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DNA Recombination

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Recombination - molecular biology

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DNA Recombination

Crossing Over

Belgian cytologist

Janssens F. A.

(1863-1924)

Janssens’s 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. 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.

2. Introduction of breaks in DNA. The breaks may occur in one DNA

strand or involve both DNA strands.

3. 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.

4. 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.

5. 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.

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.

Holliday junction cleavage (resolution)

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 (χ) 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 protein-

DNA 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

RecA Strand invasion

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

- 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.

One pair of homologous

chromosomes

Crossing-over during

meiotic prophase I

Programed generation of double-stranded DNA

breaks occur during meiosis

- Spo11 is a protein that introduces double-

strand 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 5’-

3’ 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 λ: Lytic and lysogenic cycle

Intergration of the phage λ 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 λ 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

-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.

transposase transposase

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). Viral-

like 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.