Genetic recombination

mechanism
Prabin Shrestha

 Without recombination, genomes

would be relatively static structures,
undergoing very little change.
more extensive restructuring, which
is the role of recombination, would
not occur, and the evolutionary
potential of the genome would be
severely restricted.

Recombination was first recognized as the

process responsible for crossing-over and
exchange of DNA segments between homologous
chromosomes during meiosis of eukaryotic cells

 Meiosis crossing over

Conjugation (direct transfer of dna
through cytoplasmic bridge)
Transduction (transfer of dna/rna
from virus to the infected cell)
..etc
All occur through homologous
recombination

Types of recombination mechanism

Homologous recombination
Site specific
transposition

Types of recombination mechanism

Homologous recombination
also called general recombination, is by far the
most common, most important version of
recombination in nature, being responsible for
meiotic crossing-over and the integration of
transferred DNA into bacterial genomes
It depends on extensive sequence similarity
between the participating DNA molecules, on
homologous chromosomes.
It is the type of recombination that occurs in
eukaryotic meiosis.

The Holliday model for homologous recombination

formulated by Robin Holliday.

The key features
formation of heteroduplex DNA; - the
creation of a cross bridge;
branch migration;
splicing, of the intermediate structure to
yield different types of recombinant
molecules.

Three basic steps

Holiday
junction

Holliday junction;
Branch migration;
Heteroduplex.
we can see that two homologous double
helices are aligned, although note that they
have been rotated so that the bottom strand of
the first helix has the same polarity as the top
strand of the second helix (5 3 in this case)
Then a nuclease cleaves the two strands that
have the same polarity (fig . b-parallel strands
or antiparallel strands).
The free ends leave their original
complementary strands and undergo hydrogen
bonding with the complementary strands in the
homologous double helix (Fig C).
Ligation produces the structure shown in Fig d.
This partially heteroduplex double helix is a
crucial intermediate in recombination, and has
been termed the Holliday structure.

Hetero
duplex

Branch migration
The Holliday structure creates a cross
bridge, or branch, that can move, or
migrate, along the heteroduplex .
This phenomenon of branch
migration is a distinctive property of
the Holliday structure.
Branch migration, the
movement of the
crossover point between
DNA complexes.

 There are three possible

stacking conformers:
an unstacked form and
two stacked forms.
The unstacked form
dominates in the absence
of divalent cations such as
Mg2+ due to electrostatic
repulsion between the
negatively-charged
backbones of the strands,
in the presence of Mg2+
the electrostatic repulsion
is counteracted and the
stacked structures
predominate.

RecBCD pathway
Both the RecD and RecB subunits are
helicases
The RecB subunit in addition has a
nuclease function.
RecBCD enzyme (perhaps the RecC
subunit) recognizes a specific
sequence in DNA, 5'-GCTGGTGG-3',
known as Chi (sometimes designated
with the Greek letter ) until then
keeps the unwindng the DNA
The RecD helicase travels on the
strand with a 5' end at which the
enzyme initiates unwinding, and RecB
on the strand with a 3' end.
RecB is slower than RecD, so that a
single-stranded (ss) DNA loop
accumulates ahead of RecB

 In the bacterial SOS response, it

functions in the autocatalytic
cleavage of the LexA repressor
The protein has more than one
DNA binding site, and thus can hold
a single strand and double strand
together
the RecA-ssDNA filament searches
for sequence similarity along the
dsDNA-thus recombination possible
The RecA protein catalyzes
unidirectional branch migration

LexA is a repressor enzyme

that represses SOS response
genes coding for DNA
polymerases required for
repairing DNA damage.

 Antibiotic like ciprofloxacin can

inhibit DNA gyrase (DNA
topoisomerase)
Counter productive = it also plays
role in biosynthesis of Rec A
Hence SOS response DNA repair

Resistant bacteria evolves

Branch migration does

not appear to be a
random process, but
rotating
the helices
instead
stopsin the required
manner so that the branch point
preferentially
atend
the
moves.
Two Ruv C binds after
of
branch
migration
sequence

Two pathways for Rec BCD

(1) If ATP is in excess, the enzyme simply nicks the strand with Chi (the
strand with the initial 3' end). Unwinding continues and produces a 3' ss
tail with Chi near its terminus. This tail can be bound by RecA
protein, which promotes strand exchange with an intact homologous
DNA duplex. When RecBCD reaches the end of the DNA, all three subunits
disassemble and the enzyme remains inactive for an hour or more; a
RecBCD molecule that acted at Chi does not attack another DNA
molecule.
(2) If Mg2+ ions are in excess, RecBCD cleaves both DNA strands
endonucleolytically, although the 5' tail is cleaved less often . When
RecBCD encounters a Chi site on the 3' ended strand, unwinding pauses
and digestion of the 3' tail is reduced. When RecBCD resumes unwinding,
it now cleaves the opposite strand (i.e., the 5' tail) and loads RecA protein
onto the 3-ended strand. After completing reaction on one DNA
molecule, the enzyme quickly attacks a second DNA, on which the same
reactions occur as on the first DNA.

The double-strand break model for recombination in

yeast

Although the Holliday model for

homologous recombination, either in its
original form or as modified by Meselson
and Radding, explains most of the results
of recombination in all organisms, it has a
few inadequacies, which prompted the
development of alternative schemes.

Holliday model could

not explain
gene conversion
The double-strand
break model provides
an opportunity for
gene conversion to
take place during the
recombination
process.

protein responsible for the cut is a

Type II DNA topoisomerase (
Section 13.1.2) which forms covalent
linkages with the two pieces of DNA
and hence prevents them drifting
completely apart.
The resulting heteroduplex has a pair
of Holliday structures that can be
resolved in a number of ways, some
resulting in gene conversion

 A region of extensive homology is not a prerequisite

for recombination: the process can also be initiated
between two DNA molecules that have only very short
sequences in common. This is called

site-specific recombination
and it has been extensively studied because of the
part that it plays during the infection cycle of
bacteriophage

 After injecting its DNA into an E. coli cell,

bacteriophage can follow either of two infection
pathways
lytic pathway,
results in the rapid synthesis of coat proteins,
combined with replication of the genome,
leading to death of the bacterium
release of new phages within about 20 minutes of
the initial infection.
lysogenic pathway,
new phages do not immediately appear.
The bacterium divides as normal, possibly for many
cell divisions, with the phage in a quiescent form
called the prophage.
Eventually, possibly as the result of DNA damage or
some other stimulus, the phage becomes active
again, replicating its genome, directing synthesis of

 During the lysogenic phase the genome becomes

integrated into the E. coli chromosome.
It is therefore replicated whenever the E. coli DNA is
copied,
Integration occurs by site-specific recombination
between the att sites, one on the genome and one
on the E. coli chromosome, which have at their
center an identical 15-bp sequence
Because this is recombination between two circular
molecules, the result is that one bigger circle is
formed; in other words the DNA becomes
integrated into the bacterial genome.

 The recombination event is catalyzed by a specialized

Type I topoisomerase called integrase ( Kwon et al.,
1997), a member of a diverse family of recombinases
present in bacteria, archaea and yeast.
The enzyme makes a staggered double-stranded cut
at equivalent positions in the and bacterial att sites.
The two short single-stranded overhangs - exchanged
= Holliday junction
This cleavage : forms appropriate orientation,
the DNA becomes inserted into the E. coli genome.
excision, which is also carried out by integrase, in
conjunction with a second protein= 'excisionase',

Both and E.coli

DNA have a copy
of the att site, each
one comprising an
identical central
sequence called 'O'
and flanking
sequences P and P
(for the phage att
site) or B and B
(bacterial att site).

Site-specific recombination
Based on amino acid sequence homology and
mechanistic relatedness most site-specific
recombinases are grouped into one of two
families: the tyrosine recombinase family or
the serine recombinase family.
The names stem from the conserved
nucleophilic amino acid residue that they use
to attack the DNA and which becomes
covalently linked to it during strand exchange

 Recombination between two DNA sites

begins by the recognition and binding of
these sites by the recombinase protein.
This is followed by synapsis, i.e. bringing
the sites together to form the synaptic
complex.
strand exchange takes place, as the DNA is
cleaved and rejoined by controlled
transesterification reactions

 the DNA cut at fixed points within the

crossover region of the site releases
a deoxyribose hydroxyl group, while
the recombinase protein forms a
transient covalent bond to a DNA
backbone phosphate
Though the mechanism is almost
similar in both : there are basic
differences

 Serine recombinase = gamma-delta and

Tn3 resolvase, cut all four DNA strands
simultaneously at points that are staggered
by 2bp .
During cleavage, a protein-DNA bond is
formed via a transesterification reaction in
which a

phosphodiester bond is replaced by

a phosphoserine bond between a 5
phosphate at the cleavage site and
the hydroxyl group of the conserved
serine residue (S10 in resolvase)

 Tyrosine recombinases, such as Cre or Flp,

cleave one DNA strand at a time at points
that are staggered by 6-8bp,
linking the 3 end of the strand to the
hydroxyl group of the tyrosine nucleophile
Strand exchange then proceeds via a
crossed strand intermediate analogous to
the Holliday junction in which only one
pair of strands has been exchanged

 Decreased rates of homologous recombination cause

inefficient DNA repair, which can also lead to cancer.
This is the case with BRCA1 and BRCA2, two similar
tumor suppressor genes whose malfunctioning has been
linked with considerably increased risk for breast and
ovarian cancer.
Cells missing BRCA1 and BRCA2 have a decreased rate
of homologous recombination and increased sensitivity
to ionizing radiation, suggesting that decreased
homologous recombination leads to increased
susceptibility to cancer.
Because the only known function of BRCA2 is to help
initiate homologous recombination

Cre-Lox recombination is known as

a site-specific recombinase technology
The system consists of a single enzyme, Cre
recombinase, that recombines a pair of short target
sequences called the Lox sequences.
The Cre enzyme and the original Lox site called the
LoxP sequence are derived from bacteriophage P1.
P1 phage DNA exists as a plasmid in the host.
The Cre-lox system serves several functions in the
phage:
it circularizes the phage DNA into a plasmid,
separates interlinked plasmid rings so they are passed to both
daughter bacteria equally and
may help maintain copy numbers through an alternative
means of replication

 The Cre protein (encoded by the

locus originally named as "Causes
recombination"
Lox P (locus of X-over P1) is a site on
the bacteriophage P1 consisting of
34 bp

 Placing Lox sequences appropriately

allows genes to be activated,
repressed, or exchanged for other
genes
developed by Dr. Brian Sauer initially
for use in activating gene expression
in mammalian cell lines

 The Cre-lox system is used as a

genetic tool to control site specific
recombination events in genomic
DNA.
these sites, known as loxP
sequences, contain specific binding
sites for Cre that surround a
directional core sequence where
recombination can occur.

 Cre recombinase proteins binds to the first and last 13 bp

regions of a lox site forming a dimer.
This dimer then binds to a dimer on another lox site to
form a tetramer.
Lox sites are directional and the two sites joined by the
tetramer are parallel in orientation.
The double stranded DNA is cut at both loxP sites by the
Cre protein.
The strands are then rejoined with DNA ligase in a quick
and efficient process.
The result of recombination depends on the orientation of
the loxP sites. For two lox sites on the same chromosome
arm, inverted loxP sites will cause an inversion of the
intervening DNA, while a direct repeat of loxP sites will
cause a deletion event.
If loxP sites are on different chromosomes it is possible for
translocation events to be catalysed by Cre induced
recombination.

transposition
illegitimate recombination
Transposition : not a type of recombination :
utilizes recombination, = the end result being the
transfer of a segment of DNA from one position
in the genome to another.
Transposable elements (transposons) : both in pro
and eukaryotes
Influence variation : short and long term
(evolutionary)
Each transposon codes for the enzymes that
specifically insert it into the recipient DNA
illegitimate recombination because requires no
homology

 A characteristic feature of
transposition is that the transferred
segment is flanked by a pair of short
direct repats

This particular transposon is flanked by the

tetranucleotide repeat 5-CTGG-3. Other
transposons have different direct repeat sequences

Replicative and conservative

transposition.

Retroelements
, all of which
transpose via
an RNA
intermediate.

the original transposon remaining in

place and a new copy appearing
elsewhere in the genome;

the original
transposon moving
to a new site by a
cut-and-paste
process;

Tn3-type transposon or a transposable phage

5 overhangs

Endonucleases make single-stranded

cuts either side of the transposon and
in the target site where the new copy
of the element will be inserted
Ligation of these 5 overhangs to the
free 3 ends either side of the
transposon produces a hybrid
molecule
linked together by the transposable
element flanked by a pair of
structures resembling replication
forks

DNA synthesis at these

Rather than carrying
replication forks copies the
out DNA synthesis, the
transposable element and
hybrid structure is
converts the initial hybrid into a
converted back into
co-integrate
Thisbetween
cuts the transposon out of
two separate DNA Homologous recombination
its original molecule, leaving it
molecules simply bythe two copies of the transposon
'pasted' into the target DNA.
making additional uncouples the co-integrate,
separating the original DNA molecule
single-stranded nicks
either side of the (with its copy of the transposon still in

Prokaryotic transposons
Three levels of complexity have been
characterized
Simplest : IS
Complex : Tn
Composite : combination of two IS

IS insertion sequence
insertion sequence(IS) are followed by
identifying number
Are normal constituents of bacterial
chromosomes and plasmids
Eg. E.coli has 8 copies of IS1 and 5
copies of IS2
These contain transposase gene, and in
some cases a regulatory gene flanked
by short inverted terminal repeats

ABCD12345.54321
ABCD.
TargetABCD12345.54321
sequence
Target sequence
ABCD.
IS element

serted IS element is flanked by a directly pepeated segment

DNA suggesting that it is inserted at staggered cut

Complex transposons
Carries genes not involved in the
transposition process eg. Antibiotic
resistance genes
tnpA

Transposase
lactamase

tnpR

amp

repressor and resolvase

Composite transposons
Consist of a gene containing central
region flanked by two identical or
nearly identical IS like modules that
have either the same or an inverted
Inverted repeats
relative
IS module orientation
Central region

Central region

RNA intermediates in
eukaryotes
Transposons similar to those in prokaryotes also
occur in eukaryotes including yeast maize
Drosophila and humans
~3% DNA of human contain DNA based
transposons
Most transpositions in eukaryotes involve RNA
intermediates
Such DNA based transposons in human resembles
to those of retroviruses (suggesting that these
transposons are degenerate retroviruses)
So called retro-transposons

 Transposition of Retro-transposons
occur bia pathway that resembles the
replication of retroviral DNA
Their transcription to RNA
The reverse transcriptase mediated
copying to cDNA
Largely insertion of this DNA into host
organisms genome
Mediated by enzymes integrase(resembling
cut and paste transposons)

Ty1=Transposon yeast
Most common transposon in budding
yeast (contains excised RNA
trancript)
~35 copies of 6.3~kb element

 A retroviral transposon is flanked by direct long

terminal repeats (LTRs) of 250 to 600 bp
Typically contains 3 poly proteins
Gag=which is cleaved to the proteins
comprising the viral core
Pol = which is cleaved to the above mentioned
reverse transcriptase and integrase as well as
the protease that catalyzes these cleavages
Env=which is cleaved to ciral outer envelope
proteins

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