Genetic Re Combination and Its Molecular Mechanisms

7/31/2019 Genetic Re Combination and Its Molecular Mechanisms

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GENETIC RECOMBINATION

AND ITS MOLECULARMECHANISMS


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Genetic recombination involves the rearrangement of genetic

material, usually by exchange of DNA sequences between DNA

molecules.

Recombination was first identified as the process responsible for

the exchange of segments of homologous chromosomes by

crossing over in Drosophila melanogaster (homologousrecombination or simply recombination)

It has subsequently also been implicated in integration of

transferred DNA into bacterial genomes after conjugation,

transduction or transformation .

During recombinationbases are neither added or lost.


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The types of recombination are as follows:

1. Generalized or homologous recombination: recombination

between homologous sequences of DNA, i.e. those sharingextensive nucleotide sequence similarity.

o Occurs in eukaryotes during meiosis, i.e. during

spermatogenesis in males and oogenesis in females.

o Occurs during the integration of transferred DNA into

bacterial genomes, i.e. during conjugation, transduction or

transformation.


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Figure 1. Homologous recombination during meiosis


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Figure 2. Homologous recombination processes in bacteria. Bacterial recombination

requires that a bacterial cell receive an allele obtained from another cell. (a) conjugation (b)transformation (c) transduction


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2. Site-specific recombination: recombination between specific

pairs of sequences inprokaryotes as well as eukaryotes, forexample,

o the integration of bacteriophage into a particular site in

theE. coli chromosome .

o involved in the inversion of DNA segments to alter gene

structure.


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Figure 3. bacteriophage undergoes integration into a specific-site of E. coli

chromosome during lysogeny


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A B

Recombination

sites

AB

Site-specific

recombination

Figure 4. Site-specific recombination resulting in inversion of DNA segment


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3. Transposition: recombination between a specific DNA

sequence ( transposable element) and a DNA site with which

it does not share nucleotide sequence homology.

transposon

genomic DNA

target site

Conservative

transposition

Replicative

transposition

Figure 5. Recombination between a transposon and its target site


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MECHANISM (S) OF

RECOMBINATION

The first attempts to explainthe molecular mechanism of

recombination led to the

Holliday Model

(R. Holliday, 1964)

Figure 6. Two schemes for

initiation of homologous

recombination


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In the original scheme of the Holliday model, the two molecules lined up

with one another and single-stranded nicks appeared at equivalent positions

in each helix. This produced free single-stranded ends that could be

exchanged, resulting in heteroduplex formation.

This feature of the model was criticized because no mechanism could be

proposed for ensuring that the nicks occurred at precisely the same position

on each molecule.

The Meselson-Radding modification ( Meselson and Radding, 1975)

proposes a more satisfactory scheme whereby a single-stranded nick occurs

in just one of the double helices, the free end that is produced invading' the

unbroken double helix at the homologous position and displacing one of its

strands, forming a D-loop.

Subsequent cleavage of the displaced strand at the junction between its

single-stranded and base-paired regions produces the heteroduplex.


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Figure 7. Holliday model of

homologous recombination


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1. The central feature of the model is formation of a heteroduplex by the

exchange of polynucleotide segments between the two homologous molecules

Reciprocal strand exchange in homologous recombination creates a connection

between two DNA duplexes to form a joint molecule. The point at which anindividual strand of DNA crosses from one duplex to another is called the

recombinant joint

At the site of recombination, each duplex has a region consisting of one strand from

each parental DNA molecule. This region is called hybrid DNA or heteroduplex

DNA

2. The heteroduplex is initially stabilized by base-pairing between each

transferred strand and the intact polynucleotide of the recipient molecule,

since the nucleotide sequences of the two DNA molecules are similar.

3. Subsequently the gaps are sealed by DNA ligase, giving a Holliday structure

4. This structure is dynamic, with branch migration resulting in exchange of

longer segments of DNA if the two helices rotate in the same direction.


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5. Separation, or resolution of the Holliday structure back into individual

double-stranded molecules occurs by cleavage of the three-dimensional

configuration orchi form of the Holliday structure cross the branch point

6. If a horizontal cut is made across the chi form, then the same two strands

that were originally nicked are nicked again, and only a short segment of

polynucleotide, corresponding to the distance migrated by the branch of

the Holliday structure, is transferred between the two molecules. These

products are calledpatch recombinants.

7. On the other hand, a vertical cut results in nicking of the strands that were

NOT originally nicked, leading to double-stranded DNA being transferred

between the two molecules so that the end of one molecule is exchanged

for the end of the other molecule. This is the DNA transfer seen incrossing-over , and results in the formation ofsplice recombinants.


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The mechanism of homologous recombination between phage andE. coli. has been well studied

Figure 8. The RecBCD pathway

for homologous recombination

inE. coli

Chi site consensus sequence 5-GCTGGTGG-3


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the RecBCD enzyme composed of proteins encoded by the recB, C, andD genes specifically

recognizes double-strand breaks and has helicase and exonuclease activities.

Certain regions of phage DNA, termed chi sites (8 NT-consensus sequence 5-

GCTGGTGG-3 which occurs once every 5-10 kb of DNA), undergo recombination at higher

frequencies than other regions in normalE. coli host cells

Experiments with purified RecBCD enzyme and DNA indicate that the protein complex

recognizes and binds to a free blunt end of the phage chromosome

The enzyme then moves along the DNA, its helicase activity unwinding the duplex as it goesusing its dual 53 and 3 5 exonuclease activities

However, when RecBCD encounters the first chi site, its 3 5 exonuclease activity is

inhibited and its 53 exonuclease activity is enhanced.

It makes a ss-nick approximately 56 nucleotides to the 3 side of the chi site.

Thus, after passing a chi site, RecBCD begins to generate a single-stranded 3-hydroxyl end.

the resulting recombinogenic end becomes coated with multiple RecA proteins, and can

participate in the process of strand invasion


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The enzyme RecA can mediate strand invasion

First, RecA aligns the ssDNA with its homologous target double-stranded DNAregion and forms a complex with it.

Second, RecA inserts the ssDNA into the target DNA, displacing one of the

preexisting strands and forming a heteroduplex Holliday-type structure RecA requires ss-DNA with a free 3 end and ATP

One RecA monomer binds to every 3 nucleotides.

ATP may act through an allosteric effect on RecA conformation

When ATP is bound, the DNA-binding site of Rec A has high affinity for DNA

Hydrolysis of ATP converts the binding site to low affinity, which is needed torelease heteroduplex DNA.


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Action ofE. coli proteins in branch migration and resolution of

Holliday junctions.

Branch migration does not appear to be a random process, but

instead stops preferentially at the sequenceRuv C cuts between T and G/C


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Migration of the crossover point (branch migration ) is efficiently catalyzed byE. coli

RuvA and RuvBproteins. RuvA specifically recognizes the Holliday junction, whereas

RuvB has helicase activity necessary for promoting the observed branch migration.

The active tetrameric form of RuvA binds to the center of a Holliday junction,unfolding the junction into a square planar configuration and keeping the four single-

stranded segments apart.

two ringlike hexameric RuvB proteins bind, surrounding the double-stranded DNA

exiting from opposite sides of the RuvA complex .

Deriving energy from ATP hydrolysis, the RuvB rings act as molecular pumps, pulling

two double-stranded DNAs into the RuvA complex, separating the strands, and then

extruding two double-stranded heteroduplexes out of the RuvA complex.

Following branch migration, two RuvC endonuclease proteins bind to the RuvA/ RuvBcomplex and then cut the DNA intermediate at two sites 180 apart

subsequent ligation of cleaved ends generates recombinant (or nonrecombinant)

molecules containing a segment of heteroduplex DNA


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All eukaryotic cells, including human cells, produce proteins required for

homologous recombination.

the human and yeast RAD51 proteins, which are homologous in sequence,

catalyze pairing of homologous DNA segments and DNA strand insertion

similarly to RecA.

A Topo IIlike protein encoded by the yeast Spo11 gene generates the

double-strand breaks that occur during meiotic recombination,

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Genetic Re Combination and Its Molecular Mechanisms