Genetic Re Combination and Its Molecular Mechanisms
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Transcript of 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,