ULBIS, CHRISTINE MARIE HONG (SEAT # 36)

3BIOLOGY-6
FLOW OF GENETIC INFORMATION IN THE CELL
o Replication process of duplication of DNA
o Transcription process of formation of RNA
on a DNA template
o Translation process of protein synthesis in
which the amino acid sequence of the
protein reflects the sequence of bases in the
gene that codes for that protein
o Reverse Transcriptase the enzyme that
directs the synthesis of DNA on an RNA
template
Sequence of bases in DNA encodes genetic
information.
The duplication of DNA, giving rise to new DNA
molecule with the same base sequence as the
original, is necessary whenever a cell divides to
produce daughter cells.
The actual formation of gene products requires
RNA.
The base sequence of DNA is reflected in the
base sequence of RNA.
The three kinds of RNA are involved in the
biosynthesis of proteins.
Of the three, messenger RNA (mRNA)
specifies the identity of one amino acid in
a manner directed by the genetic code.
The flow of genetic information is DNA->RNA>protein.
The only major exceptions are some
viruses (called retroviruses) in which RNA,
rather than DNA, is the genetic material.
In those viruses, RNA can direct its
own synthesis as well as that of
DNA.
Not all viruses in which RNA is the
genetic material are retroviruses,
but all retroviruses have a reverse
transcriptase.
REPLICATION OF DNA
Nucleases enzymes that hydrolyze a nucleic acid
and are specific or DNA or RNA

The process which one double-helical DNA

duplicates molecule to produce two doublestranded molecules is complex.
The cell faces three important challenges
in carrying out the necessary steps.
1. Separating the two DNA strands. The
two strands of DNA are wound around
each other in such a way that they
must be unwound if they are to be
separated. In addition to achieving
continuous unwinding of the double
helix, the cell also must protect the
unwound portions of DNA from the
action of nucleases that preferentially
attack single-stranded DNA.
2. Synthesizing of DNA from the 5 to
the 3 end. Two antiparallel strands
must be synthesized in the same

direction on antiparallel templates. In

other words, the template has one 5>3 strand and one 3->5 strand, as
does the newly synthesized DNA.
3. Guarding
against
errors
in
replication.
Ensuring
that
the
correct base is added to the
growing polynucleotide chain.
Semiconservative Replication - the mode in
which DNA reproduces itself, such that one strand
comes from parent DNA and the other strand is
newly formed
DNA replication involves separation of the
two original strands and production of two
new strands with the original strands as
templates.
Each new DNA molecule contains one strand
from the original DNA and one newly
synthesized strand.
Density-Gradient Centrifugation the technique
of separating substances in an ultracentrifuge by
applying the sample in the top of a tube that
contains a solution of varying densities
Origin of Replication the point at which the DNA
double helix begins to unwind at the start of
replication
New polynucleotide chains are synthesized
using each of the exposed strands as a
template.
Two possibilities exist for growth of the new
strands: synthesis can take place in both
directions from the origin of replication, or in
one direction only.
Replication Forks in DNA replication, the points
at which new DNA strands are formed
A bubble of newly synthesized DNA
between regions of the original DNA is a
manifestation of the advance of the two
replication forks in opposite directions.
The bubbles grow larger and eventually
merge, giving rise to two complete daughter
DNAs. This biological growth of both
polynucleotide chains represents net chain
growth.
Both
new
polynucleotide
chains
are
synthesized in the 5->3 direction.
DNA POLYMERASE
Semidiscontinuous DNA Replication
All synthesis of nucleotide chains occurs in the
5->3 direction from the perspective of the
chain being synthesized. This is due to the
nature of the reaction of DNA synthesis.
The last nucleotide added to a growing
chain has a 3-hydroxyl on the sugar.
The incoming nucleotide has 5triphosphate on its sugar.
The 3-hydroxyl group at the end of the
growing chain is a nucleophile. It attacks
the phosphorus adjacent to the sugar in
the nucleotide to be added to the
growing chain, leading to the elimination

of the pyrophosphate and the formation

of a new phosphodiester bond.
DNA Strands in Opposite Direction
One newly formed strand (leading strand) is
formed continuously from its 5 end to its 3
end at the replication fork on the exposed 3
and 5 template strand.
The other strand (lagging strand) is formed
semi discontinuously in small fragments,
sometimes called Okazaki fragments.
The 5 end of each of these fragments is
closer to the replication fork than the 3
end.
The fragments of the lagging strand are
then linked together by an enzyme
called DNA ligase.
DNA Polymerase from E.coli
The first DNA polymerase discovered was
found in E.coli. At least five DNA polymerases
are present.
Polymerase I (Pol I) consists of a single
polypeptide chain.
Polymerase II (Pol II) is not required for
replication; rather, it is strictly a repair
enzyme.
Polymerase III (Pol III) shares common
subunits in Pol II.
It consists of a core enzyme
responsible for polymerization and
3 exonuclease activity-consisting
of a-, e-, and 0-subunits-and a
number
of
other
subunits,
including a dimer of a-subunits
responsible for DNA binding, and
the y-complex-consisting of y-,
8-,8,x-, and y-subunits which
allows the b-subunits to form a
clamp that surrounds the DNA and
slides along it as a polymerization
proceeds.
It has the highest turnover
number and a huge processivity
compared to polymerase I and II.
Pol IV and Pol V are repair enzymes and
both are involved in a unique repair
mechanism called the SOS response.
Considerations regarding polymerases:
1. Speed of the synthetic reaction (turnover
number)
2. Processivity the number of nucleotides
incorporated in a growing DNA chain
before the DNA polymerase disassociates
from the template DNA
DNA polymerase catalyzes the successive
addition of each new nucleotide to the
growing chain
This
enzyme
forms
DNA
from
deoxyribonucleotides
on
a
DNA
template
Primer in DNA replication, a short stretch of
RNA hydrogen-bonded to the template DNA to

which the growing DNA strand is bonded at

the start of synthesis
DNA polymerase reaction requires all four
deoxyribonucleoside
triphosphates-dTTP,
dGTP, and dCTP.
Proffreading the process of removing
incorrect nucleotides when DNA replication is
in progress
It is done one nucleotide at a time.
Repair the enzymatic removal of incorrect
nucleotides from DNA and their replacement
by correct ones

PROTEINS REQUIRED FOR DNA REPLICATION

Replisome a complex of DNA polymerase
It is the RNA primer, primase and helicase at
the replication fork.
Two questions arise in separating the two
strands of the original DNA so that it can be
replicated.
1. How to achieve continuous unwinding of
the double helix?
2. How to protect single-stranded stretches
of DNA that are exposed to intracellular
nucleases as a result of the unwinding?
Supercoiling and Replication
o DNA gyrase (class II topoisomerase)
It is an enzyme that introduces
supercoiling into closed circular DNA.
It catalyzes the conversion of
relaxed, circular DNA with a nick in
one strand to the supercoiled form
with the nick sealed that is found in
normal prokaryotic DNA.
A light unwinding of the helix before
the nick is sealed introduces the
supercoiling.
The energy require for the process is
supplied by the hydrolysis of ATP.

In replication, the role of the gyrase is different.

The prokaryotic DNA is negatively
supercoiled in its natural state; however,
opening the helix during replication
would introduce positive supercoils
ahead of the replication fork.
DNA gyrase fights positive supercoils by
putting negative supercoils ahead of the
replication fork.
Helicase a protein that unwinds the double
helix of DNA in the process of replication
It promotes unwinding by binding at the
replication fork
This includes DnaB protein and rep
protein
Single-Strand Binding Protein (SSB) in
DNA replication, a protein that protects exposed
single-strtand sections of DNA from nucleases
It stabilizes the single-stranded regions
by binding tightly to these portions of
the molecule

 This protects single-stranded regions

which
are
very
susceptible
to
degradation from hydrolysis by the
nucleases
Primase Reaction
RNA serves as a primer in DNA replication.
A primer in DNA replication must have a
free 3-hydroxyl to which the growing
chain can attach, and both RNA and
DNA can provide this group.
Primase the enzyme that makes a short
section of RNA to act as a primer for DNA
synthesis
It responsible for copying a short stretch
of the DNA template to produce the RNA
primer sequence.
Primosome the complex at the replication
fork in DNA synthesis
It consists of the RNA primer, primase,
and helicase.
Synthesis and Linking of New DNA Strands
The synthesis of two new strands of DNA is
begun by DNA polymerase III.
The newly formed DNA is linked to the 3hydroxyl of the RNA primer, and synthesis
proceeds from the 5 end to the 3 end on both
the leading and the lagging strands.
As the replication fork moves, the RNA primer is
removed by polymerase I, using its exonuclease
activity.
The primer is replaced by deoxynucleotides,
also by DNA polymerase I, using its polymerase
activity.
None of the DNA polymerase can seal the nicks
that remain; DNA ligase is the enzyme
responsible for the final linking of the new
strand.
Summary of DNA Replication in Prokaryotes
1. DNA synthesis is bidirectional. Two replication
forks advance in opposite directions from an
origin of replication.
2. Two direction of DNA synthesis is from the 5
end to the 3 end of the newly formed strand.
One strand (the leading strand) is formed
continuously, while the other strand (the
lagging strand) is formed discontinuously. On
the lagging strand, small fragments of DNA
(Okazaki fragments) are subsequently linked.
3. Five DNA polymerase have been found in E.
coli. Polymerase III is primarily responsible for
the synthesis of new strands. The first
polymerase enzyme discovered, polymerase I,
is involved in synthesis, proofreading, and
repair. Polymerase II, IV and V function as
repair enzymes under unique conditions.
4. DNA gyrase introduces a swivel point in
advance of the movement of the replication
fork. A helix-destabilizing protein, a helicase,
binds at the replication fork and promotes
unwinding. The exposed single-stranded

regions of the template are stabilized by a

DNA-binding protein.
5. Primase catalyzes the synthesis of an RNA
primer.
6. The synthesis of new strands is catalyzed by
Pol III. The primer is removed by Pol I, which
also
replaces
the
primer
with
deoxynucleotides. DNA ligase seals the
remaining nicks.

In E. coli, the polymerizing part of Pol III is a

dimer of the B-subunit, and it forms a closed
ring, called a sliding clamp, around the DNA
chain.
This is a complication for the entire
process of replication as somehow this
ring has to get around the DNA.
The
clamp
cannot
spontaneously
surround the DNA chain because the
dimer is a closed circle. Instead, the part
of the Pol III enzyme that is called the
clamp loader opens the sliding clamp
and inserts the DNA chain.
All clamp loaders are pentameric
enzymes who are members of a family
of ATPases called the AAA+ superfamily.

PROOFREADING AND REPAIR

DNA replication takes place only one each
generation in each cell. It is essential that
fidelity of the replication process be as high as
possible to prevent mutations, which are errors
in replication.
o Mutations changes in DNA, causing
subsequent changes in an organism that
can be transmitted genetically
Errors in replication occur spontaneously only
once in every 109 to 1010 base pairs.
o Proofreading refers to the removal of
incorrect nucelotides immediately after
they are added to the growing DNA during
the replication process
DNA polymerase has three active sites.
Pol I can be cleaved into two major fragments.
One of them (the Klenow fragment)
contains the polymerase activity and
the proofreading activity.
The other contains the 5->3 repair
activity.

Errors in hydrogen bonding lead to the

incorporation of an incorrect nucleotide into a
growing DNA chain once in every 10 4 to 105
base pairs.
DNA polymerase I uses its 3 exonuclease
activity to remove the incorrect nucleotide.
Replication
resumes
when
the
correct
nucleotide is added, also by DNA polymerase I.
During replication, a cut-and-patch process
catalyzed by polymerase I takes place.
The cutting is the removal of the RNA
primer by the 5 exonuclease function of
the polymerase, and the patching is the
incorporation
of
the
required

deoxynucleotides
by
the
function of the same enzyme.

polymerase

Nick Translation a type of DNA repair that

involves polymerase I using its 5 to 3
exonuclease activity to remove primers or
replace damaged nucleotides
Mutagens agents that bring mutation; such
agents include radiation & chemical substances
that alter DNA
Ultraviolet light creates pyramidine dimers.
The pi elections from two carbons on each of
two pyramidines form a cyclobutyl ring,
which distorts the normal shape of the DNA
and
interferes
with
replication
and
transcription.
Chemical damage, which is often cause by
free radicals can lead to a break in the
phosphodiester backbone of the DNA strand.

When damage has managed to escape the

normal
exonuclease
activities
of
DNA
polymerase I and III, prokaryotes have a variety
of other repair mechanisms at their disposal.

Mismatch Repair type of DNA repair that

begins when repair enzymes find two bases that
are incorrectly paired
Area with the mismatched is removed and
DNA polymerases replicate the area again.
If there is a mismatch, the challenge for the
repair system is to know which of the two
strands is the correct one.

Base-Excision Repair a type of DNA repair

that begins with an enzyme removing a damaged
base, followed by removal of the rest of the
nucleotide
1) A base that has been damaged by oxidation
or chemical modification is removed by
DNA glycosylase, leaving an AP site, so
called because it is apurinis or apyrimidinic
(without purine or pyramidine).
2) An AP endonuclease then removes the sugar
and phosphate from the nucleotide.
3) An excision exonuclease then removes
several more bases.
4) Finally, DNA polymerase I fills in the gap, and
DNA ligase seals the phosphodiester
backbone.

Nucleotide-Excision Repair type of DNA

repair in which damaged or deformed DNA is
repaired by removal of a section of DNA
containing the damage
1) Large section of DNA containing the lesion
is removed by ABC excinuclease.
2) DNA polymerase I and DNA ligase then
work to fill the gap.

DNA RECOMBINATION
Genetic Recombination general term for several
processes
whereby
genetic
information
is
rearranged

At a molecular level, genetic recombination is

the exchange of one DNA sequence with
another or the incorporation of a DNA sequence
into another.
Homologous
Recombination
the
genetic
recombination
between
homologous DNA sequences
It also termed general recombination
because the enzymes that mediate the
exchange can use essentially any pair
of homologous DNA sequences.
Nonhomologous Recombination the
genetic
recombination
between
very
different nucleotide sequences
Recombination does not occur randomly in a
chromosome.
o Hot Spots areas on a chromosome that
are likely to have recombination events
Recombination occurs by the breakage and
reunion of DNA strands so that physical
exchange of DNA parts takes place
o Holliday Model this model was deduced
1964 by Robin Holliday
It
is
the
model
for
how
recombination occurs between the
homologous chromosomes.
1) Two
homologous
DNA
segments
align.
In
eukaryotes, this is called
chromosome pairing.
2) A nick occurs at the same
place on two homologous
strands.
3) The DNAs on the two strands
then swap places, or cross
over, at the nick by the
process of strand invasion.
4) The crossing over can then
proceed down each strand of
DNA.
5) The branch migration leads to
strand exchange between the
two homologous DNA pieces.
This leads to exchange of
genes and traits caused by
them.
Recombination is a critical process during
meiosis.
Segregation
of
chromosomes
during
formation of gametes is quite inaccurate,
with estimates indicating that abnormal
chromosomes numbers in gametes, called
aneuploidy, occur in 10%-25% of all
conceptions.

EUKARYOTIC DNA REPLICATION

Eukaryotic chromosomes accomplish DNA
synthesis by having replication begin at
multiple origins of replication, also called
replicators.
o Replicators are specific DNA sequences
that are usually between gene sequences.

The zones where replication is proceeding are

called replicons, and the size of these varies
with the species.

Only chromosomes from cells that have reached

the G1 phase are competent to initiate DNA
replication.
Many proteins are involved in the control of
replication and its link to the cell cycle.
The first proteins involved are seen during a
window of opportunity that occurs between the
early and late G1 phase.
Replication is initiated by a multi-subunit
protein called the origin recognition
complex (ORC).
It is a protein complex bound to
DNA throughout the cell cycle that
serves as an attachment site for
several proteins that help control
replication.
The next protein to bind is an activation
factor called the replication activator
protein (RAP).
It is the protein whose binding
prepares for the start of DNA
replication in eukaryotes.
After the activator protein is bound,
replication licensing factors (RLFs)
can bind.
They are proteins required for DNA
replication in eukaryotes.
Replication cannot proceed until
they are bound.
Some RLF proteins have been
found to be cystolic.
They
have
access
to
he
chromosome
only
when
the
nuclear
membrane
dissolves
during mitosis.
After RLFs bind, the DNA
competent for replication.

is

The activation of cylin-CDKs serves both to

initiate DNA replication and to prevent
formation of another pre-RC.
In the G2 phase, the DNA has been replicated.
During mitosis, the DNA is separated into the
daughter cells. At the same time, the dissolved
nuclear membrane allows entrance of the
licensing factors that are produced in the
cytosol so that each daughter cell can initiate a
new round of replication.

Eukaryotic DNA Polymerases

At least 19 different polymerases are present in
eukaryotes.
The five-best studied polymerases are called a,
B, y, 8, and E.
a, B, 8 and E are found in the
nucleus.
Y form occurs in mitochondria.

Polymerase a has the most subunits. It

has the ability to make primers, but lacks
a 3->5 proofreading activity that has low
processivity.
After making the RNA primer, Pol a,
adds about 20 nucleotides and is
then replaced by Pol 8 and E.

Polymerase 8 is the principal DNA

polymerase in eukaryotes. It interacts
with a special protein PCNA (proliferating
cell nuclear antigen).
PCNA is the eukaryotic equivalent of
the part of Pol III that functions as a
sliding clamp (B). It is a trimer of
three identical proteins that surround
the DNA.

The role of DNA polymerase E is less

clear, but is suggested to be involved in
leading strand replication. It may replase
polymerase 8 in lagging strand synthesis
DNA polymerase B appears to be a repair
enzyme.

DNA polymerase y carries out DNA

replication in mitochondria.

then

The combination of the DNA, ORC, RAP, and

RLFs constitutes the pre-replication complex
(pre-RC).
The complex of DNA, recognition protein
(ORC), activator protein (RAP), and
licensing factors (RLFs) that makes DNA
competent for replication in eukaryotes.
When cyclins combine with CDKs, they can
activate DNA replication and also block
reassembly of a pre-RC after intiation.
o Cyclins are proteins that play an important
role in control of the cell cycle by regulating
the activity of kinases. They are proteins
that are produced in one part of a cell cycle
and degraded in another.
o Cyclin-dependent
protein
kinases
(CDKs) are protein kinases that interact
with cyclins and control replication.

THE EUKARYOTIC REPLICATION FORK

DNA
replication
in
eukaryotes
is
semiconservative.
There is a leading strand with continuous
synthesis in the 5->3 direction and a
lagging strand with discontinuous synthesis
in the 5->3 direction.
An RNA primer is formed by a specific
enzyme in eukaryotic DNA replication and
the primase activity is associated with Pol a.
The formation of Okazaki fragments is
initiated by Pol a.
After the RNA primer is made and a few
nucleotides are added by Pol a, the
polymerase disassociates and is replaced by

Pol 8 and its attached PCNA protein. Another

protein, called RFC (replication factor C),
is involved in attaching PCNA to Pol 8.
The RNA primer is eventually degraded, but
since polymerases do not have 5->3
exonuclease activity, separate enzymes FEN1 and RNase H1 degrade the RNA.

Finally, DNA ligase seals the nicks that

separate the fragments.

Difference in DNA Replication in Prokaryotes and Eukaryotes

Prokaryotes
Eukaryotes
Five polymerases (I, II, III, IV, V)
Five polymerases (a, B, y, 8, E)
Functions of polymerases:
I is involved in synthesis, proofreading, repair and removal of RNA
primers
II is also a repair enzyme
III is the main polymerizing enzyme
IV, V are repair enzymes under usual conditions
Polymerases are also exonucleases
One origin of replication
Okazaki fragments 1000-2000 residues long
No proteins complexed to DNA

Functions of polymerases
a is a polymerizing enzyme
B is a repair enzyme
Y is involved in mitochondrial DNA
synthesis
8 us the main polymerizing enzyme
E is the leading strand replication
enzyme
Not all polymerases are exonucleases
Several origins of replication
Okazaki fragments 150-200 residues long
Histones complexed to DNA