January 24,

RECOMBINATION & GENECONVERSION

2011

ABSTRACT
Unless they are identical twins, which develop from a single zygote, no two offspring of
the same parents are genetically the same. As we discussed earlier, this is because, long
before the two gametes fuse at fertilization, two kinds of randomizing genetic re-assortment
have occurred in meiosis I, during the production of the gametes: the random distribution of
maternal and paternal homologs, and crossing-over. The random distribution of maternal and
paternal homologs could, in principle, produce 2n genetically different gametes, where n is
the haploid number of chromosomes. In humans, for example, each individual can produce at
least 223 = 8.4 X 10106 genetically different gametes. But the actual number of variants is very
much greater than this because of chromosomal crossing-over or simply crossing-over, which
is an outcome of homologous recombination, in which DNA segments of homologous
chromosomes are exchanged.

In meiosis, when the exchange occurs between non-sister chromatids, it mixes the genetic
constitution of each of the chromosomes. The molecular details of crossing-over are difficult
to cover here. Briefly, a conserved meiosis-specific protein called Spo11 initiates crossing-
over by creating a double-strand break in the DNA of either a maternal or a paternal
chromatid. A very large multi-enzyme recombination complex, containing double-strand
DNA repair enzymes, assembles on the break and catalyzes homologous recombination. In
most cases, these events do not result in a crossover. In some cases, however, homologous
recombination leads to a crossover, where DNA segments are exchanged between two non-
sister chromatids in a reciprocal fashion.

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Introduction:
The realization that gametes are haploid came from an observation that also suggested
that chromosomes carry genetic information. In 1883, it was discovered in a study of
roundworms that the nucleus of an unfertilized egg and that of a sperm each contain two
chromosomes, whereas the fertilized egg or zygote contains four. This led to the chromosome
theory of heredity, which explained the long-standing paradox that the maternal and paternal
contributions to the character of the progeny seem to be equal, despite the enormous
difference in size between the egg and sperm. The finding also implied that haploid germ cells
arise from a special kind of cell division in which the number of chromosomes is precisely
halved. This type of division, called meiosis (the Greek word for diminution or lessening)
begins in animals in diploid germ-line cells in the ovaries or testes.

Presynaptic alignment:
During Meiosis when pairing of homolog chromosomes is done, the paired homologs are
brought into closer juxtaposition, with their structural axes about 400 nm apart, by a
mechanism that depends in most species on the programmed double-strand DNA breaks that
occur in sister chromatids. The pulling of these axes together have presumably one possibility
that the large protein machine, called a recombination complex, which assembles on a double-
strand break in a chromatid, binds the matching DNA sequence in the nearby homolog and
helps reel them. This so-called presynaptic alignment of the homologs is followed by
synapsis, in which the axial core of a homolog becomes tightly linked to the axial core of its
partner by a closely packed array of transverse filaments to create a synaptonemal complex,
which bridges the gap, now only 100 nm, between the homologs. Although crossing-over
begins before the synaptonemal complex assembles, the final steps occur while the DNA is
held in the complex where the actual recombination process starts.

Recombination Process:
The process of recombination here is done by homologous recombination. Infact, the
homologous recombination is the only mechanism that cells have adopted in which some of the
nucleotides are missing, if, can be recovered accurately. Means, it ensures proper segregation of
chromosomes and also their rejoining.

Homologous Recombination:
The hallmark of homologous recombination is that it takes place only between DNA
duplexes that have extensive regions of sequence similarity or homology. Not surprisingly,
base-pairing underlies this requirement, and two DNA duplexes that are undergoing
homologous recombination "sample" each other's DNA sequence by engaging in extensive
base-pairing between a single strand, from one DNA duplex and the complementary single

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strand from the other. The match need not be perfect, but it must be very close for
homologous recombination to succeed.

The two strands of DNA double helix need to be separated in condition if they are to do
recombination. Single-strand DNA-binding proteins, that are essential for DNA replication
as well as for homologous recombination, they bind tightly and cooperatively to the
sugar-phosphate backbone of all single-stranded DNA regions of DNA, holding them in
an extended conformation with the bases exposed. In this extended conformation, a DNA
single strand can base-pair efficiently either with a nucleoside triphosphate molecule e.g. in
DNA replication or with a complementary section of another DNA single strand e.g. as part of
a genetic recombination process. Because extensive base-pair interactions cannot occur
between two intact DNA double helices, the DNA hybridization that is critical for
homologous recombination can begin only after a DNA strand from one DNA helix is freed
from pairing with its complementary strand, thereby making its nucleotide available for
pairing with a second DNA helix.

The single-strand at the 3' DNA end is acted upon by several specialized proteins that
direct it to invade a homologous DNA duplex. Of central importance is the RecA protein, its
name in E.coli, and its homolog Rad5l, its name in virtually all eucaryotic organisms. Like a
single-strand DNA-binding protein, the RecA type of protein binds tightly and in long
cooperative clusters to single-stranded DNA forming a nucleoprotein filament. Because each
RecA monomer has more than one DNA-binding site, a RecA fllament can hold a single
strand and a double helix together. This arrangement allows RecA to catalyze a multistep
DNA synapses reaction that occurs between a DNA double helix and a homologous region of
single-stranded DNA. In the first step, the RecA protein intertwines the DNA single
strand and the DNA duplex in a sequence-independent manner.

Next, the DNA single strand "searches” the duplex for homologous sequences. Exactly
how this searching and eventual recognition occurs is not understood, but it may involve
transient base pairs formed between the single strand and bases that flip out from the duplex
DNA. Once a homologous sequence has been located, a strand invasion occurs: the single
strand displaces one strand of the duplex a sit forms conventional base pairs with the other
strand. The result is a hetero-duplex, a region of DNA double helix formed by the pairing
of two DNA strands that were initially part of two different DNA molecules.

The search for homology and the invasion of a single strand into a DNA duplex are the
critical reactions that initiate homologous recombination. They require, in addition to RecA-
like proteins and single-strand binding proteins, several proteins with speciaiized functions.
For example, Rad52 protein displaces the single-strand binding proteins allowing the
binding of Rad5l molecules, and in addition, promotes the annealing of complementary
single-strands.

Next, DNA hybridization creates a region of DNA helix formed from strands that
originate from two different DNA molecules. The formation of such a region, known as a
hetero-duplex, is an essential step in any homologous recombination process.

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Once a strand invasion reaction has occurred, the point of strand exchange called the
"branch point” can move through a process called branch migration. In this reaction, an
unpaired region of one of the single strands displaces a paired region of the other single
strand, moving the branch point without changing the total number of DNA base pairs.
Although spontaneous branch migration can occur, it proceeds equally in both directions, so it
makes little net progress. Specialized DNA helicases, however, can catalyze unidirectional
branch migration, readily producing a region of hetero-duplex DNA that can be thousands of
base pairs long.

In a related reaction, DNA synthesis catalyzed by DNA polymerase can drive a

unidirectional branch migration process through which the newly synthesized DNA is
displaced as a single strand, mimicking the way that a newly synthesized RNA chain is
released by RNA polymerase. This form of DNA synthesis appears to be used in several
homologous recombination processes, including the double-strand break repair processes.

Homologous recombination can be viewed as a group of related reactions that use

single-strand invasion, branch migration, and limited DNA synthesis to exchange DNA
between two double helices of similar nucleotide sequence. Having discussed its role in
accurately repairing damaged DNA, we now turn to homologous recombination as a means
to generate DNA molecules of novel sequence. During this process a special DNA
intermediate often forms that contains four DNA strands shared between two DNA helices.
In this key intermediate, known as a Holliday junction, or cross-strand exchange, two DNA
strands switch partners between two double helices. The Holliday junction can adopt multiple
conformations, and a special set of recombination proteins binds to, and thereby stabilizes, the
open, symmetric isomer. By using the energy of ATP hydrolysis to coordinate two branch
migration reactions, these proteins can move the point at which the two DNA helices are
joined rapidly along the two helices.

The four-stranded DNA structures produced by homologous recombination are only

transiently present in cells. Thus, to regenerate two separate DNA helices, and thus end
the recombination process, the strands connecting the two helices in a Holliday junction
must eventually be cut, a process referred to as resolution In bacteria, where we understand
this process the best, a specialized endonuclease (called RuvC) cleaves the Holliday
junctions leaving nicks in the DNA that DNA ligase can seal easily. However, during the
meiotic processes that produce germ cells in eucaryotes e.g. sperm and egg in animals, the
resolution mechanisms appears to be much more complicated. Extensive homologous
recombination occurs as an integral part of the process whereby chromosomes are parceled
out to germ cells during meiosis. Both chromosome have crossed over and gene conversion
result from these recombination events, producing hybrid chromosomes that contain genetic
information from both the maternal and paternal homologs.

Gene Conversion:
In sexually reproducing organisms, it is a fundamental law of genetics that each parent
makes an equal genetic contribution to an offspring, which inherits one complete set of

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nuclear genes from the father and one complete set from the mother. Underlying this law is
the highly accurate parceling out of chromosomes to the germ cells that takes place during
meiosis. Thus, when a diploid cell undergoes meiosis to produce four haploid germ cells,
exactly half of the genes distributed among these four cells should be maternal and the other
half paternal.

In some organisms e.g. fungi, it is possible to recover and analyze all four of the haploid
gametes produced from a single cell by meiosis. Studies in such organisms have revealed rare
cases in which the parceling out of genes violates the standard rules of genetics. Occasionally,
for example, meiosis yields three copies of the maternal version of a gene and only one copy
of the paternal allele. Alternative versions of the same gene are called alleles, and the
divergence from their expected distribution during meiosis is known as gene conversion.
Genetic studies show that only small sections of DNA typically undergo gene conversion, and
in many cases only a part of a gene is changed.

Several pathways in the cell can lead to gene conversion. First, the DNA synthesis that
accompanies the early steps of homologous recombination will produce regions of the
double Holliday junction where three copies of the sequence on one homolog are present,
these will produce sites of gene conversion once the Holliday junction is resolved. In
addition, if the two strands that make up a hetero-duplex region do not have identical
nucleotide sequences, mismatched pairs will result. These can be repaired by the cell's
mismatch repair system when used during recombination.

However the mismatch repair system makes no distinction between the paternal and
maternal strand and will randomly choose which strand to repair. As a consequence of
this repair, one allele will be "lost" and the other duplicated, resulting in net "conversion
“of one allele to the other. Thus, gene conversion, originally regarded as a mysterious
deviation from the rules of genetics, can be seen as a straightforward consequence of
the mechanisms of homologous recombination and DNA repair.

Regulation of Recombination:
Recombination has two distinct functions in meiosis; it helps hold homologs together so
that they are properly segregated to the two daughter cells produced by meiosis I, and it
contributes to the genetic diversification of the gametes that are eventually produced. As
might be expected, therefore, crossing-over is highly regulated. The number and location of
double-strand breaks along each chromosome is controlled, as is the likelihood that a break
will be converted into a crossover. Although the double-strand breaks that occur in meiosis I
can be located almost anywhere along the chromosome, they are not distributed uniformly.
They cluster at “hot spots”, where the chromatin is accessible, and occur only rarely in “cold
spots”, such as the heterochromatin regions around centromeres and telomeres.

At least two kinds of regulation influence the location and number of crossovers that
form, neither of which is well understood. Both operate before the synaptonemal complex
assembles. One ensures that at least one crossover forms between the members of each
homolog pair, as is necessary for normal homolog segregation in meiosis I. In the other,

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called crossover interference, the presence of one crossover event inhibits another from
forming close by, perhaps by locally depleting proteins required for converting a double-
strand DNA breaks into a stable crossover.

Effects of Recombination & Gene Conversion:

The sorting of chromosomes that takes place during meiosis is a remarkable feat of
intracellular bookkeeping. In humans, each meiosis requires that the starting cell keep track of
92 chromatids, or 46 chromosomes, each of which has duplicated, distributing one complete
set of each type of chromosome to each of the four haploid progeny cells. Not surprisingly,
mistakes can occur in allocating the chromosomes during this elaborate process. Mistakes are
especially common in human female meiosis, which arrests for years after diplotene: meiosis
I is completed only at ovulation, and meiosis II only after the egg is fertilized. Indeed, such
chromosome segregation errors during egg development are the commonest cause of both
spontaneous abortion and mental retardation in humans.

When homologs fail to separate properly, a phenomenon called non-disjunction; the

result is that some of the haploid gametes produced lack a particular chromosome, while
others have more than one copy of it. Cells with an abnormal number of chromosomes are
said to be aneuploid, whereas those with the correct number are said to be euploid. Upon
fertilization, aneuploid gametes form abnormal embryos, most of which die. Some survive,
however.

Down syndrome in humans, for example, which is the leading single cause of mental
retardation, is caused by an extra copy of chromosome 21, usually resulting from non-
disjunction during meiosis I in the female ovary. Segregation errors during meiosis I increase
greatly with advancing maternal age. Despite its fallibility, almost all eukaryotes use meiosis,
intermittently at least, to shuffle their genetic information before passing it on to the next
generation. Crossing-over makes a major contribution to this genetic shuffling process.

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2011

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i) And

MOLECULER BIOLOGY OF CELL BY ALBERTS (5th EDITION)

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