts from repair
Ixr1p is a protein that contains HMG domain and represses transcription

of the COX5b gene by binding to its upstream regulatory region [40]. One of
the common features of HMG-domain proteins is their ability to bind to
irregular DNA structures, and several of them including Ixr1 have been
demonstrated to bind to cisplatin adducts [30]. Deletion of IXR1 results in a
twofold increase in cisplatin resistance, accompanied by a threefold decrease
in the level of adduct [6]. No change in cisplatin resistance is observed in
cells deleted for ROX1, another repressor of COX5b, indicating that the
increase in resistance observed in the ixr1∆ mutant is not due to
overexpression of COX5b [46]. The increased resistance observed in an
ixr1∆ mutant is dependent on intact excision repair:deletion of IXR1 in cells
defective in nucleotide excision repair, the major repair mechanism that
removes cisplatin adducts, does not make them more resistant to cisplatin,
i.e., a rad2∆ ixr1∆ strain is as sensitive to cisplatin as a rad2∆ strain [45]. It
has thus been proposed that binding of Ixr1p to cisplatin adducts shields
them from being repaired by the excision repair.
In mammals, human HMG2 confers increased cisplatin sensitivity when
overexpressed in a human non-small cell lung cancer cell line [2]. In these
cells, the level of cellular platinum is increased. Two HMG proteins, HMG1
and the human mitochondrial transcription factor (mtTFA), inhibit
nucleotide excision repair of cisplatin-damaged DNA fragments in vitro
[24]. HMG-domain proteins thus appear to play an important role in both
yeast and mammals in limiting access to cisplatin adducts by the excision
repair machinery.
3.3.2 III.3.2 Ctr1p mediates cisplatin uptake
Ctr1p is a high-affinity copper transporter residing in the plasma

membrane. Deletion of CTR1 results in a twofold increase in cisplatin
resistance and a twofold decrease in cellular accumulation of cisplatin [26].
The increase in resistance in the ctr1∆ mutant is not likely to be a secondary
effect of intracellular copper deficiency due to the ctr1∆ mutation, since
deletions of other copper transporter genes or genes involved in intracellular
copper trafficking and utilization do not result in increased cisplatin
resistance. Furthermore, addition of copper to the ctr1∆ mutant, at a
concentration that restores copper uptake and growth to this mutant, does not
increase sensitivity to cisplatin, suggesting that the resistance observed in the
ctr1∆ mutant is not due to copper starvation. Addition of copper to wild-
type cells increases cisplatin resistance and decreases cisplatin accumulation,
both of which are not observed in the ctr1∆ mutant, indicating that the
effects of copper on reducing cisplatin uptake are mediated by the copper
transporter Ctr1p [26]. Interestingly, cisplatin treatment results in
delocalization and degradation of Ctr1p [26]. Taken together, these findings
support a hypothesis that the copper transporter Ctr1p mediates uptake of
cisplatin in yeast.
Mammals contain functional homologs of the yeast CTR1 gene, hCTR1
in humans and mCTR1 in mice, which complement a yeast ctr1 mutant for
intracellular copper deficiency [41, 58]. Mouse cell lines that are wild-type,
heterozygous, or homozygous for a knockout allele of mCTR1 display a
graded increase in cisplatin resistance and decrease in cisplatin accumulation
[26]: heterozygous cells are fourfold more resistant to cisplatin and exhibit a
35% decrease in cisplatin accumulation compared to wild-type cells;
homozygous cells show an eightfold increase in resistance and a 70%
decrease in drug accumulation. These observations in mice suggest that
hCtr1 in humans, which is 92% identical to mCtr1, may play a key role in
cisplatin uptake and resistance.
The mechanism of cisplatin uptake has been unclear. Inability to saturate
the rate of cisplatin uptake supports a simple diffusion model, whereas the
existence of agents and conditions that modulate cisplatin accumulation,
such as pH, osmolarity, sodium, potassium, protein kinase C (PKC), protein
kinase A (PKA), and the calcium/calmodulin pathway, suggests that some
component of cisplatin uptake is mediated by a transport mechanism [22]. It
has been proposed that about 50% of the intial rate of uptake is due to
passive diffusion and that the remaining 50% is due to facilitated diffusion
through an as yet unidentified gated channel. Ctr1p might be responsible for
the facilitated uptake of cisplatin. Cisplatin uptake is not completely
abolished in the ctr1∆ mutants in both yeast and mice [26]. The residual
cisplatin uptake observed in the ctr1∆ mutants might be due to diffusion
across the plasma membrane or to additional transport proteins.
3.3.3 Other yeast genes whose mammalian homologs are implicated

in cisplatin resistance
All of the yeast genes identified in the screens have mammalian

homologs or are involved in functions that are present in mammals. In
addition to IXR1 and CTR1, mammalian homologs of the yeast PDE2 and
SKY1 genes may have roles in cisplatin resistance in mammals.
Overproduction in yeast of Pde2p, a phosphodiesterase that functions in the
PKA pathway, confers increased cisplatin resistance to mutants defective in
excision repair [7]. Studies in mammalian cells also suggest potential links
between the PKA pathway and cisplatin sensitivity [11]. Deletion of the
SKY1 gene in the excision repair-deficient rad4∆ mutant increases cisplatin

resistance [53]. Antisense experiments in human ovarian carcinoma cell
lines demonstrate that inactivation of its human homolog, SRPK1, leads to
increased cisplatin resistance [53]. The basis for increased cisplatin
resistance in cells overexpressing PDE2 or in cells deleted for SKY1 is not
known. Having yeast models should facilitate elucidation of the functions of
these mammalian gene products in mediating cisplatin sensitivity.
3.4 Limitations of the use of yeast in understanding

cisplatin resistance in mammals
Although mismatch repair deficiency has been associated with cisplatin

resistance in mammalian cells [17], deletion of mismatch repair genes in
yeast – MSH2, MSH3, MSH6, MLH1, or PMS1 – does not result in
increased cisplatin resistance [26]. In mammals, mismatch repair deficiency
is also associated with resistance to other DNA-damaging agents [39]. It has
been postulated that detection of DNA damage by the mismatch repair
system triggers a pathway that leads to apoptosis. Cisplatin-induced death of
mammalian cells exhibits certain features of apoptosis, such as chromatin
condensation, DNA fragmentation, and activation of key regulators of
apoptosis such as p53 and p73 [13, 28]. In yeast, however, there has been no
clear evidence for the existence of an active cell death pathway such as
apoptosis. Lack of involvement of mismatch repair genes in cisplatin
resistance in yeast may reflect differences in the way mammalian cells and
yeast cells die after exposure to toxic doses of cispatin.
4 CONCLUSIONS AND PERSPECTIVES
Cisplatin is one of the most widely used anticancer drugs effective in the
treatment of a variety of cancers. Intrinsic or acquired resistance to cisplatin
reduces its efficacy, however, which limits its curative potential.
Considerable effort has been made to define the cellular and molecular
mechanisms responsible for cisplatin resistance in mammals. Most of the
mammalian genes governing cisplatin resistance were identified due to their
biochemical properties – cMOAT/MRP2 and ATP7B are involved in efflux
of drugs and metals, γ-glutamylcysteine synthetase, and metallothionein
increase intracellular thiols that react with cisplatin, and mismatch repair
proteins and HMG domain proteins bind to platinated DNA.
Genetic selections in yeast have identified a number of genes that affect
cisplatin resistance, most of which either have mammalian homologs or are
involved in functions that are also present in mammals. Genetic analyses of
the yeast ixr1∆ mutants indicated that HMG-domain proteins confer

sensitivity to cisplatin by shielding cisplatin adducts from repair. These
findings complement biochemical studies of the role of human HMG
proteins in cisplatin resistance. Increased cisplatin resistance and decreased
cisplatin uptake of yeast ctr1∆ mutants led to the identification of the
mammalian copper transporter Ctr1 as a mediator in cisplatin uptake.
Although deletion and overexpression studies in yeast and mammalian
cells have clearly demonstrated the role of some genes in cisplatin
resistance, whether these genes are involved in cisplatin resistance of tumors
in patients remains to be determined. To understand clinical mechanisms of
resistance, analysis of specimens from patients is necessary. Studies of
genetically modified cell lines and mice will provide complementary
information.
Studies with yeast have identified additional candidate genes that should
be closely monitored in clinical settings. Mutations in these genes or changes
in their expression levels may influence the efficacy of cisplatin and thus
could be a crucial factor that may determine the prognosis of the patient.
Because Ctr1 is a cell-surface protein, it may be a good target for developing
drugs that would antagonize cisplatin uptake to protect normal tissues from
damage or that would facilitate cisplatin uptake to sensitize tumor cells to
cisplatin. Localized administration of such Ctr1 modifiers might greatly
enhance the efficacy of cisplatin against tumors and minimize its side
effects.
REFERENCES
1. Aebi, S., D. Fink, R. Gordon, H. K. Kim, H. Zheng, J. L. Fink, and S. B. Howell.
1997. Resistance to cytotoxic drugs in DNA mismatch repair-deficient cells. Clin.
Cancer Res. 3:1763–1767.
2. Arioka, H., K. Nishio, T. Ishida, H. Fukumoto, K. Fukuoka, T. Nomoto,
H. Kurokawa, H. Yokote, S. Abe, and N. Saijo. 1999. Enhancement of cisplatin
sensitivity in high mobility group 2 cDNA-transfected human lung cancer cells. Jpn.
J. Cancer Res. 90:108–115.
3. Beck, D. J., and R. R. Brubaker. 1973. Effect of cis-platinum(II)diamminedichloride
on wild type and deoxyribonucleic acid repair-deficient mutants of Escherichia coli.
J. Bacteriol. 116:1247–1252.
4. Beck, D. J., S. Popoff, A. Sancar, and W. D. Rupp. 1985. Reactions of the UvrABC
excision nuclease with DNA damaged by diamminedichloroplatinum(II). Nucl. Acids
Res. 13:7394–7412.
5. Botstein, D., S. A. Chervitz, and J. M. Cherry. 1997. Yeast as a model organism.
Science 277:1259–1260.
6. Brown, S. J., P. J. Kellet, and S. J. Lippard. 1993. Ixr1, a yeast protein that binds to
platinated DNA and confers sensitivity to cisplatin. Science 261:603–605.
7. Burger, H., A. Capello, P. W. Schenk, G. Stoter, J. Brouwer, and K. Nooter. 2000.

A genome-wide screening in Saccharomyces cerevisiae for genes that confer resistance
to the anticancer agent cisplatin. Biochem. Biophys. Res. Comm. 269:767–774.
8. Burns, N., B. Grimwade, P. B. Ross-Macdonald, E.-Y. Choi, K. Finberg, G. S.
Roeder, and M. Snyder. 1994. Large-scale analysis or gene expression, protein
localization, and gene disruption in Saccharomyces cerevisiae. Genes and Dev. 8:1087–
1105.
9. Cenni, B., H.-K. Kim, G. J. Bubley, S. Aebi, D. Fink, B. A. Teicher, S. B. Howell,
and R. D. Christen. 1999. Loss of DNA mismatch repair facilitates reactivation of a
reporter plasmid damaged by cisplatin. Br. J. Cancer 80:699–704.
10. Cui, Y., J. Konig, U. Buchholz, H. Spring, I. Leier, and D. Keppler. 1999. Drug
resistance and ATP-dependent conjugate transport mediated by the apical multidrug
resistance protein, MRP2, permanently expressed in human and canine cells. Mol.
Pharmacol. 55:929–937.
11. Cvijic, M. E., W.-L. Yang, and K.-V. Chin. 1998. Cisplatin resistance in cyclic AMP-
dependent protein kinase mutants. Pharmacol. Ther. 78:115–128.
12. Duckett, D. R., J. T. Drummond, A. I. H. Murchie, J. T. Reardon, A. Sancar, D. M.
J. Lilley, and P. Modrich. 1996. Human MutSα recognizes damaged DNA base pairs
containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct. Proc.
13. Eastman, A. 1990. Activation of programmed cell death by anticancer agents:cisplatin
as a model system. Cancer Cells 2:275–280.
14. Ferry, K. V., D. Fink, S. W. Johnson, S. Nabel, T. C. Hamilton, and S. B. Howell.
1999. Decreased cisplatin damage-dependent DNA synthesis in cellular extracts of
mismatch repair deficient cells. Biochem. Pharmacol. 57:861–867.
15. Fichtinger-Schepman, A. M. J., J. L. van der Veer, J. H. J. den Hartog, P. H. M.
Lohman, and J. Reedijk. 1985. Adducts of the antitumour drug cis-
diamminedichloroplatinum (II) with DNA:formation, identification and quantitation.
Biochemistry 24:707–713.
16. Fink, D., S. Nebel, S. Aebi, H. Zheng, B. Cenni, A. Nehme, R. D. Christen, and S. B.
Howell. 1996. The role of DNA mismatch repair in platinum drug resistance. Cancer
Res. 56:4881–4886.
17. Fink, D., H. Zheng, S. Nebel, P. S. Norris, S. Aebi, T.-P. Lin, A. Nehme, R. D.
Christen, M. Haas, C. L. MacLeod, and S. B. Howell. 1997. In vitro and in vivo
resistance to cisplatin in cells that have lost DNA mismatch repair. Cancer Res.
57:1841–1845.
18. Fox, M. E., B. J. Feldman, and G. Chu. 1994. A novel role for DNA
photolyase:binding to DNA damaged by drugs is associated with enhanced cytotoxicity
in Saccharomyces cerevisiae. Mol. Cell. Biol. 14:8071 – 8077.
19. Fram, R. J., P. S. Cusick, J. M. Wilson, and M. G. Marinus. 1985. Mismatch repair
of cis-diamminedichloroplatinum(II)-induced DNA damage. Mol. Pharmacol. 28:51–55.
20. Fraval, H. N. A., C. J. Rawlings, and J. J. Roberts. 1978. Increased sensitivity of UV-
repair-deficient human cells to DNA bound platinum products which unlike thymine
dimers are not recognized by an endonuclease extracted from Micrococcus luteus.
Mutat. Res. 51:121–132.
21. Furuchi, T., H. Ishikawa, N. Miura, M. Ishizuka, K. Kajiya, S. Kuge, and
A. Naganuma. 2001. Two nuclear proteins, Cin5 and Ydr259c, confer resistance to
cisplatin in Saccharomyces cerevisiae. Mol. Pharmacol. 59:470–474.
22. Gately, D. P. and S. B. Howell. 1993. Cellular accumulation of the anticancer agent
cisplatin:a review. Br. J. Cancer 67:1171–1176.
23. Herskowitz, I. 1988. The life cycle of the budding yeast, Saccharomyces cerevisiae.
Microbiol. Rev. 52:536–553.
24. Huang, J-.C., D. B. Zamble, J. T. Reardon, S. J. Lippard, and A. Sancar. 1994.
HMG-domain proteins specifically inhibit the repair of the major DNA adduct of the
anticancer drug cisplatin by human excision nuclease. Proc. Natl. Acad. Sci. USA
91:10394–10398.
25. Iida, T., H. Kijima, Y. Urata, S. Goto, Y. Ihara, M. Oka, S. Kohno, K. J. Scanlon,
and T. Kondo. 2001. Hammerhead ribozyme against gamma-glutamylcysteine
synthetase sensitizes human colonic cancer cells to cisplatin by down-regulating both
the glutathione synthesis and the expression of multidrug resistance proteins. Cancer
Gene Ther. 8:803–814.
26. Ishida, S. 2001. Mechanisms of cisplatin resistance in yeast and mammals Ph.D. thesis.
University of California, San Francisco.
27. Ishikawa, T., and F. Ali-Osman. 1993. Glutathione-associated cis-
diamminedichloroplatinum(II) metabolism and ATP-dependent efflux from leukemia
cells. J. Biol. Chem. 268:20116–20125.
28. Jordan, P., and M. Carmo-Fonseca. 2000. Molecular mechanisms involved in
cisplatin cytotoxicity. Cell. Mol. Life Sci. 57:1229–1235.
29. Jungmann, J., H. A. Reins, J. Lee, A. Romeo, R. Hassett, D. Kosman, and
S. Jentsch. 1993. MAC1, a nuclear regulatory protein related to Cu-dependent
transcription factors is involved in Cu/Fe utilization and stress resistance in yeast.
EMBO J. 12:5051–5056.
30. Kartalou, M., and J. M. Essigmann. 2001. Recognition of cisplatin adducts by cellular
proteins. Mutat. Res. 478:1–21.
31. Kawabe, T., Z.-S. Chen, M. Wada, T. Uchimi, M. Ono, S. Akiyama, and M.
Kuwano. 1999. Enhanced transport of anticancer agents and leukotriene C4 by the
human canalicualr multispecific organic anion transporter (cMOAT/MRP2). FEBS Lett.
456:327–331.
32. Kelley, S. L., A. Basu, B. A. Teicher, M. P. Hacker, D. H. Hamer, and J. S. Lazo.
1988. Overexpression of metallothionein confers resistance to anticancer drugs. Science
241:1813–1815.
33. Koike, K., T. Kawabe, T. Tanaka, S. Toh, T. Uchimi, M. Wada, S. Akiyama, M.
Ono, and M. Kuwano. 1997. A canalicular multispecific organic anion transporter
(cMOAT) antisense cDNA enhances drug sensitivity in human hepatic cancer cells.
Cancer Res. 57:5475–5479.
34. Komatsu, M., T. Sumizawa, M. Mutoh, Z.-S. Chen, K. Terada, T. Furukawa, X.-L.
Yang, H. Gao, N. Miura, T. Sugiyama, and S. Akiyama. 2000. Copper-transporting
P-type adenosine triphosphate (ATP7B) is associated with cisplatin resistance. Cancer
Res. 60:1312–1316.
35. Kondo, Y., E. S. Woo, A. E. Michalska, K. H. A. Choo, and J. S. Lazo. 1995.
Metallothionein null cells have increased sensitivity to anticancer drugs. Cancer Res.
55:2021–2023.
36. Kraker, A., J. Schmidt, S. Krezoski, and D. H. Petering. 1985. Binding of cis-
dichlorodiammine platinum(II) to metallothionein in Ehrlich cells. Biochem. Biophys.
Res. Comm. 130:786–792.
37. Kurokawa, H., T. Ishida, K. Nishio, H. Arioka, M. Sata, H. Fukumoto, M. Miura,
and N. Saijo. 1995. γ-glutamylcysteine synthetase gene overexpression results in
increased activity of the ATP-dependent glutathione S-conjugate export pump and

cisplatin resistance. Biochem. Biophys. Res. Comm. 216:258–264.
38. Labbe, S., Z. Zhu, and D. J. Thiele. 1997. Copper-specific transcription of yeast genes
encoding critical components in the copper transport pathway. J. Biol. Chem.
272:15951–15958.
39. Lage, H., and M. Dietel. 1999. Involvement of the DNA mismatch repair system in
antineoplastic drug resistance. J. Cancer Res. Clin. Oncol. 125:156–165.
40. Lambert, J. R., V. W. Bilanchone, and M. G. Cumsky. 1994. The ORD1 gene
encodes a transcription factor involved in oxygen regulation and is identical to IXR1, a
gene that confers cisplatin sensitivity to Saccharomyces cerevisiae. Proc. Natl. Acad.
Sci. USA 91:7345–7349.
41. Lee, J., J. R. Prohaska, S. L. Dagenais, T. W. Glover, and D. J. Thiele. 2000.
Isolation of a murine copper transporter gene, tissue specific expression and functional
complementation of a yeast copper transport mutant. Gene 254:87–96.
42. Liu, J., Y. Liu, S. S. M. Habeebu, and C. D. Klaassen. 1998. Metallothionein (MT)-
null mice are sensitive to cisplatin-induced hepatotoxicity. Toxicol. App. Pharmacol.
149:24–31.
43. Loehrer, P. J., and L. H. Einhorn. 1984. Cisplatin. Ann. Intern. Med. 100:704–713.
44. Martins, L. J., L. T. Jensen, J. R. Simons, G. L. Keller, and D. R. Winge. 1998.
Metalloregulation of FRE1 and FRE2 homologs in Saccharomyces cerevisiae. J. Biol.
Chem. 273:23716–23721.
45. McA’Nulty M. M., and S. J. Lippard. 1996. The HMG-domain protein Ixr1 blocks
excision repair of cisplatin-DNA adducts in yeast. Mutat. Res. 362:75–86.
46. McA’Nulty, M. M., J. P. Whitehead, and S. J. Lippard. 1996. Binding of Ixr1, a
yeast HMG-domain protein, to cisplatin-DNA adducts in vitro and in vivo. Biochemistry
35:6089–6099.
47. Mello, J. A., S. Acharya, R. Fishel, and J. M. Essigmann. 1996. The mismatch-repair
protein hMSH2 binds selectively to DNA adducts of the anticancer drug cisplatin.
Chem. Biol. 3:579–589.
48. Nagata, J., H. Kijima, H. Hatanaka, S. Asai, H. Miyachi, A. Takagi, T. Miwa,
T. Mine, H. Yamazaki, M. Nakamura, T. Kondo, K. J. Scanlon, and Y. Ueyama.
2001. Reversal of cisplatin and multidrug resistance by ribozyme-mediated glutathione
suppression. Biochem. Biophys. Res. Comm. 286:406–413.
49. Rosenberg, B., L. Van Camp, and T. Krigas. 1965. Inhibition of cell division in
Escherichia coli by electrolysis products from a platinum electrode. Nature 205:698–
699.
50. Rosenberg, B., L. Van Camp, E. B. Grimley, and A. J. Thomson. 1967. The
inhibition of growth or cell division in Escherichia coli by different ionic species of
platinum complexes. J. Biol. Chem. 242:1347–1352.
51. Rosenberg, B., L. Van Camp, J. E. Trosko, and V. H. Mansour. 1969. Platinum
compounds: a new class of potent antitumor agents. Nature 222:385–386.
52. Rosenberg, B. 1985. Fundamental studies with cisplatin. Cancer 55:2303–16.
53. Schenk, P. W., A. W. M. Boersma, J. A. Brandsma, H. den Dulk, H. Burger,
G. Stoter, J. Brouwer, and K. Nooter. 2001. SKY1 is involved in cisplatin-induced
cell kill in Saccharomyces cerevisiae, and inactivation of its human homologue, SRPK1,
induces cisplatin resistance in a human ovarian carcinoma cell line. Cancer Res.
61:6982–6986.
54. Schenk, P. W., A. W. M. Boersma, M. Brok, H. Burger, G. Stoter, and K. Nooter.
2002. Inactivation of the Saccharomyces cerevisiae SKY1 gene induces a specific
modification of the yeast anticancer drug sensitivity profile accompanied by a mutator

phenotype. Mol. Pharmacol. 61:659–666.
55. Takahara, P. M., A. C. Rosenzweig, C. A. Frederick, and S. J. Lippard. 1995.
Crystal structure of double-stranded DNA containing the major adduct of the anticancer
drug cisplatin. Nature 377:649–652.
56. Vaisman, A., M. Varchenko, A. Umar, T. A. Kunkel, J. I. Risinger, J. C. Barrett,
T. C. Hamilton, and S. G. Chaney. 1998. The role of hMLH1, hMSH3, and hMSH6
defects in cisplatin and oxaliplatin resistance:correlation with replicative bypass of
platinum-DNA adducts. Cancer Res. 58:3579–3585.
57. Yamaguchi-Iwai, Y., M. Serpe, D. Haile, W. Yang, D. J. Kosman, R. D. Klausner,
and A. Dancis. 1997. Homeostatic regulation of copper uptake in yeast via direct
binding of MAC1 protein to upstream regulatory sequences of FRE1 and CTR1. J. Biol.
Chem. 272:17711–17718.
58. Zhou, B., and J. Gitschier. 1997. hCTR1:a human gene for copper uptake identified by
complementation in yeast. Proc. Natl. Acad. Sci. USA 94:7481–7486.
Chapter 16
USING YEAST TOOLS TO DISSECT THE
ACTION OF ANTICANCER DRUGS:
MECHANISMS OF ENZYME INHIBITION
AND CELL KILLING BY AGENTS TARGETING
DNA TOPOISOMERASES
Anna T. Rogojina, Zhengsheng Li, Karin C. Nitiss and John L. Nitiss

Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, Memphis,
TN 38105
1 INTRODUCTION
Resistance to anticancer agents is one of the defining problems of cancer

pharmacology. Drug resistance is clearly a major obstacle to the cure of
many neoplastic diseases. Through the years we have come to understand
that anticancer drug resistance has different biochemical and molecular
mechanisms depending on the class of drug. Early studies centered on trying
to understand “acquired drug resistance”, alterations in cancer cells that were
originally sensitive to one or more chemotherapeutic agents, but which
acquired changes that attenuated the cell cytotoxicity of the original agent,
and also frequently resulted in “multidrug resistance”, resistance to multiple
classes of anticancer agents. While prevention of acquired drug resistance,
and a detailed understanding of mechanisms leading to drug resistance are
still critical concerns of cancer pharmacology, we now understand that many
cancer cells accumulate changes that result in inherent drug resistance.
Alternatively, some of the changes leading to the development of neoplastic
disease can make cancer cells inherently more sensitive to specific classes of
anticancer agents. Current experimental approaches are providing a detailed
description of the molecular changes that occur in many types of cancers.
409
© 2007 Springer.
410 A. T. Rogojina, Z. Li, K. C. Nitiss and J. L. Nitiss
One approach to converting this description of the molecular changes that

occur in cancer into effective therapeutic strategies entails the design of new
agents that specifically inhibit the proteins that have become essential for the
survival and proliferation of cancer cells. A second approach is to optimize
drugs acting against established anticancer targets. Both approaches require
a detailed understanding of how anticancer drugs can lead to cell death,
starting with a precise understanding of the target(s) of a drug, the cellular
process affected by drug action, and the consequences of target inhibition.
DNA topoisomerases are an important target of many clinically active
anticancer agents. Both type I and type II topoisomerases are targeted by
various anticancer drugs. Type I topoisomerases are targeted by the campto-
thecins, which have demonstrated substantial clinical activity in a wide range
of tumors. Topoisomerase II is the target of many chemically diverse agents
including epipodophyllotoxins such as etoposide [74] and anthracyclines
such as doxorubicin [64]. While these agents have substantial antitumor
activity, a wide range of questions remains to be answered. For example,
while it is well established that drugs such as etoposide target topoisomerase
II, the biochemical mechanisms leading to enzyme inhibition remain to be
identified. This is an important issue for topoisomerase II-targeting agents
such as mAMSA that have been clinically disappointing. While many
properties of a drug can influence its clinical activity, differences in
biochemical mechanisms of drugs affecting the same target may be of
decisive importance. Since drugs targeting topoisomerases are typically used
in combination with drugs affecting other targets, a comprehensive
understanding of cell-killing mechanisms is an important tool for devising
optimal multidrug schedules. Finally, as is the case for most types of
cytotoxic chemotherapy, drugs targeting topoisomerases can lead to a wide
range of undesirable side effects. For topoisomerase II-targeting drugs, this
includes the induction of translocations that can cause secondary
malignancies [38].
Yeast has been a powerful model system for studying both the biological
roles of DNA topoisomerases, and for studying the mechanism of action of
drugs targeting these enzymes [41, 42]. Since topoisomerases play roles in
so many different DNA transactions, higher eukaryotes do not readily
tolerate mutations that impair topoisomerase activity. By contrast, intro-
duction of defined topoisomerase alterations has proven to be a productive
approach to understanding topoisomerase biology as well as drug action.
This review concentrates on using yeast to study antitopoisomerase drug
action. Several recent reviews have provided a detailed description of the
roles of topoisomerases in different biological processes [41, 42, 71].
Chapter 16: Topo-Targeting Agents 411
2 HOW TOPOISOMERASE-TARGETING
DRUGS KILL CELLS: USING YEAST
FOR MECHANISM-BASED STUDIES
OF DRUG ACTION AND DRUG SPECIFICITY
The reaction mechanism of all topoisomerases includes the transient

cleavage of DNA by a tyrosine participating in a transesterification reaction,
resulting in the formation of a covalent phosphotyrosine linkage with DNA,
and concomitant breaking of the phosphodiester backbone of DNA. Type I
topoisomerases make single-strand breaks in DNA while type II enzymes in
eukaryotic cells are homodimeric enzymes that introduce double-strand
breaks using one tyrosine from each subunit. DNA topoisomerases use these
strand breaks as DNA gates for carrying out strand passage and altering
DNA topology. After strand passage has occurred, a reverse reaction reseals
the DNA break, using the energy of the phosphotyrosine bond. A full
description of mechanistic and structural aspects of topoisomerase
biochemistry can be found in recent reviews [11, 72]. Most drugs targeting
DNA topoisomerases act by increasing the level of the cleaved intermediate
where the enzyme is covalently bound to DNA. Typically, such agents
reversibly inhibit the DNA ligation reaction that seals DNA breaks after
strand passage. Because the enzyme remains trapped on the DNA, drugs
targeting topoisomerases will inhibit enzyme activity, as well as generating
enzyme-mediated DNA damage.
Although topoisomerases are critical for many DNA transactions that take
place during replication, transcription, and chromosome segregation, the
major killing mechanism for these agents arises from the generation of
enzyme-mediated DNA damage. This hypothesis was demonstrated using
yeast strains with alterations in DNA topoisomerases. The idea behind the
experiments is as follows: if topoisomerase-targeting drugs kill cells by
depriving cells of an activity important for survival, then decreasing the level
of the targeted topoisomerase should lead to enhanced drug sensitivity. By
contrast, if enzyme-mediated DNA damage is responsible for cell killing,
then loss of topoisomerase activity will decrease the amount of enzyme-
mediated DNA damage, and cell survival will be enhanced.
The first experiments testing this experimental scheme were applied to
topoisomerase I. Topoisomerase I is not required for viability in Saccharomyces
cerevisiae, and cells carrying a deletion of the TOP1 gene have subtle
phenotypes, with minimal effects on cell viability [22, 65, 66]. Therefore, a
drug targeting topoisomerase I such as camptothecin could result in cell
killing by two possible mechanisms. The first mechanism is enzyme-
mediated DNA damage, which would be abolished upon deletion of the
TOP1 gene. The second mechanism would occur if camptothecin affects (an
essential) target besides topoisomerase I. In the latter case, deletion of
topoisomerase I would not prevent camptothecin-induced cell killing. When

yeast cells lacking top1 were treated with camptothecin, drug-induced
cytotoxicity was completely eliminated [18, 39]. This result eliminated the
possibility that camptothecin targets some other important function, and
established that the sole cytotoxic target of camptothecin (in yeast) is
topoisomerase I.
A similar series of experiments were carried out for drugs that target DNA
topoisomerase II. Since yeast topoisomerase II is essential for viability [15,
23], the approach described above required modification. First, a simple test
for poisoning of topoisomerase II could be carried out by assessing the effect
of enzyme overexpression on drug sensitivity [45] This type of experiment
can show that poisoning, i.e., generation of enzyme-mediated DNA damage
plays a role in drug sensitivity. However, it cannot demonstrate whether
inhibition of enzyme activity also contributes to cell killing. To test this
point, drug sensitivity can be examined in contexts where enzyme activity is
reduced, but not completely eliminated. Since several temperature-sensitive
alleles of TOP2 have been described, one way to reduce enzyme activity is
to grow strains at a semi-permissive temperature. For example, the top2-1
allele, has normal enzyme activity at 25°, greatly reduced activity (∼10% of
wild type) at 30°, and minimal activity at 35° [15]. Importantly, cells are still
able to grow at 30°, albeit with a substantially reduced growth rate. Thus, if
cell killing depends entirely on generation of enzyme-mediated DNA
damage, the reduced enzyme activity at 30° should result in drug resistance.
Conversely, if reduction of enzyme activity also plays a role in cell killing,
loss-of-enzyme activity due to the thermolabile enzyme should enhance the
effects of drug-mediated enzyme inhibition. Growing top2-1 cells at 30°
clearly leads to high levels of resistance to both etoposide and mAMSA,
indicating that generation of DNA damage is the most important determinant
of cell killing [46]. Furthermore, since changing Top2 activity abolished cell
killing, cytotoxicity by these agents in yeast depends only on the drugs
action against Top2. While this experimental strategy for testing drug
specificity can work for any drug causing cell killing in yeast, the
identification of alleles conferring resistance to multiple classes of Top2
poisons has proven to be a simpler experimental strategy. In practice, one
compares the sensitivity of a compound in an isogenic pair of yeast strains
that differ only at the Top2 gene, with one strain carrying a wild-type Top2
gene, and the other a drug resistant allele [29, 50].
The experimental systems described above have clearly demonstrated the
importance of generation of enzyme-mediated DNA damage in cell killing by
drugs targeting either topoisomerase I or topoisomerase II. One implication of
these results is that since cell killing does not depend significantly on
inhibition of enzyme activity, there may not be a strong rationale for
combining drugs targeting both topoisomerases. This combination has been
investigated in both preclinical models and in several clinical trials.

Frequently the combination of drugs targeting topoisomerase I and
topoisomerase II leads to high levels of toxicity, but little clinical efficacy
[24, 40, 56]. While elevated levels of DNA damage may be achievable by
combining topoisomerase I and topoisomerase II targeting agents, there is no
other mechanistic rationale for this type of combination.
In addition to testing the general mechanism of action of drugs targeting
topoisomerases, the strains described above have been very useful in
demonstrating the targets of novel agents. For example, fluoroquinolones
targeting eukaryotic topoisomerase II were shown to be very specific for
topoisomerase II [17]. In some cases, the yeast model systems clarified
mechanisms of drug action in cases where biochemical data were ambiguous
or contradictory [47, 50]. Finally, this distinct pattern of sensitivity can be
used for screens for identifying new topoisomerase-targeting agents.
Another important aspect of drugs targeting DNA topoisomerases is that
drug action is typically freely reversible. Removal of drug leads to a rapid
reversal of DNA cleavage both in cells, and with in vitro reactions with
purified topoisomerases [10]. Liu and colleagues proposed a model,
illustrated in Figure 1, that can explain how reversible topoisomerase
cleavage can be converted into irreversible DNA damage [13, 76]. Proteins
tracking along DNA, such as a replication fork, can collide with a covalent
complex. A collision between a replication fork and a Top1 covalent
complex can lead directly to a double strand break (that does not have
protein covalently attached) and a gapped double helix that has protein
covalently bound at the 3′ end of the gap (see Figure 1). The fate of a
collision between a replication fork and a Top2 covalent complex is less
clear. Since Top2 forms a very stable dimer [63], it is unclear whether the
collision might break the dimer apart, leading to a double strand break with
protein attached at each 5′ end. One possibility is that the stalling of the
replication fork provokes a processing reaction (discussed in section 4),
leading to conversion of the reversible
covalent complex into an irreversible lesion.
Figure 1. Topoisomerase - mediated DNA damage is

responsible for cell killing. Covalent Top1 or Top2
complexes acting in front of replication forks can be
converted into irreversible DNA damage. Top1 covalent
complexes can be converted into double-strand breaks
without additional processing, while the mechanism of
generating double-strand breaks by Top2 covalent
complexes remains poorly understood.
It is also instructive to consider the fate of collisions between covalent

complexes and tracking proteins besides replication forks. In the case of
Top2 covalent complexes, a protein tracking along DNA such as RNA
polymerase may provoke similar effects as a replication fork; stalling of the

tracking process, and breaking of the covalent complex or stimulating of
processing reactions. By contrast, a collision of a tracking RNA polymerase
with a Top1 covalent complex may lead to a single-strand break, but will not
readily generate a double-strand break. These considerations would predict
that ongoing DNA replication would be required for double-strand break
generation with Top1-targeting drugs, but not required for Top2-targeting
agents. Indeed, arresting replication prevents cell killing by camptothecin
both in yeast and in mammalian cells, while ongoing DNA replication is not
required for cell killing by Top2-targeting agents.
3 RESISTANCE TO TOPOISOMERASE-
TARGETING DRUGS: IDENTIFYING CHANGES
IN TOPOISOMERASE PROTEINS THAT ALTER
DRUG SENSITIVITY
A general question in cancer pharmacology concerns how cancer cells can

become resistant to anticancer agents. It is appreciated that substantial
information can be obtained by identifying and characterizing mutations in
drug targets that confer resistance. For drugs that act as inhibitors of enzyme
activity, resistance-conferring mutations are typically dominant. Therefore,
the resistant mutations can be readily isolated even in mammalian cells, and
proof that the mutation in the gene encoding the drug target is causally
related to drug resistance is usually straightforward. Mutations leading to
resistance typically occur close to drug-binding sites. Therefore, identifycation
of a spectrum of drug-resistant mutants can be valuable for understanding
drug mechanisms. The unique mechanism of topoisomerase poisons com-
plicates the identification of drug-resistant forms of topoisomerases, and
makes it much more difficult to demonstrate that the identified mutation
causes drug resistance. As described in the previous section, resistance to
topoisomerase poisons is recessive [46, 48]. Interpretation of mutations
leading to drug resistance is also more complicated, since as noted above,
reduction of enzyme activity is sufficient to confer drug resistance. Because of
these technical challenges, yeast has proven to be uniquely useful for iden-
tifying mutant forms of topoisomerases with altered drug sensitivity, and for
demonstrating whether specific mutations cause altered drug sensitivity.
The first camptothecin resistant mutant in topoisomerase I was cha-
racterized by Andoh and colleagues. This mutation was present in the coding
sequence of topoisomerase I in a mammalian cell line that had been
selected for camptothecin resistance [2]. One strategy for demonstrating that
the mutation is responsible for resistance is to reconstitute the identified
mutation in the cDNA of Top1, and following expression in E. coli, showing

that the mutation resulted in a protein that is camptothecin resistant [21].
This laborious approach showed that the resistance in the cell line (at least in
part) arose from mutation(s) in Top1. Similar types of experiments were
carried out using mammalian cell lines selected for resistance to either
etoposide or mAMSA. While several mutations were identified in the coding
sequence of Top2α [6, 25, 32], the inability to easily express eukaryotic
Top2 in E. coli prevented a direct demonstration that the identified mutations
resulted in drug-resistant proteins. Direct demonstration that the identified
mutations altered drug sensitivity first took advantage of the strong homology
between yeast and human topoisomerases. Introduction of the mutations
seen in mammalian cells into the coding sequence of yeast Top2 was suf-
ficient to confer resistance to Top2-targeting drugs [48]. Subsequently, a
more rigorous demonstration that the mutations confer drug resistance was
achieved by complementing yeast top2 mutations by expression of human
topoisomerase II [27]. Expression of alleles of human Top2 carrying muta-
tions identified in drug-resistant mammalian cell lines still complemented a
deficiency of yeast Top2, indicating that the alleles encoded active enzymes.
Furthermore, expression of these alleles in yeast also resulted in resistance to
Top2-targeting drugs, efficiently demonstrating that the identified mutations
alter the drug sensitivity of topoisomerase II [27]. Since the mutations confer
recessive drug resistance, simple introduction of the drug-resistant Top2
allele will not confer drug resistance. Extinguishing expression of wild-type
drug-sensitive alleles of Top2, along with expression of a drug-resistant
allele of Top2 is required to reconstitute drug resistance. While RNA
interference technologies now make such (difficult) experiments plausible,
an additional issue is that ectopic expression of topoisomerases is normally
deleterious [36], further complicating the use of mammalian cells for this
type of experiment.
In addition to characterizing mutations selected in mammalian cells, yeast
represents an ideal system for selecting a broad spectrum of mutants in
topoisomerase genes that alter drug sensitivity (reviewed in [5, 43, 44, 58].
In practice, the experiments in yeast have involved introduction of libraries
carrying a topoisomerase gene that has been subjected to in vitro
mutagenesis, followed by screening or selection for drug-resistant (or hyper-
sensitive) clones. As suggested above, a common class of mutations will be
mutations that reduce topoisomerase activity. To reduce this concern, a
common approach has been to express the topoisomerase gene from a strong
promoter [30, 35]. This approach can also be readily adapted to the
examination of human topoisomerases by mutagenizing a human topoi-
somerase gene, followed by selection for altered drug sensitivity in yeast [3,
52].
Following selection for mutations with alterations in drug sensitivity, a
detailed biochemical analysis of the mutant proteins to establish the
mechanisms of resistance is required. The details of biochemical changes

associated with topoisomerases with altered drug sensitivities is beyond the
scope of this review (see [20, 55, 62, 72] for a discussion of the molecular
mechanisms of drug sensitivity as revealed by biochemical analysis).
However, it is important to note that using this approach to identify
mutations conferring drug resistance has significant shortcomings. Notably,
mutations conferring resistance can arise from reduced enzyme activity.
Identification of some of the determinants of binding of topoisomerase I-
targeting drugs has been made largely because of the determination of three
dimensional x-ray structures of the ternary complex of topotecan (a
camptothecin derivative), DNA, and topoisomerase I [9, 62]. Structures of
Top2:DNA:drug have not yet been reported, nor have plausible detailed
models for drug:protein been proposed.
An alternate approach to understanding drug-protein interactions has been
to identify mutations in Top2 that lead to enhanced drug sensitivity [16, 26].
Mutations conferring drug hypersensitivity probably do not occur solely due
to reduced enzyme activity. Several mutations have been identified leading
to high levels of hypersensitivity to Top2-targeting agents (Rogojina and
Nitiss, unpublished data). This approach may be particularly useful to
generate models for drug binding, and potentially to generate reagents that
can be used to obtain details of drug-protein interactions.
4 EVENTS DOWNSTREAM OF TOPOISOMERASE

INHIBITION: GENES ALTERING SENSITIVITY
TO TOPOISOMERASE-TARGETING DRUGS
An important strength of yeast genetic systems is the ability to identify

genes that are required for survival under stress conditions. Yeast systems
have been particularly important in identifying and characterizing DNA
repair and tolerance pathways. Importantly, much of the work in DNA repair
pathways in yeast has proven to be directly relevant to mammalian repair
pathways [60]. There have been two broad approaches to studying the
genetic control of sensitivity to topoisomerase-targeting agents in yeast. The
first approach takes advantage of knowledge of the type of lesion that is
generated by trapping a topoisomerase covalent complex, and assesses the
roles of relevant repair pathways. More recently, it has been possible to
undertake genome-wide examination of genes required for surviving
topoisomerase-mediated damage. Since camptothecin generates lethal damage
preferentially during S phase [49], and since sensitivity to camptothecin can be
observed without additional mutations that enhance drug accumulation,
several genome-wide screens have identified camptothecin hypersensitive
mutants [4, 8, 14]. The poor accumulation of most topoisomerase II-targeting
agents in yeast has made genome-wide screens more difficult for this class
of drugs. These two approaches are clearly complementary, and both
approaches have led to the identification of important pathways for repairing
topoisomerase-mediated DNA damage. This section will first discuss some
of the targeted approaches, and then summarize some of the interesting
results from genome-wide approaches.
The repair of topoisomerase-mediated DNA damage requires the
collaboration of several distinct DNA repair pathways. As shown in Figure
1, replication in the presence of Top1 poisons leads directly to double-strand
breaks. As expected, cells lacking double-strand break repair pathways such
as homologous recombination are hypersensitive to camptothecin [39]. The
repair of camptothecin-induced double-strand breaks includes recruitment
of the histone variant H2AX to the site of double-strand breaks, with
subsequent recruitment of other repair proteins [7, 53, 57]. Since the
generation of double-strand breaks following collisions with topoisomerase
II may require additional processing steps, the role of H2AX and other
signaling processes are poorly understood with topoisomerase II poisons.
Nonetheless, double-strand break repair pathways also play essential roles in
cell survival following exposure to topoisomerase II poisons. Interestingly,
the nonhomologous end-joining pathway of double-strand break repair is
important for cell survival following exposure to the clinically important
Top2 poison etoposide [31], which may relate to the genesis of translocations
leading to secondary cancers. By contrast, yeast cells lacking nonhomologous
end-joining are not hypersensitive to camptothecin (unpublished data),
consistent with results obtained with DT-40 (chicken) cells [1].
In addition for the need to repair double-strand breaks, there are other
aspects of topoisomerase/DNA covalent complexes that require other DNA
repair and stress tolerance pathways. For example, in addition to double-
strand break repair, recombination pathways and replication functions are
also required for restarting of blocked or collapsed replication forks [12, 37].
Topoisomerase: DNA covalent complexes have been shown to impede
replication fork progression, leading to a requirement for functions needed
for fork restart. Mutants have been isolated that are hypersensitive to Top1
poisons and are defective in functions that are important for lagging strand
DNA synthesis [59]. These results indicate that the aberrant DNA replication
that occurs in the presence of Top1 poisons results in an enhanced
requirement for specific DNA replication functions that may play roles in
restarting blocked or broken replication forks. In addition to replication
restart mechanisms, a variety of checkpoint functions are also needed to
ensure that compete and accurate completion of replication occurs. Mutants
defective in both S-phase and DNA damage checkpoints are hypersensitive
to camptothecin [70] (see below).
A third aspect of repair of topoisomerase-mediated DNA damage is the

repair of DNA that has protein covalently bound to a DNA end. In principle,
a variety of repair nucleases, possibly in conjunction with proteolytic enzymes
could repair this type of damage. Nash and colleagues have identified a
specific repair enzyme that participates in the repair of Top1-mediated DNA
damage [54, 75]. This enzyme, termed tyrosyl-DNA phosphodiesterase
(Tdp1) can remove peptides covalently bound to DNA via a 3′ phosphor-
tyrosine linkage. Tdp1 was originally isolated from yeast by looking directly
for a biochemical activity able to disjoin a 3′ phosphotyrosine linkage from
an oligonucleotide [75]. The yeast gene was subsequently cloned based on
its ability to confer sensitivity to camptothecin. Interestingly, tdp1– single
mutants have only modest sensitivity to camptothecin, but show synergistic
sensitivity in combinations with mutations in other repair genes (such as the
RAD9 checkpoint function) [54].
The observation that tdp1– single mutants have modest camptothecin
sensitivity has led to a search for other proteins that may participate in the
removal of proteins covalently bound to DNA. Several groups have shown
that the mutation in either component of the Rad1–Rad10 nuclease greatly
enhances the camptothecin sensitivity of tdp1– mutants [14, 34, 68]. The
nuclease activity of Rad1–Rad10 complex is consistent with removal of a
protein covalently bound to the 3′ end of DNA. Similarly, loss-of-function of
the Mms4–Mus81 nuclease also enhances the sensitivity of tdp1– mutants to
camptothecin [14, 34]. However, both Rad1–Rad10 and Mms4–Mus81 also
play critical roles in homologous recombination. Therefore, it will be
important to directly demonstrate that these nuclease complexes are able to
process 3′ phosphotyrosine adducts on DNA.
Removal of Top1 covalent complexes by Tdp1-dependent pathways is
likely to be more complicated than the simple action of the Tdp1 protein.
Removal of Top1 protein by Tdp1p is highly inefficient unless Top1p is
denatured or proteolyzed prior to Tdp1 action. Furthermore, it is unclear how
Tdp1p is recruited to Top1 covalent complexes. One plausible model is that
covalent complexes are first recognized by unknown proteins, leading to
ubiquitination and proteolysis of Top1p which would create a substrate for
Tdp1 or other proteins that can complete the removal of the phosphotyrosyl
peptide from DNA.
As indicated above, double-strand break repair pathways play critical
roles in the repair of Top2-mediated DNA damage [61]. There is less
information concerning possible roles of other repair pathways. As indicated
below, some of the genomic approaches are beginning to yield information
concerning pathways important for surviving Top2-mediated DNA damage.
Since Top2p forms a 5′ phosphotyrosyl linkage with DNA, it was
predicted that Tdp1p would not be active against lesions involving Top2p.
Recently, we reported that deletion of the TDP1 gene in yeast confers
hypersensitivity to Top2-targeting agents. Combining tdp1 mutations with

deletions of genes involved in nonhomologous end-joining, excision repair,
or post-replication repair enhanced sensitivity to Top2-targeting drugs over
the level seen with single mutants, suggesting that Tdp1 may function in
collaboration with multiple repair pathways. Deletion of tdp1 can sensitize
yeast cells to drugs targeting Top2 even when TOP1 is deleted, excluding
models where Top2 covalent complexes can lead to trapping of Top1 (with a
requirement that Tdp1 function to repair consequences of Top1 being
trapped). A critical experiment showed that bacterially expressed yeast
Tdp1p is able to remove a peptide derived from yTop2 that is covalently
bound to DNA by a 5′ phosphotyrosyl linkage. Additionally, mutation of one
of the essential histidines of Tdp1 to alanine completely ablated activity
against 5′ phosphotyrosyl substrates, demonstrating that the enzyme
mechanism against 5′ and 3′ modified substrates is probably identical [51].
These results establish Tdp1 as part of a pathway for removing both Top1-
and Top2-mediated DNA damage. Furthermore, some excision repair
functions (such as the Rad2p) may also define other pathways for removing
Top2 covalently bound to DNA.
The development of a complete set of yeast strains that carries deletions
of each yeast open reading frames represents an outstanding tool for genetic
analysis on a genome-wide scale [67]. This resource allows identification of
all (nonessential) genes that confer a particular phenotype, and eliminates
much of the tedious mutant purification required in conventional genetic
screens. Several genomic screens have been carried out for yeast mutants
that are hypersensitive to DNA damaging agents. Some of these screens
have used camptothecin sensitivity as part of a secondary characterization [4,
8, 73]. These screens led to the identification of more than 100 genes that
confer hypersensitivity to camptothecin. These include many repair proteins
described above, such as recombination and checkpoint functions. They also
include proteins required for a variety of other processes including
transcription, and chromatin structure that may plausibly function in repair
processes. An intriguing set of mutants has no obvious connection to DNA
metabolism. Further experiments will be needed to determine the genetic
pathways that these proteins represent.
There have also been efforts to use the deletion collection to identify
mutants required for repairing Top2 mediated damage. Osheroff and
colleagues screened the yeast deletion collection using high concentrations
of etoposide. This screen yielded mutants required for homologous
recombination as well as a novel gene, MMS22 that is also required for
survival following exposure to alkylating agents and ionizing radiation. The
small number of genes identified probably stems from the poor accumulation
of etoposide in wild-type yeast cells. To overcome this difficulty, we have
taken advantage of mutations in the TOP2 coding sequence (described in
section 3) that confer hypersensitivity to mAMSA. A useful property of
these mutants is that they confer dominant hypersensitivity to mAMSA;

hence they can be readily transformed into yeast cells. In a preliminary
screen, we analyzed the mAMSA sensitivity of the approximately 300
deletion strains previously shown to be sensitive to either MMS or ionizing
radiation. Approximately 100 strains also showed hypersensitivity to
mAMSA (Jeffrey Berk and JL Nitiss, in preparation). These experiments
demonstrate that the genetic control of sensitivity to Top2-targeting drugs is
also complex, with many of the same proteins participating in the repair
of both Top1 and Top2 damage. Interestingly, several deletions confer
camptothecin sensitivity but do not affect sensitivity to mAMSA. Other
deletions show hypersensitivity to mAMSA, but wild-type sensitivity to
camptothecin. These results indicate that repair of Top1 and Top2 damage
differ in some details in yeast, as would be expected from the differences in
the lesions generated.
While the genomic approaches using the yeast deletion strains are very
powerful, there are some shortcomings. First, the screens that have been
carried out identify only genes that are not essential for viability. Several
genes that participate in DNA repair also carry out essential functions. Second,
it is unclear how many relevant mutants have been missed in the screens that
have been carried out. While this is a shortcoming of any genetic screen, the
fact that limited amounts of camptothecin are accumulated in wild-type yeast
cells suggests that mutants that are sensitive to small amounts of damage are
most likely to be isolated.
An alternate way of dealing with the issues described above is to devise a
way of mimicking drug action by mutations in target proteins. Mutations in
topoisomerases that result in enzymes that are partly defective in religation
or that exhibit elevated levels of DNA cleavage would generate the same
effects in cells as treatment of cells with a topoisomerase-targeting agent
[33]. Levin et al. isolated this type of mutation in yeast Top1. Bjornsti
and colleagues have subsequently used these Top1 mutants to identify
temperature-sensitive mutants that are inviable at a nonpermissive temperature
only when the cells express an elevated cleavage mutant of Top1 [33, 59].
Importantly, they showed that this type of screen could identify mutations in
genes that are essential for viability [19]. For example, the mutant screen
identified mutations in CDC45, a protein involved in both replication
initiation and elongation [59]. Mutations were also identified in UBC9, an
E2 specifically involved in conjugation of the ubiquitin homolog SUMO to a
variety of proteins [28].
The approach taken by Bjornsti and colleagues may also be applicable to
studying Top2-targeting drugs. We identified a mutation in human Top2α
changing Asp48 to Asn that also has the property of failing to transform
yeast cells deficient in recombination repair [69]. In repair proficient strains,
the Asp48Asn mutant can be expressed and complements a temperature
sensitive top2 mutation. Purified Asp48Asn Top2α has relaxation activity

similar to the wild-type enzyme, but the purified protein exhibited appro-
ximately fivefold increase in levels of drug-independent cleavage compared
to the wild-type enzyme [69]. The lethality exhibited by the mutant in recom-
bination deficient mutants is likely due to its enhanced drug independent
cleavage. Since the mutant can introduce Top2-mediated DNA damage in
the absence of drug, it can be exploited in screens for mutants that are
deficient in repairing Top2-mediated DNA damage.
The genetic approaches outlined above provide important tools for
understanding the repair of DNA damage by both Top1- and Top2-targeting
drugs. There are additional tools that are needed to construct biochemically
testable models of repair of topoisomerase mediated DNA damage. We still
lack the capability to accurately measure topoisomerase mediated damage in
yeast cells. Efficient measurement of topoisomerase covalent complexes in
yeast cells could be used to determine the processes that convert covalent
complexes into irreversible damage, and then to measure the rates of
disappearance of the protein that is covalently bound to DNA.
Figure 2. Pathways for the repair of Top2 mediated DNA damage. Following recognition of
Top2 covalent complexes (perhaps by collision with replication forks as shown in Figure 1),
repair can be initiated either by proteolysis or by nucleolytic processing. Proteolysis will not
completely remove the protein, since the phosphotyrosyl linkage to DNA cannot be removed
by proteases. Therefore, after proteolysis, a nucleolytic processing step is still required. The
product of nucleolytic processing is a DNA molecule containing a double-strand break.
Figure 2 shows a conceptual model for repairing Top2 mediated damage;

similar models can be proposed for the repair of Top1 damage. Trapped
covalent complexes are recognized by (unknown) proteins, leading to initial
processing. The first step may be proteolysis of the bound protein, or

nucleolytic removal of the protein, and we suggest that there may separate
pathways depending on proteolysis or nucleolytic removal. Proteolysis is
insufficient to completely remove the protein, so a nucleolytic processing
step is required after proteolysis. The advantage of proteolytic pathways is
likely the more efficient removal of a small peptide bound to DNA
compared to a large protein. For example, Top2 generates a footprint on
DNA of about 30 nucleotides, which may substantially hinder access of
nucleolytic repair activities. The product of complete removal of Top2 is a
DNA containing a double-strand break. Pathways such as homologous
recombination or nonhomologous end-joining would then process this
broken DNA. An interesting speculation is that specific processing pathways
may preferentially use specific double-strand break repair pathways. For
example, a proteolysis-dependent pathway may be preferentially commit
substrates to homologous recombination.
5 FUTURE PROSPECTS FOR UNDERSTANDING

THE ACTION OF DRUGS TARGETING
TOPOISOMERASES USING YEAST
Drugs targeting DNA topoisomerases are highly active against a variety

of tumors. This provides a strong incentive for continuing investigations into
the mechanisms of drug action. As this review highlights, most of the critical
questions regarding the action of topoisomerase-targeting drugs have not yet
been answered. The following questions continue to fascinate us, and it is
likely that yeast will continue to be an important tool in answering the
questions.
First, how does the binding of a topoisomerase targeting drug lead to
trapping of covalent complexes? Can this information be used to rationally
design new topoisomerase targeting drugs with improved spectra of activity?
Clearly, these questions will require many different approaches, and the
tools described in section 3 will certainly play a role
Second, what are the different ways that topoisomerase-mediated damage
can be repaired? For topoisomerase II, the repair pathways are particularly
critical, since treatment of patients with drugs such as etoposide can lead to
oncogenic translocations. Presumably, topoisomerase-mediated translocations
arise from one or more repair pathways. Knowledge of the different repair
pathways may indicate which repair pathways are error-prone (i.e., prone to
generating interchromosomal translocations). This may lead to approaches
that minimize the translocations without compromising clinical efficacy.
Finally, there are a broad range of specific biochemical and pharmacological

questions about currently used topoisomerase targeting agents, and new
experimental drugs. The clear ability of yeast systems to answer questions
not accessible with other experimental systems will hopefully make it easier
to bring novel, safe therapies to a clinical setting more rapidly and
efficiently.
ACKNOWLEDGMENTS
Work in our laboratory is supported by grants CA 52814 and CA82313

from the National Cancer Institute, and by the American Lebanese Syrian
Associate Charities (ALSAC).
REFERENCES
1. Adachi, N., S. So, and H. Koyama. 2004. Loss of nonhomologous end joining confers
camptothecin resistance in DT40 cells. Implications for the repair of topoisomerase
I-mediated DNA damage. J. Biol. Chem. 279:37343–37348.
2. Andoh, T., K. Ishii, Y. Suzuki, Y. Ikegami, Y. Kusunoki, Y. Takemoto, and
K. Okada. 1987. Characterization of a mammalian mutant with a camptothecin-resistant
DNA topoisomerase I. Proc. Natl. Acad. Sci. USA 84:5565–5569.
3. Benedetti, P., P. Fiorani, L. Capuani, and J. C. Wang. 1993. Camptothecin resistance
from a single mutation changing glycine 363 of human DNA topoisomerase I to
cysteine. Cancer Res. 53:4343–4348.
4. Bennett, C. B., L. K. Lewis, G. Karthikeyan, K. S. Lobachev, Y. H. Jin, J. F.
Sterling, J. R. Snipe, and M. A. Resnick. 2001. Genes required for ionizing radiation
resistance in yeast. Nat. Genet. 29:426–434.
5. Bjornsti, M. A., A. M. Knab, and P. Benedetti. 1994. Yeast Saccharomyces cerevisiae
as a model system to study the cytotoxic activity of the antitumor drug camptothecin.
Cancer Chemother. Pharmacol. 34 Suppl:S1–5.
6. Bugg, B. Y., M. K. Danks, W. T. Beck, and D. P. Suttle. 1991. Expression of a mutant
DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to
teniposide. Proc. Natl. Acad. Sci. USA 88:7654–7658.
7. Celeste, A., S. Petersen, P. J. Romanienko, O. Fernandez-Capetillo, H. T. Chen,
O. A. Sedelnikova, B. Reina-San-Martin, V. Coppola, E. Meffre, M. J.
Difilippantonio, C. Redon, D. R. Pilch, A. Olaru, M. Eckhaus, R. D. Camerini-
Otero, L. Tessarollo, F. Livak, K. Manova, W. M. Bonner, M. C. Nussenzweig, and
A. Nussenzweig. 2002. Genomic instability in mice lacking histone H2AX. Science
296:922–927.
8. Chang, M., M. Bellaoui, C. Boone, and G. W. Brown. 2002. A genome-wide screen for
methyl methanesulfonate-sensitive mutants reveals genes required for S phase progression in
the presence of DNA damage. Proc. Natl. Acad. Sci. USA 99:16934–16939.
9. Chrencik, J. E., B. L. Staker, A. B. Burgin, P. Pourquier, Y. Pommier, L. Stewart,
and M. R. Redinbo. 2004. Mechanisms of camptothecin resistance by human
topoisomerase I mutations. J. Mol. Biol. 339:773–784.
10. Corbett, A. H., and N. Osheroff. 1993. When good enzymes go bad: conversion of
topoisomerase II to a cellular toxin by antineoplastic drugs. Chem. Res. Toxicol. 6:585–597.
11. Corbett, K. D., and J. M. Berger. 2004. Structure, molecular mechanisms, and
evolutionary relationships in DNA topoisomerases. Annu. Rev. Biophys. Biomol. Struct.
33:95–118.
12. Cox, M. M., M. F. Goodman, K. N. Kreuzer, D. J. Sherratt, S. J. Sandler, and K. J.
13. D’Arpa, P., C. Beardmore, and L. F. Liu. 1990. Involvement of nucleic acid synthesis
in cell killing mechanisms of topoisomerase poisons. Cancer Res. 50:6919–6924.
14. Deng, C., J. A. Brown, D. You, and J. M. Brown. 2005. Multiple endonucleases
function to repair covalent topoisomerase I complexes in Saccharomyces cerevisiae.
Genetics. 170: 591–600.
15. DiNardo, S., K. Voelkel, and R. Sternglanz. 1984. DNA topoisomerase II mutant of
Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter
molecules at the termination of DNA replication. Proc. Natl. Acad. Sci. USA 81:2616–2620.
16. Dong, J., J. Walker, and J. L. Nitiss. 2000. A mutation in yeast topoisomerase II that
confers hypersensitivity to multiple classes of topoisomerase II poisons. J. Biol. Chem.
275:7980–7987.
17. Elsea, S. H., N. Osheroff, and J. L. Nitiss. 1992. Cytotoxicity of quinolones toward
eukaryotic cells. Identification of topoisomerase II as the primary cellular target for the
quinolone CP- 115,953 in yeast. J. Biol. Chem. 267:13150–13153.
18. Eng, W. K., L. Faucette, R. K. Johnson, and R. Sternglanz. 1988. Evidence that
DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin. Mol.
Pharmacol. 34:755–760.
19. Fiorani, P., and M. A. Bjornsti. 2000. Mechanisms of DNA topoisomerase I-induced
cell killing in the yeast Saccharomyces cerevisiae. Ann. N. Y. Acad. Sci. 922:65–75.
20. Fortune, J. M., and N. Osheroff. 2000. Topoisomerase II as a target for anticancer
drugs: when enzymes stop being nice. Prog. Nucleic Acid Res. Mol. Biol. 64:221–253.
21. Fujimori, A., W. G. Harker, G. Kohlhagen, Y. Hoki, and Y. Pommier. 1995.
Mutation at the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell line
resistant to camptothecin. Cancer Res. 55:1339–1346.
22. Goto, T., and J. C. Wang. 1985. Cloning of yeast TOP1, the gene encoding DNA
topoisomerase I, and construction of mutants defective in both DNA topoisomerase I
and DNA topoisomerase II. Proc. Natl. Acad. Sci. USA 82:7178–7182.
23. Goto, T., and J. C. Wang. 1984. Yeast DNA topoisomerase II is encoded by a single-
copy, essential gene. Cell 36:1073–1080.
24. Hammond, L. A., J. R. Eckardt, R. Ganapathi, H. A. Burris, G. A. Rodriguez, S. G.
Eckhardt, M. L. Rothenberg, G. R. Weiss, J. G. Kuhn, S. Hodges, D. D. Von Hoff,
and E. K. Rowinsky. 1998. A phase I and translational study of sequential
administration of the topoisomerase I and II inhibitors topotecan and etoposide. Clin.
Cancer Res. 4:1459–1467.
25. Hinds, M., K. Deisseroth, J. Mayes, E. Altschuler, R. Jansen, F. D. Ledley, and
L. A. Zwelling. 1991. Identification of a point mutation in the topoisomerase II gene
from a human leukemia cell line containing an amsacrine-resistant form of
topoisomerase II. Cancer Res. 51:4729–4731.
26. Hsiung, Y., S. H. Elsea, N. Osheroff, and J. L. Nitiss. 1995. A mutation in yeast TOP2
homologous to a quinolone-resistant mutation in bacteria. Mutation of the amino acid
homologous to Ser83 of Escherichia coli gyrA alters sensitivity to eukaryotic
topoisomerase inhibitors. J. Biol. Chem. 270:20359–20364.
27. Hsiung, Y., M. Jannatipour, A. Rose, J. McMahon, D. Duncan, and J. L. Nitiss.
1996. Functional expression of human topoisomerase II alpha in yeast: mutations at
amino acids 450 or 803 of topoisomerase II alpha result in enzymes that can confer
resistance to anti-topoisomerase II agents. Cancer Res. 56:91–99.
28. Jacquiau, H. R., R. C. van Waardenburg, R. J. Reid, M. H. Woo, H. Guo, E. S.
Johnson, and M. A. Bjornsti. 2005. Defects in SUMO (small ubiquitin-related
modifier) conjugation and deconjugation alter cell sensitivity to DNA topoisomerase
I-induced DNA damage. J. Biol. Chem. 280:23566–23575.
29. Jannatipour, M., Y. X. Liu, and J. L. Nitiss. 1993. The top2-5 mutant of yeast
topoisomerase II encodes an enzyme resistant to etoposide and amsacrine. J. Biol.
Chem. 268:18586–18592.
30. Jiang, X. 2005. Random mutagenesis of the B'A' core domain of yeast DNA
topoisomerase II and large-scale screens of mutants resistant to the anticancer drug
etoposide. Biochem. Biophys. Res. Commun. 327:597–603.
31. Jin, S., S. Inoue, and D. T. Weaver. 1998. Differential etoposide sensitivity of cells
deficient in the Ku and DNA-PKcs components of the DNA-dependent protein kinase.
32. Lee, M. S., J. C. Wang, and M. Beran. 1992. Two independent amsacrine-resistant
human myeloid leukemia cell lines share an identical point mutation in the 170 kDa
form of human topoisomerase II. J. Mol. Biol. 223:837–843.
33. Levin, N. A., M. A. Bjornsti, and G. R. Fink. 1993. A novel mutation in DNA
topoisomerase I of yeast causes DNA damage and RAD9-dependent cell cycle arrest.
Genetics 133:799–814.
34. Liu, C., J. J. Pouliot, and H. A. Nash. 2002. Repair of topoisomerase I covalent
complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1. Proc. Natl.
Acad. Sci. USA 99:14970–14975.
35. Liu, Y. X., Y. Hsiung, M. Jannatipour, Y. Yeh, and J. L. Nitiss. 1994. Yeast
topoisomerase II mutants resistant to anti-topoisomerase agents: identification and
characterization of new yeast topoisomerase II mutants selected for resistance to
etoposide. Cancer Res. 54:2943–2951.
36. McPherson, J. P., and G. J. Goldenberg. 1998. Induction of apoptosis by deregulated
expression of DNA topoisomerase IIalpha. Cancer Res. 58:4519–4524.
37. Michel, B., M. J. Flores, E. Viguera, G. Grompone, M. Seigneur, and V. Bidnenko.
2001. Rescue of arrested replication forks by homologous recombination. Proc. Natl.
Acad. Sci. USA 98:8181–8188.
38. Mistry, A. R., C. A. Felix, R. J. Whitmarsh, A. Mason, A. Reiter, B. Cassinat,
A. Parry, C. Walz, J. L. Wiemels, M. R. Segal, L. Ades, I. A. Blair, N. Osheroff,
A. J. Peniket, M. Lafage-Pochitaloff, N. C. Cross, C. Chomienne, E. Solomon,
P. Fenaux, and D. Grimwade. 2005. DNA topoisomerase II in therapy-related acute
promyelocytic leukemia. N. Engl. J. Med. 352:1529–1538.
39. Nitiss, J., and J. C. Wang. 1988. DNA topoisomerase-targeting antitumor drugs can be
studied in yeast. Proc. Natl. Acad. Sci. USA 85:7501–7505.
40. Nitiss, J. L. 2002. DNA topoisomerases in cancer chemotherapy: using enzymes to
generate selective DNA damage. Curr. Opin. Investig. Drugs 3:1512–1516.
41. Nitiss, J. L. 1998. DNA topoisomerases in DNA repair and DNA damage tolerance, pp.
517–537. In J. A. Nickoloff, and Hoekstra, M.F. (ed.), DNA Damage and Repair, Vol. 2;
DNA Repair in Higher Eukaryotes. Humana Press, Totawa, NJ.
42. Nitiss, J. L. 1998. Investigating the biological functions of DNA topoisomerases in
eukaryotic cells. Biochimica. Biophysica. Acta 1400:63–81.
43. Nitiss, J. L. 1996. Mutational analysis of topoisomerase II drug action: the yeast test
tube. Anticancer drugs 7 Suppl 3:27–34.
44. Nitiss, J. L. 1994. Yeast as a genetic model system for studying topoisomerase
inhibitors. Adv. Pharmacol. 29B:201–226.
45. Nitiss, J. L., Y. X. Liu, P. Harbury, M. Jannatipour, R. Wasserman, and J. C.

Wang. 1992. Amsacrine and etoposide hypersensitivity of yeast cells overexpressing
DNA topoisomerase II. Cancer Res. 52:4467–4472.
46. Nitiss, J. L., Y. X. Liu, and Y. Hsiung. 1993. A temperature sensitive topoisomerase II
allele confers temperature dependent drug resistance on amsacrine and etoposide: a
genetic system for determining the targets of topoisomerase II inhibitors. Cancer Res.
53:89–93.
47. Nitiss, J. L., P. Pourquier, and Y. Pommier. 1997. Aclacinomycin A stabilizes
topoisomerase I covalent complexes. Cancer Res. 57:4564–4569.
48. Nitiss, J. L., P. M. Vilalta, H. Wu, and J. McMahon. 1994. Mutations in the gyrB
domain of eukaryotic topoisomerase II can lead to partially dominant resistance to
etoposide and amsacrine. Mol. Pharmacol. 46:773–777.
49. Nitiss, J. L., and J. C. Wang. 1996. Mechanisms of cell killing by drugs that trap
covalent complexes between DNA topoisomerases and DNA. Mol. Pharmacol.
50:1095–1102.
50. Nitiss, J. L., J. Zhou, A. Rose, Y. Hsiung, K. C. Gale, and N. Osheroff. 1998. The
bis(naphthalimide) DMP-840 causes cytotoxicity by its action against eukaryotic
topoisomerase II. Biochemistry 37:3078–3085.
51. Nitiss, K. C., M. Malik, X. He, S. W. White, and J. L. Nitiss. 2006. Tyrosyl-DNA
phosphodiesterase (Tdp1) participates in the repair of Top2-mediated DNA damage.
52. Patel, S., B. A. Keller, and L. M. Fisher. 2000. Mutations at arg486 and glu571 in
human topoisomerase IIalpha confer resistance to amsacrine: relevance for antitumor
drug resistance in human cells. Mol. Pharmacol. 57:784–791.
53. Paull, T. T., E. P. Rogakou, V. Yamazaki, C. U. Kirchgessner, M. Gellert, and
W. M. Bonner. 2000. A critical role for histone H2AX in recruitment of repair factors
to nuclear foci after DNA damage. Curr. Biol. 10:886–895.
54. Pouliot, J. J., K. C. Yao, C. A. Robertson, and H. A. Nash. 1999. Yeast gene for a
Tyr-DNA phosphodiesterase that repairs topoisomerase I complexes. Science 286:552–
555.
55. Rasheed, Z. A., and E. H. Rubin. 2003. Mechanisms of resistance to topoisomerase
I-targeting drugs. Oncogene 22:7296–7304.
56. Reck, M., G. Groth, E. Buchholz, E. Goetz, U. Gatzemeier, and C. Manegold. 2005.
Topotecan and etoposide as first-line therapy for extensive disease small cell lung
cancer: a phase II trial of a platinum-free regimen. Lung Cancer 48:409–413.
57. Redon, C., D. Pilch, E. Rogakou, O. Sedelnikova, K. Newrock, and W. Bonner.
2002. Histone H2A variants H2AX and H2AZ. Curr. Opin. Genet. Dev. 12:162–169.
58. Reid, R. J., P. Benedetti, and M. A. Bjornsti. 1998. Yeast as a model organism for
studying the actions of DNA topoisomerase-targeted drugs. Biochim. Biophys. Acta
1400:289–300.
59. Reid, R. J., P. Fiorani, M. Sugawara, and M. A. Bjornsti. 1999. CDC45 and DPB11
are required for processive DNA replication and resistance to DNA topoisomerase
I-mediated DNA damage. Proc. Natl. Acad. Sci. USA 96:11440–11445.
60. Resnick, M. A., and B. S. Cox. 2000. Yeast as an honorary mammal. Mutat. Res.
451:1–11.
61. Sabourin, M., J. L. Nitiss, K. C. Nitiss, K. Tatebayashi, H. Ikeda, and N. Osheroff.
2003. Yeast recombination pathways triggered by topoisomerase II-mediated DNA
breaks. Nucleic Acids Res. 31:4373–4384.
62. Staker, B. L., K. Hjerrild, M. D. Feese, C. A. Behnke, A. B. Burgin, Jr., and
L. Stewart. 2002. The mechanism of topoisomerase I poisoning by a camptothecin
analog. Proc. Natl. Acad. Sci. USA 99:15387–15392.
63. Tennyson, R. B., and J. E. Lindsley. 1997. Type II DNA topoisomerase from
Saccharomyces cerevisiae is a stable dimer. Biochemistry 36:6107–6114.
64. Tewey, K. M., T. C. Rowe, L. Yang, B. D. Halligan, and L. F. Liu. 1984.

Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II.
Science 226:466–468.
65. Thrash, C., A. T. Bankier, B. G. Barrell, and R. Sternglanz. 1985. Cloning,
characterization, and sequence of the yeast DNA topoisomerase I gene. Proc. Natl.
Acad. Sci. USA 82:4374–4378.
66. Thrash, C., K. Voelkel, S. DiNardo, and R. Sternglanz. 1984. Identification of
Saccharomyces cerevisiae mutants deficient in DNA topoisomerase I activity. J. Biol.
Chem. 259:1375–1377.
67. Tong, A. H., M. Evangelista, A. B. Parsons, H. Xu, G. D. Bader, N. Page,
M. Robinson, S. Raghibizadeh, C. W. Hogue, H. Bussey, B. Andrews, M. Tyers,
and C. Boone. 2001. Systematic genetic analysis with ordered arrays of yeast deletion
mutants. Science 294:2364–2368.
68. Vance, J. R., and T. E. Wilson. 2002. Yeast Tdp1 and Rad1-Rad10 function as
redundant pathways for repairing Top1 replicative damage. Proc. Natl. Acad. Sci. USA
99:13669–13674.
69. Walker, J. V., K. C. Nitiss, L. H. Jensen, C. Mayne, T. Hu, P. B. Jensen,
M. Sehested, T. Hsieh, and J. L. Nitiss. 2004. A mutation in human topoisomerase II
alpha whose expression is lethal in DNA repair-deficient yeast cells. J. Biol. Chem.
279:25947–25954.
70. Wan, S., H. Capasso, and N. C. Walworth. 1999. The topoisomerase I poison
camptothecin generates a Chk1-dependent DNA damage checkpoint signal in fission
yeast. Yeast 15:821–828.
71. Wang, J. C. 2002. Cellular roles of DNA topoisomerases: a molecular perspective. Nat.
Rev. Mol. Cell Biol. 3:430–440.
72. Wang, J. C. 1998. Moving one DNA double helix through another by a type II DNA
topoisomerase: the story of a simple molecular machine. Q. Rev. Biophys. 31:107–144.
73. Westmoreland, T. J., J. R. Marks, J. A. Olson, Jr., E. M. Thompson, M. A.
Resnick, and C. B. Bennett. 2004. Cell cycle progression in G1 and S phases is CCR4
dependent following ionizing radiation or replication stress in Saccharomyces
cerevisiae. Eukaryot. Cell 3:430–446.
74. Yang, L., T. C. Rowe, and L. F. Liu. 1985. Identification of DNA topoisomerase II as
an intracellular target of antitumor epipodophyllotoxins in simian virus 40-infected
monkey cells. Cancer Res. 45:5872–5876.
75. Yang, S. W., A. B. Burgin, Jr., B. N. Huizenga, C. A. Robertson, K. C. Yao, and
H. A. Nash. 1996. A eukaryotic enzyme that can disjoin dead-end covalent complexes
between DNA and type I topoisomerases. Proc. Natl. Acad. Sci. USA 93:11534–11539.
76. Zhang, H., P. D’Arpa, and L. F. Liu. 1990. A model for tumor cell killing by
topoisomerase poisons. Cancer Cells 2:23–27.
INDEX
ATPases 5, 114, 142–144, 152, bisubstrate 103

157, 158, 162, 163, 165, 318, bleomycins 319, 327, 331
395, 399
AtMRPs 302 CaaX 101–112, 114, 115
Atr 18, 33, 34, 53–55, 59, 182, Caenorhabditis 155, 179, 196
187, 351 Cdk 3, 4, 6–8, 17, 38–40, 42–44,
Atr/Atrip 53 46–49, 55–57, 92, 142,
Atrip 33, 53, 182 160–161, 165, 301, 321, 341
acquisition 233, CHIP 142, 161, 239–241, 260,
adozelesin 17 375, 386
alkylative 330 CKI’s 44, 43
aminoacylated 87 Claspin 18
angio-genicinhibitors 376 Clbs 44–46, 49, 55, 56, 61, 76,
ansamycins 157–159, 165 88, 89, 91
antifolates 327, 333 Clns 44
aquated 394 CsA 348, 349, 387, 388
ars 2 Cter 235, 236, 238, 240, 248,
aureobasidin 195, 197 255, 262, 263, 287
autophosphorylated 322 camptothecin 332, 333, 335,
aza 332 410–412, 414–420
carboxylmethylation 101
bax 242, 250, 252, 253, 259, caspase 45
266, 267 cDNAs 130, 184, 214, 215, 220,
benzimidazoles 58, 321, 326, 328 221, 223, 244, 245, 247, 248,
benzoquinoid 157, 158 249, 252, 255, 264, 265, 286,
bHLH 146 383, 384, 386, 395, 415
bilayer 296 cdc 4, 6–10, 14, 15, 17–19, 33, 39
bioreductive 337 40, 41, 43–46, 49, 50, 52, 55, 60
biosynthesis 102, 109, 61, 62, 76, 88–92, 102, 104, 106
192–198, 200, 201, 203, 246, 132, 142, 146, 149, 159,
299, 303, 354 160–162, 164, 165, 179–184,
bis-trihalocarbinol 329 199, 200, 321, 322, 361, 420
429
430 Index
centromeres 1, 14, 20, 42, 248 etoposide 201, 316, 410, 412,
centromeric 2, 10, 245, 264, 266 415, 417, 419, 422
ceramidases 195, 198 exocyst 107
cerulenenin 293
chemotypes 322, 323 FKBPs 142, 144, 147, 151, 153,
chromatinized 240, 256, 260 154, 161–163, 347–350, 362,
cisplatin 319, 331, 393–404 365, 383–388
cMOAT 395, 399, 403 FOA 213–217, 246, 251, 256,
cohesins 14, 20, 42, 45 257, 265
counterselection 212, 213, 215, Fraumeni 19, 235
246 farnesylated 103, 105–110, 115,
cross-linking 330, 338 116
cyclin 3, 6, 7, 38–40, 42–46, 48, farnesylation 101–109, 115–116
49, 55, 57, 76, 88–92, 161, farnesyltransferase 101–105, 110,
165, 180, 316, 321, 323, 329, 115
330, 341 forkhead 356
cyclohexanone 329 fumonisins 195, 196
cyclophilin 151, 153, 154, 162,
387, 388 GeneChip 241, 249
cyclosome 44, 55, 89 GGTase 102–105
Gleevec 341
DnaJ 107–109, 158 GPCRs 203
DnaK 158 GTPase 46, 60, 80–84, 109, 110,
DSBs 13, 186, 187, 257, 315, 361, 363
319, 320, 322, 326, 327, 332, geldanamycin 143, 148, 164
333, 337, 411, 413, 414, 418, gemciytabine 201
421, 422 geminin 4
dideoxynucleotides 241 geranylgeranylation 102
dihydroceramidase 198 geranylgeranyltransferase
dihydroceramide 195 102
dimer 5, 144, 154, 155, 160, 234, glutamylcysteine 396, 399, 403
237, 239, 259, 358, glutamylcysteinyl 396
382–384, 399, 413 glycerophospholipid 197
dissociators 211–218, 220, 221,
223–226, 256 hyperrecombination 19
downregulated 18, 20, 90, 92, 257 helicase 5, 9, 11, 33, 87, 318, 320
downregulation 199 hematopoetic 125
dTOR 363 heterodimers 192, 195, 298, 318,
319
Euplotes 128, 129 hexadecanal 194
Exportin 79–81, 87 hnRNPs 76, 87, 88
ellipticine 336, 339 homodimeric 300, 411
enzymology 9 hTERT 129–132, 149
epipodophyllotoxin 332, 410 hus 183
Index 431
hydroxyurea 8, 10, 182, 183, 317, mAMSA 410, 412, 415, 419,
331 420
hyperacetylation 256 Microsatellite 316, 318
hyperphosphorylated 56, 60, 90 MRP 289, 292–296, 300, 302
hyperrecombinant 11 manumycin 105
mcm 4–6, 9, 12, 15–19, 33
imatinib 341 mec 51, 52
immunophilins 147, 151, 153, metabolomics 304
154, 156, 161, 164, 387 metastatic 20
immunosupressant 192 microarray 299, 376, 386–388
importin/karyopherin 78, 80, 81 microdomains 201
importins 78, 79, 81–83, 87, microsequence 128
89–91, 93, 357 minichromosome 4
inositolphosphoceramides 192, misexpression 91
197, 198 mislocalization 91, 110, 219
interactors 257 mislocalized 220
intercalaters 333 mispaired 396, 397
intercalators 327 mTOR 350, 351, 359–362, 364,
intergenic 2 365, 366
initiation 3, 4, 6–9, 14–16, 18, mulitdrug 114, 289, 340, 395,
33, 34, 39, 41, 47, 233, 399, 409, 410
352–354 multicellular 179, 181, 243
intrastrand 394, 396 multichaperone 156
intronic 239 mutator 12
myriocin 192, 195
KRPs 362
Kss/Fus 105 Nibrin 52, 53
karyopherins 78–83, 86, 87 nitroaromatic 331, 332
kinases 3, 4, 6–8, 10, 11, 14, nitrosoguanidine 183, 318
17–19, 33, 34, 38–40, 43, 44, noncomplementation 350
46, 47, 51–55, 57–64, 77, 78, nucleoporins 76, 77, 81, 85, 86
89–91, 105, 106, 142, 145,
147–149, 152, 159–162, 164, overexpression 18, 58, 114, 145,
165, 182, 184, 185, 194–197, 149, 153, 154, 159, 160, 186,
199, 203, 235, 236, 238, 240, 212, 219, 221, 245, 263, 287,
244, 258, 321–323, 341, 347, 291, 293, 295, 301, 321, 342,
351–354, 358–366, 382, 389, 361, 395, 396, 398, 400, 401,
399, 400, 402 404, 412
kinetochores 13, 42, 60, 61, 321 oncogene 17, 19, 78, 106, 161,
165, 199, 212, 233
LacZ 152, 309, 353, 383 orthologue 45, 53, 55, 56, 63,
LexA 152, 383, 386 165, 289
ligase 9, 12, 18, 44, 89, 183, 238 ovalicin 376–378
lysophospholipids 295 overexpress 321, 341
432 Index
p53 75, 126, 142, 145, 146, 148, RecQ 11, 318, 320
157, 165, 211–226, 233–268, RFC 8, 9, 14, 53, 54, 57, 59,
284–288, 379, 403 182
PDREs 297, 298, 300 Rheb 103, 107, 109, 110–116,
Pdr 289, 291–301, 304, 340 352, 363, 364
Pedicin 328 RhoA 102
PolII 355 Rnr 50, 51, 56, 58
Polymorhphism 286, 295 Rothmund 18, 19, 320
PxxP 236 Rpa 8, 9, 18, 54, 59, 319, 320
palmitaldehyde 194 rad 10, 19, 49–51, 64, 182, 183,
palmitoyltranferase 192 184, 185, 186
pastoris 291, 303 rDNA 10, 379
peptidomimetic 103, 104 recombinogenic 124
perfetto 264, 265 reductase 10, 15, 50, 56, 193,
phosphorylation 3, 4, 6–8, 11, 17, 329, 330, 386
20, 33, 40, 41, 43, 46, 48, reductional 15
51–57, 60, 61, 63, 78, 89–93, reinitiation 8, 47, 62
147, 157, 165, 180, 184, 185, relocalized 89
194, 201, 212, 218, 236, 238, replicative 5, 233
300, 351–354, 359, 360, 382 replication 7, 33
phytoceramidase 198 reveromycin 292
phytoceramide 195, 198 rhbA 364
polymerase, DNA 7–9, 12, 14, rheostatable 245, 253, 259, 261
15, 19, 47–49, 59, 125, 155, ribonucleotide 10, 15, 50, 56,
184, 185, 341 185, 329, 330
premeiotic 15, 16 rRNAs 86, 87, 355
prenyltransferases 102
preRC 3–5, 7, 8, 18 Schizosaccharomyces pombe
prereplicative 3, 33 1–20, 33, 39, 43, 52, 53, 56, 64,
primase 9, 54, 59 76, 80, 88, 91, 92, 103, 104,
processivity 9, 184 108–116, 154, 155, 157, 165,
proteasome 89, 90, 147, 238, 321 179, 181, 182, 184, 186, 195,
215, 257, 350
Rab 103 ScRheb 111, 113–115
Rac 103 SpRas 109
RAD52 51, 128, 129, 316, 320, SpRheb 111, 113–115
322, 332–339 securins 19, 42, 45, 46, 49, 56, 57
Raf 112, 113, 148, 160, 162 separase 42, 45, 49
Ral 107 separin 56
RanGAP 80, 82–84 sevenless 160
RanGDP 82–84, 86, 87, 90 signaling 46, 53–55, 60, 63, 64, 92,
RanGEF 80, 82, 84 141, 145–149, 151–153, 155,
RanGTP 82–87, 90 158–160, 181, 185, 186, 191,
Rapamycin 347–366 199, 201, 203, 241–243, 299,
Index 433
349, 351, 352, 353, 354–365, 220, 222, 223, 224, 239, 240,
382, 389, 397, 417 244, 250, 256, 261, 284, 286,
snRNPs 87 290, 297, 298, 354, 355, 361,
sphingomyelinases 195, 198 365, 366, 376, 379–383
spingholipid 299 triapine 329
splitomicin 379, 380 tribrid 382
sporulate 15 transactivation 213, 214, 217,
staurosporine 293 218, 222, 234–237, 239–242,
subcomplexes 5 244–253, 255–268, 284–287,
subtelomeric 359 382, 386
sumolation 236, 238, tumourigenesis 20, 124, 127, 129,
supertrans 253, 259, 268 130, 133, 141, 145, 155, 165
synaptobrevin 108 tumourigenic 123, 130, 131, 148,
synthase 195–198 165, 166
syringomycin 193, 195, 351–366 tumours 7, 17–20, 63, 75, 82, 88,
91, 103, 105, 113, 124, 127,
Tor 113, 347, 348, 350, 351, 352, 130, 131, 148, 155, 161, 165,
353, 354, 355, 356, 357, 358, 187, 203, 211, 221, 225, 226,
359, 360, 361, 362, 363, 364, 233–238, 240–245, 247–254,
365, 366 257, 259, 260, 262–264, 266–
TPRs 77, 142, 144, 147, 268, 290, 315, 316, 320, 322,
151–157, 161, 164 323, 329, 338, 340, 341, 363–
TRanscription/EXport 88 365, 379, 396, 404, 410, 422
Transportin 80, 395, 399
Trrap 351 Ub 142
taxanes 328 ubiquitin 4, 43–45, 48, 55, 57,
telangiectasia 18, 19, 53, 186, 351 89, 201, 212, 214–217, 238,
tetramerization 220, 222, 235, 319, 420
236, 255, 262, 263, 287 ubiquitinate 45
tetratricopeptide 144 ubiquitinated 321
thialysine 107, 113, 114 ubiquitination (Ubi) 41, 45, 46,
topoisomerase 315, 332–334, 49, 61, 108, 236, 418
337, 409–414, 416–418, ubiquitylation 202
420–423
topoisomerase I 327, 410–416 Xenopus 4, 6, 9, 38, 43, 44, 48,
topoisomerase II 327, 410, 412, 54, 91, 110, 162, 179, 181
413, 415, 417, 422 XPA 260, 319
tracrolimus 348 XPC 260
transactivate 249, 250, 263 xeroderma 18, 19, 316, 319
transactivational 158
transactivators 356, 358 YACs 124
transactive 250
transcriptional 50, 56, 58, 87, 92, zebrafish 110
127, 153, 155, 202, 217, 218,

Yeast As A Tool in Cancer Research 2007

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Yeast As A Tool in Cancer Research 2007

Uploaded by

Copyright:

Available Formats

YEAST AS TOOL IN CANCER RESEARCH

Yeast as Tool in Cancer Research

JOHN L.. NITISS

ISBN 978-1-4020-5962-9 (HB)

Printed on acid-free paper

All Rights Reserved

Yeast has proved to be the most useful single-celled organism for

JULIE M. BAILIS ANITA H. CORBETT

RICHARD F. GABER KARL KUCHLER

CARMELA PALERMO CHRISTOPH SCHÜLLER

MARIJA TESIC NANCY C. WALWORTH

In 1970, Lee Hartwell reported a series of genetic experiments showing

areas of cancer research. Accordingly, this volume is intentionally directed

Julie M. Bailis1 and Susan L. Forsburg2

The genetic integrity of cells depends on the complete, accurate

organization of origins of DNA replication, the proteins that assemble at

1.1 DNA replication origins and the origin recognition

replication origins has been estimated at 1 per 20 kb [114] to 1 per 55 kb

2 THE PREREPLICATIVE COMPLEX

The preparation for S phase begins as early as mitosis (M phase) or G1,

3 REPLICATION INITIATION REQUIRES TWO

Although several of the proteins required for DNA replication are

Rum1 [126]. These observations demonstrated that increased levels of CDK

4 ADDITIONAL REPLICATION FACTORS

maintenance of chromosome integrity during S phase, perhaps through a

5 TIMING OF REPLICATION ORIGIN FIRING

Coordination of multiple replication origins on multiple chromosomes

6 RESPONSE TO REPLICATION BLOCKS

During S phase progression, active replication forks may encounter

7 RECOMBINATION PROTEINS IN S PHASE

Several replication mutants display increased levels of recombination,

8 COORDINATION OF S PHASE EVENTS

close apposition of sister chromatids throughout G2 and M phase likely also

passing replication fork or other aspects of S-phase progression remains to

9 DNA REPLICATION IN FISSION YEAST

Meiosis is a specialized cell cycle that generates recombinant, haploid

function in premeiotic DNA replication. However, MCM proteins are not

10 REPLICATION: A CONSERVED PROCESS

Most, if not all, of the proteins involved or implicated in DNA replication

11 PATHWAYS TO CANCER FROM REPLICATION

The recent completion of the S. pombe genome [205] revealed an

Table 1. Fission yeast replication-related proteins and their human homologs

Overexpression of certain replication proteins, such as Cdt1, can promote

important consequence of these findings is that the presence of the MCM

normally prevents premature sister-chromatid separation, can induce cell

1. Adachi, Y., J. Usukura, and M. Yanagida. 1997. A globular complex formation by

38. Dotan, Z. A., A. Dotan, T. Litmanovitch, Y. Ravia, N. Oniashvili, I. Leibovitch,

113. Masutani, C., R. Kusumoto, A. Yamada, N. Dohmae, M. Yokoi, M. Yuasa,

131. Murray, J. M., H. D. Lindsay, C. A. Munday, and A. M. Carr. 1997. Role of

166. Shirahige, K., Y. Hori, K. Shiraishi, M. Yamashita, K. Takahashi, C. Obuse,

183. Tan, D. F., J. A. Huberman, A. Hyland, G. M. Loewen, J. S. Brooks, A. F. Beck,

repair disorders [16] is associated with mutation of the checkpoint protein

10. Kanemaki, M., A. Sanchez-Diaz, A. Gambus, and K. Labib. 2003. Functional

Jennifer S. Searle 1 and Yolanda Sanchez 2

1 THE MANY LAYERS OF CELL CYCLE

1.1 The cell cycle engine is composed of a cyclin

For many years, scientists sought to identify the molecular components

2 LAYERS OF REGULATION: PART I. HOW

2.1 Phosphorylation and proteolysis regulate the activity

Using genetic and biochemical analyses, researchers in the last 15 years

Following activation of MPF and chromosome condensation, and prior to

Figure 2. Progression through mitosis. Following activation of MPF and chromosome