3/28/2012

Recombination and Transposition

(Watson, Baker, Bell, Gann,
Levine and Losick bab 11, Yuwono
bab 13, Weaver Bab 22 &23)

Dr. Ratna Megawati Widharna, SKG, MFT

Mutation, Recombination

Mutation: DNA structural/ composition

change in genome of individual that can
cause phenotipic change in the individu,
although not every mutation can change
the phenotype
Genetic recombination : genetic element
exchange process that occur between
different DNA strand (interstrand) or
between gen parts located in 1 DNA
strand (intrastrand)

Recombination

Started by both homolog chromosom

DNA strand cutting on the same site by
nuclease activity
In Escherichia coli, nuclease is coded
by gen recB and recC which are
responsible in exonuclease V (135
kDalton) and exonuclease V (125
kDalton)
Cutting only occur in one of both
chromosom DNA double strand

3 genetic recombinant
General/Homolog recombinant
Site-specific recombination
Transposition/replicative
recombination

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Recombinant
DNA construction

Trudy McKee,
Biochemistry : The
molecular Basis of
Life

Homolog recombination

Homolog recombination
Cause the exchange DNA
intermolecule that has quite big
homology of nucleotide sequence
Specific character : the process can
occur in every point in homology
area
Recombination occur through the
DNA strand direct breakage which is
followed by the rejoining process

Interchromosom recombination involves

physical exchange process between
chromosom parts
Recombination process takes place
accurately no single nucleotide base pair
is missing or included in recombinant
chromosom
Structure seen as chiasma in meiosis
Chiasma = cutting and splicing DNA strand,
when 2 non-sister chromatids is cut and
joined one another

Key steps of homolog

recombination
1.

Allignment of 2 homologous DNA

molecules (DNA sequences are
identical / nearly identical for a
region of at least 100 base pairs /
so). Despite this high degree of
similarity, DNA molecules can have
small regions of sequence
difference and may carry different
sequence variants, known as
alleles, of the same gene

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Key steps of Homologous

recombination (2)

Key steps of Homologous

recombination (4)

2. Introduction of breaks in the DNA

Breaks may occur in 1 DNA strand /
involve both DNA stands
The nature of these breaks =
feature that largely distinguishes the
2 models (site specific and
transposition recombination)

4. Movement of the Holliday junction

A Holliday junction can move along the
DNA by the repeated melting and
formation of base pairs
Each time the junction moves, base
pairs are broken in the parental DNA
molecules while identical base pairs
are formed in the recombination
intermediate Branch migration

Key steps of Homologous

recombination (3)

Key steps of Homologous

recombination (5)

3. Formation of initial short regions of base

pairing between the 2 recombining DNA
molecules. This pairing occurs when a
single stranded region of DNA originating
single-stranded
from one parental molecule pairs with its
complementary strand in the homologous
duplex DNA molecule Strand invasion
the 2 DNA molecules become
connected by crossing DNA strands
Holliday junction

5. Cleavage of the Holliday junction

Cutting the DNA strands within the Holliday
junction regenerates 2 separate duplex
DNA molecules finishes genetic
exchange RESOLUTION
Which of the 2 pairs of DNA strands in the
Holliday juction are cut during resolution
has a large impact on the extent of DNA
exchange that occurs between the 2
recombining molecules

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Homolog recombination
Started when 2 homolog chromosom
is located near each other so that
the homolog nucleotide sequence
can be exchanged.
Synapsis = contact between the 2
chromosom pairs in Prophase
Model Holliday

Recombination is initiated by the

introduction of a nick in each DNA
molecule at an identical location (Fig 101b)
DNA strands near the nick site can then
be peeled away from their
complementary strands, freeing these
strands to invade, and ultimately basepair with, the homologous duplex (Fig 101c)

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Finishing recombination
(resolution of the Holliday
junction)

Invasion is symmetrical: the same

region of DNA sequence is swapped
between the 2 molecules
Strand invasion generates the Holliday
junction, the key recombination
intermediate
Holliday junction generated by strand
invasion can then move along the DNA
by branch migration increases the
length of DNA exchanged

Heteroduplex DNA

If the 2 DNA molecules are not identicalbut, e.g., carry a few small sequence
differences, as is true often between 2
alleles of the same gene-branch
gene branch migration
through these regions of sequence
difference generates DNA duplexes
carrying 1 or a few sequence mismatches
(B and b alleles in Fig 10-1d & inset)
HETERODUPLEX DNA

Fig 10-2: alternative pairs of DNA

cut sites occur on the branched DNA
To make these cut sites easier to
visualize, the Holliday junction is
rotated to give a square-planner
structure with no crossing strands
The 2 alternative choices for
cleavage sites : 1 and 2 (Fig 10-2)

Splice recombination

The cut sites marked 1 occur in the 2 DNA strands

that were not broken during the initiation reaction
(Fig 10-1b)
If these strands are now cut, ant then covalently
joined (the 2nd reaction catalyzed by DNA ligase), the
resulting DNA molecules will have the structure and
sequence shown in the left bottom of the figure
Splice recombination products because the 2 original
duplexes are now spliced together such that
regions from the parental DNA molecules are
covalently joined together by a region of hybrid
duplex

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Holliday junction cleavage

Crossover product

Generation of splice products results

in reassortment of genes that flank
the site of recombination
crossover product within this DNA
molecule, crossing over has
occurred between the A and C genes

Holliday junction cleavage

Patch products

In contrast, the alternative pair of cut sites in

the Holliday junction (marked 2 in Fig 10-2) is
in the 2 DNA strands that were broken to
initiate recombination. After resolution and
covalent jjoining
g of the strands at these sites,,
the resulting DNA molecules contain a region
or patch of hybrid DNA Patch products
Recombination does not result in reassortment
of the genes flanking the site of initial cleavage
(see fate of the A/a and C/c allele markers)
non-crossover products

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Double-Strand Break Repair Model More

Accurately Describes Many Recombination
Events

Homologous recombination is often

initiated by double-stranded breaks in
DNA double-stranded break repair
pathway (Fig 10-3)
Holliday model : starts with aligned
homologous chromosomes
Initiating event : introduction of a
double-stranded break (DSB) in 1 of
the 2 DNA molecules (Fig 10-3a). The
other DNA duplex remains intact

After introduction of the DSB, a DNAcleaving enzyme sequentially degrade the

broken DNA molecule to generate the
g
of single-strand
g
stranded DNA ((Fig
g
regions
10-3b) creates single-strand extensions
(ssDNA tails) on the broken molecules
terminate with 3 ends
In some cases, both strands at a DSB r
processed, whereas in other cases, only
the 5-terminating strand is degraded

The ssDNA tails generated then invade the

unbroken homologous DNA duplex (Fig 10-3c)
Fig: 1 strand invasion, as likely occurs initially
Next panel : 2 invading strands
I each
In
h case, the
th invading
i
di
strand
t
d base-pairs
b
i
with its complementary strand in the other
DNA molecules
Because the invading strands ends with 3
termini, they can serve as primers for new
DNA synthesis

Elongation from these DNA endsusing the complementary strand in

the homologous duplex as a
template serves to regenerate the
regions of DNA that were destroyed
during the processing of the strands
at the break site (Fig 10-3d,e)

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gene conversion event

If the 2 original DNA duplexes were not identical in

sequence near the site of the break (e.g, having
single base-pair changes), sequence information
could be lost during recombination by the DSB-repair
pathway.
Fig10 3: sequence information lost from the gray
Fig10-3:
DNA molecule as a result of DNA processing is
replaced by the sequence present on the blue duplex
as a result of DNA synthesis
These nonreciprocal step in DSB-repair sometimes
leaves a genetic trace-giving rise to a gene
conversion event

The 2 Holliday junctions found in the

recombination intermediates generated by
this model move by branch migration and
ultimately are resolved to finish
recombination
Once again, the strands that are cleaved
during resolution of these Holliday
junctions determine whether the product
DNA molecules will contain reassorted
genes in the regions flanking the site of
recombination (i.e result in crossing over)
or not

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In contrast, when 2 Holliday junction are

cleaved using different sites, crossover
products are generated (panel C of figure)
Junction x was cleaved at site 1 whereas
junction y was cleaved at site 2
Notice that now gene A is linked to gene b,
whereas gene a is linked to gene B; thus
reassortment of the flanking gene has
occurred.
Cleavage of junction x at site 2 and junction y
at site 1 also generates crossover products

Simple rule (1)

For each junction, there are 2 possible

cleavage sites (site 1 and site 2)
If both junctions are cleaved in the same
way, that is either both at site 1 or both at
site 2,
2 then non-crossover
non crossover products will be
generated (panel b of the figure).
Notice, the allele markers A/B and /b are
still on the same DNA molecules as they
were in the parental chromosomes.
Cleavage of both junctions at site 1 : noncrossover products

Cleavage at both junctions at site 2

will give a patch product (patch +
patch, non-crossover products)
Cleavage at both junctions at site 1
also gives a patch product (splice +
splice = patch because the 2nd
splice-type resolution essentially
underdoes the rearrangement
caused by the 1st cleavage

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Homologous recombination in
Eucaryotes

Simple rule (2)

Cleavage of 1 junction at site 1, but

the other at site 2 therefore
generates crossover products (splice
+ patch = splice) because the
arrangement caused by the site 1
cleavage is retained in the final
product

Function of homologous
recombination in bacteria
To repair double-stranded breaks in
DNA ( ~ eucaryotic cell)
To
o restart
esta t collapsed
co apsed replication
ep cat o forks
o s
( ~ eucaryotic cell)
To allow a cells chromosomal DNA
to recombine with DNA that enters
via phage infection/conjugation

Cells with defects in the proteins that promote

recombination are hypersensitive to DNA damaging
agents, esp those that break DNA strands
Animal carrying mutations that interfere with
homologous recombination are predisposed to
certain types of cancer
Critical for meiosis required for proper
chromosome pairing maintaining integrity of the
genome
Recombination reshuffles genes between the
parental chromosomes, ensuring variation in the
sets of genes passed to the next generation

Homologous Recombination is
Required for Chromosome Segregation
during Meiosis

Before division: cell has 2 copies of each

chromosome (the homologs) 1 each that was
ingerited from its 2 parents
During the S phase, these chromosomes are
replicated to give a total DNA content of 4N
The products of replication i.e. sister
chromatids-stay together
Then, in the preparation for the 1st nuclear
division, these duplicated homologous
chromosomes must pair & align & then
separate sister chromatin remain paired

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3/28/2012

Then, in the 2nd nuclear division, it is the

sister chromatids that separate product :
4 gametes, each with 1 copy of 2
chromosome ((i.e. the 1N DNA content))
Without recombination, chromosomes often
fail to align properly for the first meiotic
division a high incidence of chromosome
loss improper segregation of
chromosomes: NONDISJUNCTION

Nondisjunction

Leads to a large number of gametes without

the correct chromosome complement
Gametes with either too few / too many
chromosomes cannot develop properly once
fertilized failure in homologous
recombination poor fertility
Homologous recombination events that occur
during meiosis : Meiotic recombination
frequently give rise to crossing over between
genes on the 2 homologous parental
chromosomes

11

3/28/2012

Although DNA replication,

repair, homologous
recombination occur with high
fidelity to ensure the genome
identity between generations,

there are genetic processes

that rearrange DNA
sequences and thus lead to
a more dynamic genome
structure

Site-Specific Recombination &

Transposition of DNA
Dr. Ratna Megawati Widharna, SKG, MFT
Molecular biology
gy
References
Watson, Baker, Bell, Gann, Levine and Losick bab
11, Yuwono bab 13, Weaver Bab 22 &23

Two classes of genetic recombination

for DNA rearrangement:
Conservative site-specific
recombination (CSSR):

recombination between two defined

sequence elements

Transpositional recombination
(Transposition): recombination

between specific sequences and

nonspecific DNA sites

12

3/28/2012

Figure 11-1

Topic 1: Conservative SiteSpecific Recombination

1.Exchange of non-homologous
sequences at specific DNA
sites(what)
2.Mediated by proteins that
recognize specific DNA sequences.
(how)

1.
2.

ConservativeSiteSpecific
Recombination
BiologicalRolesofSiteSpecific
Recombination( phage
i
integration/excision,multimericgenome
i / ii
li
i
resolution)

3.

Transposition (concepts,learningfromB.
McClintock,DNAtranposons.Virallike
retrotransposons/retroviruses,polyA
retrotransposons)

ConservativeSiteSpecificR
Recombination

OUTLINE

11 Sitespecificrecombinationoccursat
specificDNAsequences inthetargetDNA

CSSR (conserved site-specific

recombination) is responsible
for many reactions in which a
defined segment of DNA is
rearranged.

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3/28/2012

CSSR can generate three different

types of DNA rearrangements

Figure 11-3

If the two sites at which recombination will take

place are oriented oppositely to one another in
the same DNA molecule then the site-specific
reacombination results in inversion of the
segment of DNA between the two recombination
sites

If the two sites at which recombination will take

place are oriented in the same direction in the
same DNA molecule, then the segment of DNA
between the two recombinogenic sites is deleted
from the rest of the DNA molecule and appears
as a circular molecule. Insertion is the reverse
reaction of the deletion

recombination at direct repeats

causes a deletion

Figure 11-4
Structures
involved
in CSSR

recombination at inverted repeats

causes an inversion

14

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Serine Recombinases
Tyrosine Recombinases

ConservativeSiteSpecificR
Recombination

ConservativeSiteSpecificR
Recombination

12 Sitespecificrecombinases cleaveand
rejoin(join)DNAusingacovalentprotein
DNAintermediate

13 Serinerecombinasesintroduce
doublestrandedbreaksinDNAandthen
swapstrandstopromoterecombination

Figure 11-5

Conservative Site-Specific Recombination

The covalent protein-DNA intermediate

conserves the energy of the cleaved
phosphodiester bond within the
protein-DNA linkage, which allows
the cleaved DNA strands to be
rejoined/resealed by reversal of the
the cleavage process
This mechanistic feature contributes
the conservative to the CSSR name,
because every DNA bond that is
broken during the reaction is
resealed by the recombinase without
consuming any external energy.

Figure 11-6

15

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ConservativeSiteSpecificR
Recombination

ConservativeSiteSpecificR
Recombination

14 Tyrosinerecombinasesbreakand
rejoinonepairofDNAstrandsatatime

15 Structure oftyrosinerecombinases
boundtoDNArevealthemechanismof
DNAexchange
Cre is a tyrosine recombinase
Cre is an phage P1-encoded
protein, functioning to
circularize the linear phage
genome during infection
The recombination sites of Cre is
lox sites. Cre-lox is sufficient for
recombination
Read Box11-1 for Cre application

Figure 11-8

Figure 11-7

16

3/28/2012

Many phage insert their DNA into

the host chromosome during
infection using this recombination
mechanism. Example: phage
Alter gene expression. Example:
Salmonella Hin recombinase (the
details are not discussed in the
class)
Maintain the structural integrity of
circular DNA molecules during
cycles of DNA replication. Example:
resolvase that resolves dimer to
monomer

Topic 2 Biological roles of

site-specific recombination

Thegeneralthemesofsitespecific
recombination
1.

2.

All reactions depend critically on the

assembly of the recombinase protein on
the DNA and bring together of the two
recombination sites
Some recombination requires only the
recombinase and its recognition
sequence for such an assembly; some
requires accessory proteins including
Architectural Proteins that bind specific
DNA sequences and bend the DNA.

17

3/28/2012

Biologicalrolesofsitespeccificrecombination

21&2 integraseworkswithIHFandXisto
integrate/excisethephagegenomeinto/fromthe
bacterialchromosome
The outcome of bacteriophage
infection of a host bacterium
Establishment of the lysogenic state:
requires the integration of phage
DNA into host chromosome
lytic growth is the growth stage of
multiplication of the independent
phage DNA that requires the excision
of the integrated phage DNA from
the host genome.

Phage genome

Crossover regions

Bacterial genome
IHF integrationhostfactor
g
encodedbybacteria)

Int encodedintegrase)
Xis(encodedexcisionase)

Figure 11-9

-encoded integrase (Int)

Figure 11-2: genome integration.

Recombination always occurs at exactly
the same sequence within two
recombination sites, one on the phage
DNA, and the other on the bacterial DNA.

catalyzes recombination between two

attachment (att) sites. attP site is on the
phage DNA and attB site is on the
bacterial genome
Is a tyrosine recombinase, and the
mechanism of strand exchange is similar
to that catalyzed by Cre recombinase.
recombinase
Requires accessory proteins to assemble
the integrase on the att sites. Both IHF
and Xis are architectural proteins. IHF
binds to DNA to bring together the Int
recognition sites. Xis binds to the
integrated att sites to stimulate excision
and to inhibit integration (see 22).

18

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Biologicalrolesofsitespeccificrecombination

25RecombinaseconvertsmultimericcircularDNA
moleculesintomonomers
The chromosomes of most bacteria,
plasmids and some viral genomes
are circular.
During the process of homologous
recombination, these circular DNA
sometimes form dimers and even
multimeric forms, which can be can
be converted back into monomer by
site specific recombination.
Site-specific recombinases also called
resolvases catalyze such a process.

Xer recombinase catalyzes the

monomerization of bacterial
chromosomes and of many bacterial
plasmids.
Xer recombinase is a member of the
tyrosine recombinase family
Xer is a heterotetramer containing two
subunits of XerC and two subunits of
XerD. Both XerC and XerD are tyrosine
recombinases but recognize different
DNA sequence.
The recombination sites in bacterial
chromosomes, called dif sites have
recognition sites for both XerC and
XerD.

Figure 11-15

Figure 11-14 Circular DNA molecules

can form multimers

The dimer only resolves when XerD is activated by

the presence of FtsK

19

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Topic 3 Transposition ()

Transposition is a specific form of

genetic recombination that moves
certain genetic elements from one DNA
site to another.
These mobile genetic elements are
called transposable elements or
transposons.
Movement occurs through
recombination between the DNA
sequences at the ends of the
transposons and a sequence in the host
DNA with little sequence selectivity.

Box 11-3 Example

of corn cob
showing color
variegation due
to transposition

FIGURE 11-17 Transposition of a

mobile genetic element to a new
site in host DNA, which occurs
with or without duplication of the
element.

Because transposition has little

sequence selectivity in their choice of
insertion sites, the transposons can
insert within genes or regulatory
sequence of a gene, which results in
the completely disruption of gene
function or the alteration of the
expression of a gene. These disruption
l d tto the
leads
th discovery
di
off transposable
t
bl
elements by Barbara McClintock.

It was actually the ability of

transposable elements to break
chromosomes that first came to
McClintock attention (late 1940s). She
found that some maize () strains
experienced frequent chromosomebroken, and the hotspots for
chromosome breaks varied among
diff
different
t strains
t i
and
d among different
diff
t
chromosomal locations in the
descendents () of an individual
plant, which leads to the concept that
genetic elements could
move/transpose

plant genomes are very rich in

functional transposons

20

3/28/2012

BarbaraMcClintock Maize
Thebiologicalrelevanceoftransposons

2. The genetic recombination

mechanisms of transposition are
also used for other functions than
the movement of transposons, such
as integration of some virus into
the host genome and some DNA
rearrangement to alter gene
expression [V(D)J recombination].

Transposition

1.Transposons are present in the

genomes of all life-forms. (1)
transposon-related sequences can
make up huge fractions of the
genome of an organism (50% of
human and maize genome). (2)
the transposon content in
different genomes is highly
variable (Fig 11-18).

3(16)Therearethreeprincipleclassesof
transposableelements
1.
2.

3.

DNA transposons
Viral-like retrotransposons
including
g the retrovirus,, which
are also called LTR
retrotransposons
Poly-A retrotransposons, also
called nonviral
retrotransposons.

FIGURE 11-19 Genetic organization

of the three classes of transposable
elements

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32DNAtransposonscarryatransposase gene,
flankedbyrecombinationsites

2.

3.

Recombination sites are at the two

ends of the transposon and are
inverted repeated sequences
varying in length from 25 to a few
hundred bp.
The recombinase responsible for
transposition are usually called
transposases or integrases.
Sometimes they carry a few
additional genes. Example, many
bacterial DNA transposons carry
antibiotic resistance gene.

Transposition

1.

2.

Autonomous transposons: carry a

pair of terminal inverted repeats
and a transposase gene; function
independently
Nonautonomous transposons:
carry the terminal inverted
repeats but not the functional
transposase; need the transposase
encoded by autonomous
transposons to enable
transposition

1.

2.
3.

Transposition

33Transposonsexistasbothautonomousand
nonautonomouselements

Transposition

Transposition

1.

34Virallikeretrotransposons andretroviruses
carryterminalrepeatsequencesandtwogenes
importantforrecombination

1.
2.

3.

Inverted terminal repeat sequences

for recombinase binding are
embedded within long terminal
repeats (LTRs), being organized on
the two ends of the elements as
direct repeats.
reverse transcriptase (RT), using an
RNA template to synthesize DNA.
integrase (the transposase)

35PolyAretrotransposons looklikegenes
Do not have the terminal inverted
repeats.
On end is called 5 UTR (untranslated
region), the other end is 3 UTR followed
b a stretch
by
h off A-T base
b
pairs
i called
ll d the
h
poly-A sequence. Flanked by short target
site duplication.
Carry two genes. ORF1 encodes an RNAbinding proteins. ORF2 encodes a protein
with both reverse transcriptase (RT) and
endonuclease activity. Truncated
elements lacking complete 5 UTR??

22

3/28/2012

4.

3(79)DNAtranspositionbyacutandpaste
mechanism(nonreplicativemechanism)
Transposition

Multimers of transposase binds to

the terminal inverted repeats of the
transposons, and bring two ends
together to form a stable proteinDNA complex called the synaptic
complex/transpososome.
This complex ensures the DNA
cleavage and joining reaction,
which is called strand transfer and
is similar to the recombinase

1.

2.

The transposase first cleaves one DNA

strand at each end of the transposon,
resulting in free 3-OH groups
Different transposons use different
mechanism to cleave the second
strands, resulting in 5 ends at the
transposons. The mechanism including
using a secondary enzyme (Tn7), using
an unusual DNA transesterification
mechanism to generate a DNA hairpin
structure subsequently resolved by
transposases (Tn10, Tn5) (3-9, Fig 1121)

3.

5.

The 3OH ends of the transposon attack

the DNA phosphodiester bonds at the
sites of the new insertion/target DNA,
resulting in transposon insertion. This
DNA rejoining reactions occurs by a
one-step transesterification reaction
called DNA strand transfer.
The intermediate with two nicks is
finished by gap repair. The old
insertion site having a double-stranded
break are repaired by DSB repair (3-8)

FIGURE 1120 The cutand-paste

mechanism
of
transposition
One-step
transesterification

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310DNAtranspositionbyareplicative
mechanism/replicativetransposition

FIGURE 11-22
Replicative
transposition

Transposition

The mechanism is similar to the

cut-and-paste transposition.
The assembly of the transposase
protein on the two ends of the
transposon DNA to generate the
transpososome.
2.

The transposase first cleaves one

DNA strand at each end of the
transposon, resulting in two 3OH
ends. BUT NO cleavage occurs at the
second strand.

The 3OH ends of the transposon DNA

are then joined to the target sites by the
DNA strand transfer reaction, resulting
in a doubly branched DNA molecule.
4. The two branches within this
intermediate have the structure of a
replication fork, which recruits the
replication proteins for strand synthesis.
synthesis
As a result, the donor DNA is duplicated
in the host DNA.
Replicative transposition frequently causes
chromosomal inversions and deletions
that can be highly detrimental ()
to the host cell.
3.

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1st step of replicative transposition :

assembly of the transposase protein on the
2 ends of the transposon DNA to generate
a transpososome essential to coordinate
the DNA cleavage and joining reactions on
the 2 ends of the transposons DNA
2nd step : DNA cleavage at the ends of the
transposons DNA catalyzed by the
trasnposase within the transpososome

The transposase introduces a nick

into the DNA at each of the 2
junctions between the transposon
sequence and the flanking host DNA
liberates 2 3OH DNA ends on the
transposon sequence
In contrast to cut-and-paste
transposition, the transposon DNA is
not excised from the host sequences
at this stage major difference

The 3OH ends of the trasnposon DNA are then

joined to the target DNA site by the DNA
strand transfer reaction. The mechanism is the
same as cut-and-paste transposition
However the intermediate generated by DNA
However,
strand transfer is in this case a doubly
branched DNA molecule
In this intermediate, the 3ends of the
transposon are covalently joined to the new
target sitre, while the 5ends of the transposon
sequence remain joined to the old flanking
DNA

The 2 DNA branches within this

intermediate have the structure of a
replication fork
After DNA strand transfer, the DNA
replication proteins from the host
cell can assemble at these forks
Replicative transposition phage Mu :
this assembly specifically occurs at
only 1 of the 2 forked structures

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311VirallikeRetrotransposons&Retroviruses
moveusinganRNAintermediate
Transposition

The 3OH end in the cleaved target DNA

serves as a primer for DNA synthesis
Replication
p
p
proceeds through
g the
transposon sequence and stops at the
second fork
This replication reaction generates 2
copies of the transposon DNA
These copies are flanked by the short
direct target site duplications

The mechanism is similar to the DNA

transposons (Cut-and-Paste). The
major difference is the involvement
of an RNA intermediate.

Transcription of the retrotransposon

(or retroviral) DNA sequence into
RNA by cellular RNA polymerase,
which is initiated at a promoter
sequence within one of the LTRs.

2.

Replicative transposition frequently causes

chromosomal inversions and deletions that can
be highly detrimental to the host cell
This propensity to cause rearrangements may
put replicative transposons at a selective
disadvantage
Perhaps this is why so many elements have
developed ways to excise completely from
their original DNA location prior to joining to a
new DNA sire
By excision, transposons avoid generating
these major disruptions to the host genome

3.

4.

5.

The RNA is then reverse transcribed

to cDNA (dsDNA) that is free from
any flanking host DNA sequences,
resulting in the excised form of
transposon
Integrase assembles on the ends of
cDNA and cleaves a few nucleotides
off the 3
3ends
ends, generating 3
3OHs
OHs.
Integrase inserts the transposon into
target site using the DNA strand
transfer reaction.
Gap repair and ligation complete the
recombination and generate targetsite duplications.

26

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Figure 11-23
Mechanism of
retroviral
integration
and
transposition
of viral-like
retrotranspos
ons.

FIGURE 11-24 Similarity of catalytic domains

of transposases and integrases. (b) Scematic
of the domain organization of the above three
proteins

312DNAtransposasesandretroviral
integrasesaremembersofaprotein
superfamily

313PolyARetrotransposition movebya
reversesplicingmechanism
Transposition

Transposition
Tn5

MuA

RSV integrase

FIGURE 11-24 Similarity of catalytic domains

of transposases and integrases. (a) structure
of the conserved core domains of three
transposases and integrase

Using an RNA intermediate but a

different mechanism from that of the
viral-like retrotransposons
The mechanism used is called target site
primed reverse transcription.
1. Transcription of the integrated DNA
2. The newly synthesized RNA is
exported to cytoplasm to produce
ORF1 and ORF2 proteins, which
remain to bind the RNA

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3/28/2012

3.

4.

5.

The protein-RNA complex

reenters the nuclease and
associate with the chromosomal
DNA
The endonuclease activity of
ORF2 introduce a nick on the
chromosomal DNA at the T-rich
sites.
The 3OH generated on the
target DNA serves as the primer
for reverse transcription of the
element RNA (ORF2)

Key points
1.

ConservativeSiteSpecificRecombination
(concept,threetypes,mechanismsserineandtyrosine
recombinases)

2.

BiologicalRolesofSiteSpecific
Recombination ( phageintegration/excision,
Recombination
phage integration/excision
multimericgenomeresolution)

3.

Transposition (concepts,learningfromB.
McClintock,DNAtranposons.Virallike
retrotransposons/retroviruses,polyA
retrotransposons)

28