DNA Recombination

Crossing Over

Belgian cytologist
Janssens F. A.
(1863-1924)

Janssenss hypothesis of crossing over (1909):

- At the start of meiosis, through the process of
synapsis, the holologous chromosome form pairs
with their long axes paralellel.
- Two of the chromatids break at a corresponding
place on each, then rejoin crossways
- In this manner, recombinant chromatids might be
produced that contain a segment derived from each of
the original homologous chromosome.

Homologous recombination
Homologous recombination (general recombination): Genetic exchange
between a pair of homologous DNA sequences, typically those located on two
copies of the same chromosome

Homologous recombination in
Prokaryotes

Homologous recombination in
Eukaryotes

Genetic exchange;
The reassortment of genes along chromosomes;
The repair of broken DNA strand (DSB repair)

Key steps of homologous recombination

1.

2.
3.

4.

5.

Alignment of two homologous DNA molecules (very similar DNA

molecules). Despite of this high degree of similarity, DNA molecules can
have small regions of sequence difference and may carry different
sequence variants (alleles) of the same gene.
Introduction of breaks in DNA. The breaks may occur in one DNA
strand or involve both DNA strands.
Formation of initial short regions of base pairing between the two
recombining DNA molecules. This step is called strand invasion. As a
result of strand invasion, the two DNA molecules become connected by
crossing strand. This cross structure is called Holliday junction.
Movement of 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.
This process is called branch migration.
Cleavage the Holliday junction. Cutting the DNA strands within the
Holliday junction regenerates two separate duplex DNA molecules, and
therefore finishes genetic exchange. This process is called resolution.

Holliday model
(named after Robin Holliday who proposed this model in 1964)

Holliday model

The Holliday junction generated by strand invasion can move along the DNA by branch
migration. This migration increases the length of the DNA exchanged. If the two DNA
molecules are not identical, branch migration generates DNA duplexes carrying one or a
few sequence mismatches. Such regions are called heteroduplex DNA

Holliday junction

The Holliday junction is rotated to give a square-planner structure with no crossing

strands. A cross-exchange (holliday junction) can be seen in electron micrograph.

Holliday junction cleavage (resolution)

Two strands with the same sequence

and polarity must be cleaved. There
are two alternative choices for
cleavage sites:
- Site 1: occur in the two strands that
were not broken during the initiation
reaction. Cleavage at site 1 generates
the splice or crossover product, as,
within this DNA molecule, crossing
over has occurred between the A and
C genes.
- Site 2: occur in the two strands that
were broken during the initiation
reaction. Cleavage at site 2 generates
the patch or non-crossover
product.

Double-stranded break (DSB) repair model

Two possible ways of resolving a recombination intermediate

with two Holliday junctions from the DSB repair pathway

- Cleavage of both junctions at site 2: patch + patch = patch, non-crossover products

- Cleavage of both junctions at site 1: splice + splice = patch
- Cleavage of one junction at site 1, but the other at site 2: splice + patch = splice,
crossover products

Homologous recombination protein machine

Homologous recombination in E. coli (RecBCD pathway)

- Homologous recombination in E. coli via
RecBCD pathway requires a DSB on one
of the recombining two DNA molecules.
- Chi () site: crossover hotspot instigator
- RecBCD: a protein complex which is
composed of three subunits (RecB, RecC
and recD). RecBCD has both DNA
helicase and nuclease activities.
- RecBCD enters the DNA at the site of
the DSB and moves along the DNA,
unwinding the strands. The nuclease
activities of RecBCD frequently cleave
each strand during unwinding and thereby
destroy the DNA.
- Upon encountering the chi site, RecD is
lost. RecBC continues unwinding DNA
and cleaving only the 5-3 strand. The
DNA molecule is converted into one with
a 3 single-stranded extension terminating
with the chi sequence at the 3 end.

RecBCD pathway: RecA protein promotes strand invasion

- RecA is the founding member of a family

of enzymes called strand-exchange
proteins.
- RecA binds to the single-stranded
region of DNA terminating with the chi
sequence at the 3 end and promotes
strand invasion.
- The active form of RecA is a proteinDNA filament. The filaments that contain
approximately 100 subunits of RecA and
300 nucleotides of DNA are common.

RecBCD pathway: RuvAB complex specifically recognizes

Holliday junctions and promotes branch migration

Strand invasion

RecA

Branch migration

- After the strand invasion, the two recombining DNA molecules are connected by
Holliday juctions.
- RuvA protein is a Holliday junction specific DNA-binding protein. RuvA recognizes and
binds to Holliday junction and recruits RuvB protein to thic site
- RuvB is a hexameric ATPase that provides the energy to drive the exchange base pairs
that move the DNA branch.

Structure of the RuvA-DNA complex and the schematic model

of the RuvAB complex bound to Holliday junction DNA

RecBCD pathway: RuvC cleavage specific DNA strands at

Holliday junction to finish recombination

Patch recombination products

(non-crossover products)

Splice recombination products

(crossover products)

Structure of the RuvC recsolvase and the schematic model of

the RuvC dimer bound to Holliday junction DNA

Homologous recombination in eukaryotes

One pair of homologous
chromosomes

Crossing-over during
meiotic prophase I

- In
eukaryotic
cell,
homologous
recombination is also required for DNA
repair and the restarting of collapsed
replication fork (as same as in bacteria)
- Homologous
recombination
plays
important addition roles in eukaryotes.
Most
importantly,
homologous
recombination is critical for meiosis.
During
meiosis,
homologous
recombination is required for proper
chromosome pairing and, thus, for
maintaining the integrity of the genome.
- The homologous recombination events
that occur during meiosis are called
meiotic recombination.

Programed generation of double-stranded DNA

breaks occur during meiosis

- Spo11 is a protein that introduces doublestrand breaks in chromosomal DNA to

initiate meiotic recombination
- A specific Tyr side chain in the
Spo11protein attacks the phosphodiester
backbone to cut the DNA
- Two subunits of Spo11 cleave the DNA
two nucleotide apart on the two DNA
strands to make a staggered double-strand
break.

Overview of meiotic recombination pathway

- Spo11 protein introduces DSB formation
- MRX protein processes the cleaved DNA
ends. MRX is a multi-subunit DNA
nuclease (composed of Mre11, Rad50 and
Xrs2). This DNA-processing reaction is 53 resection.
- Dmc1 and Rad51 are RecA-like proteins
which promote strand invasion
- The proteins that promote branch
migration are still unknown
- The Mus81 protein may function as a
Holliday junction resolvase

Summary
- Homologous recombination occur in all organisms, allowing for genetic
exchange , the reassortment of gene along chromosome and the repair of broken
DNA strands.
- The recombination process involves the breaking and rejoining DNA molecules:
+ Initiation of exchange requires that one of two homologous DNA
molecules have a double-strand break
+ The broken DNA ends are processed by DNA-degrading enzymes to
generate single-stranded DNA segments
+ These single-stranded regions participate in DNA pairing with the
homologous partner DNA. Then, two DNA molecules are joined by a branch
structure in the DNA called Holliday junction
+ Cutting the DNA at the Holliday junction resolves the junction and
terminate recombination. Holliday junction can be cut in two alternative ways.
One way generates crossover products. The other way generates non-crossover
products.
- Cells encode enzymes that catalyze all the steps in homologous recombination.
In bacteria, the proteins (enzymes) are RecBCD, RecA, RuvA, RuvB and RuvC.
In eukaryotes, the proteins are Spo11, MRX, Dmc1, Rad51, Mus81.

Site-specific recombination
and
Transposition of DNA

Recombination can also occur between

nonhomologous DNA sequences
Genetic recombination: The processes by which a new genotype is formed by
re-assortment of genes resulting in gene combinations different from those
that were present in the parents

Conservative site-specific
recombination

Transpositional recombination
(transposition)

Conservative site-specific
recombination (CSSR)

Bacteriophage : Lytic and lysogenic cycle

Intergration of the phage genome into the bacterial

chromosome: An example of site-specific recombination

- A key feature of this reaction is that the segment of DNA that will be moved carries
specific short sequences elements, called recombination sites, where DNA exchange
occurs.
- During intergration, recombination always occurs at exactly the same nucleotide
sequence within two recombination sites, one on the phage DNA and the other on the
bacterial DNA.
- Recombination sites carry two classes of sequence elements: sequences specifically
bound by the enzyme recombinase, and sequences where DNA cleavage and rejoining
occur. Recombination sites are often quite short, 20bp or so.

Structure involved in conservative site-specific recombination

- Recombination site is organized as a
pair of recombinase recognition
sequences, positioned symmetrically.
These recognition sequences flank a
central short asymmetric sequences
sequence, known as the crossover
region, where DNA cleavage and
rejoining occurs.
- The subunits of the recombinase
recognize the specific sequences and
bring these sites together to form a
protein-DNA complex bridging the
DNA sites, known as the synaptic
complex.
- Within the synaptic complex, the
recombinase catalyzes the cleavage and
rejoining of the DNA molecules

Site-specific recombinases cleavage and rejoin DNA using a

covalent protein-DNA intermediate

- There are two families of conservative site-specific recombinases: the serine recombinase
and the tyrosine recombinase. Fundamental to the mechanism used by both families is that
when they cleavage DNA, a covalent protein-DNA intermediate is generated.
- For the serine recombinase, the side chain of Ser residue within the protein active site
attacks a specific phosphodiester bond in the recombination site. This reaction introduces a
single strand break and simultaneously generate a covalent linkage between Ser and phosphate
at this DNA cleavage site.
- The covalent protein-DNA intermediate conserves the energy of the cleaved phosphodiester
bond within the protein-DNA linkage. As a result, the DNA strands can be rejoined by reversal
of the cleavage process.

Recombinases by Family and by Biological function

The insertion of a circular bacteriophage lambda DNA

chromosome into the bacterial chromosome

Three types of conservative site-specific recombination

Transposition of DNA

Transposition of a mobile genetic element to a new site

in the host DNA

- 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 transposon.
- Transposon can insert within genes, often completely disrupting gene function. They can
also insert within the regulatory sequences of a gene where their presence may lead to
changes in how that gene is expressed. Therefore, transposons are the most common source
of new mutation in many organisms.

Three major classes of transposable elements

DNA-only transposon

DNA transposons carry both DNA sequences that function as

recombination sites (terminal inverted repeats) and genes encoding
protein that participate in recombination (transposase or intergrase).

Bacteria contain many types of mobile genetic

elements, three of which are shown

DNA transposition by a cut-and-paste mechanism

transposase

transposase

-To initiate recombination, the transposase

binds to the terminal inverted repeats at
end of the transposon.
- Once the transposase recognizes these
sequences, it brings the two ends of the
transposon DNA together to generate a
stable protein-DNA complex (synaptic
complex or transpososome).
- The transposases subunits excised the
transposon from its original location in the
genome.
- After excision of the transposon, the 3-OH
ends of the transposon DNA attack the DNA
phosphodiester bonds at a site of target
DNA.
- The transposon is covalently joined to the
DNA at the target site. This reaction is called
DNA strand transfer.
- The remaining recombination steps are
carried out by cellular DNA repair proteins.

DNA transposition by a replicative mechanism

transposase

transposase

Viral-like retrotransposons/ retroviruses

Viral-like retrotransposons/ retroviruses also carry terminal inverted repeat.

The terminal inverted repeats are embedded within longer repeated
sequences. These sequences are called long terminal repeats (LTRs). Virallike retrotransposons encode two protein needed for their mobility: intergase
(transposases) and reverse transcriptase (RT).

The life cycle of a retroviruses

Viruses are fully mobile genetic elements that can escape from cells

Viral-like retrotransposons and retroviruses move using an

RNA intermediate

The cDNA is recognized by the

intergrase protein for recombination
with a new target DNA site.

Transposition by a nonretroviral retrotransposon

Example of transosable elements:

Ac/ Ds family of transposons in maize

Movement of Ds element gives mottled corn

Summary
- Two classes of genetic recombination: conservative site-specific recombination
and transposition are responsible for many types of DNA rearrangements.
- Conservative site-specific recombination (CSSR) occurs at defined sequence
elements in the DNA. Recombinase protein recognize these sequence elements
and act to cleave and join DNA strands to rearrange DNA segments containing
the recombination sites.
- There are two families of conservative site-specific recombinase (serine
recombinase and tyrosine recombinase).
-Transposition is a class of recombination that moves mobile genetic elements,
called transposon, to new genomic sites.
- There are three major classes of transposons: DNA transposons, Viral-like
retrotransposons/ retroviruses and Nonretroviral retrotransposons
- The DNA transposons move by two mechanisms: cut-and-paste mechanism and
replicative mechanism
- The two classes of retrotransposons move using an RNA intermediate. These
retro elements require reverse transcriptase as well as a recombinase protein for
mobility.