Mechanisms of Genetic Change II:
Recombination
Recombination
• The most important features of organisms are to adapt in
the environment and to maintain their DNA sequence in
the cells generation to generations with very little
alterations.
• In long term survival of organisms depends on genetic
variations, a key feature through which the organism can
adapt to an environment which changes with time.
• This variability among the organisms occurs through the
ability of DNA to undergo genetic rearrangements resulting
in a little change in gene combination. Rearrangement of
DNA occurs through genetic recombination.
• Thus, recombination is the process of formation of new
recombinant chromosome by combining the genetic
material from two organisms. The new recombinants show
changes in phenotypic characters.
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• Mechanism of Recombination
• Basically, there are three theories viz.,
breakage and reunion, breakage and copying
and complete copy choice that explain the
mechanism of recombination (Fig.8.23).
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(i) Breakage and Reunion:
• Two homologous duplex of chromosome
laying in paired form breaks between the gene
loci a and b, and a+ and b+ (Fig. 8.23A). The
broken segments rejoin crosswise and yield
recombinants containing a and b+ segment,
and a+ and b segment. This type of
recombination does not require the synthesis
of new DNA. This concept has been used to
explain genetic recombination.
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• (ii) Breakage and Copying:
• One helix of paired homologous chromosome
(ab and a+ b+) breaks between a and b (Fig.
8.23B).
• Segment b is replaced by a newly synthesized
segment copied from b+ and attached to a
section. Thus the recombinants contain and
ab+ and a+ b+.
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• (iii) Complete Copy Choice:
• In, 1931, Belling proposed this theory for
recombination of chromosome in higher animals.
However, it has been questioned by several workers.
• According to this theory a portion of one parental
strand of homologous chromosome acts as template
for the synthesis of a copy of its DNA molecule. The
process of copying shifts to the other parental strand.
Thus, the recombinants contain some genetic
information of one parental strand and some of the
other strand (Fig. 8.23 C).
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Types of Recombination
• Many kinds of recombination occur in
microorganisms.
• These are classified basically into the
following three groups:
• (i) General/ homologous recombination,
• (ii) Non-reciprocal recombination, and
• (iii) Site specific recombination.
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1.Homologous recombination
• General recombination occurs only between the complementary
strands of two homologous DNA molecules. Smith (1989) reviewed
the homologous recombination in prokaryotes. General
recombination in E. coli is guided by base pairing interactions
between the complementary strands of two homologous DNA
molecules.
• Double helix of two DNA molecules breaks and the two broken
ends join to their opposite partners to reunite to form double helix.
The site of exchange can occur anywhere in the homologous
nucleotide sequence where a strand of one DNA molecule becomes
base paired to the second strand to yield heteroduplex just
between two double helices (Fig. 8.24).
• In the heteroduplex no nucleotide sequences are changed at the
site of exchange due to cleavage and rejoining events. However,
heteroduplex joints can have a small number of mismatched base
pairs.
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• General recombination is
also known as homologous
recombination as it
requires homologous
chromosomes.
• In bacteria and viruses
general recombination is
carried out by the products
of rec genes such as RecA
protein. The RecA protein
is very important for DNA
repair; therefore, it is recA
dependent recombination.
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Holliday Model for General
Recombination
• Holliday (1974) presented a model to show the
general recombination
• According to this model recombination occurs in
five steps such as
– strand breakage,
– strand pairing,
– strand invasion/assimilation,
– chiasma (crossing over) formation,
– breakage and reunion and mismatch repair.
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2-Non-reciprocal Recombination
(Gene Conversion)
• The fundamental law of genetics is that the two partners contribute the
equal amount of genes to the offsprings. It means that the offsprings
inherit the half complete set of genes from the male and half from the
female. One diploid cell undergoes meiosis producing four haploid cells;
therefore, the number of genes contributed by male gets halved and so
the genes of female.
• In higher animals like man it is not possible to analyse these genes taking a
single cell. However, in certain organisms such as fungi it is possible to
recover and analyse all the four daughter cells produced from a single cell
through meiosis.
• Occasionally, three copies of maternal allele and only one copy of paternal
allele is formed by meiosis. This indicates that one of two copies of
parental alleles has been altered to the maternal allele. This gene
alteration is of non-reciprocal type and is called gene conversion. Gene
conversion is thought to be an important event in the evolution of certain
genes and occurs as a result of the mechanism of general recombination
and DNA repair.
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mechanism for gene conversion
• Non-reciprocal general recombination is given
in Fig. 8.27. Kobayashi (1992) has discussed
the mechanism for gene conversion and
homologous recombination.
• This process starts when a nick is made in one
of the strands (a).
• From this point DNA polymerase synthesizes
an extra copy of a strand and displaces the
original copy as a single strand (b).
• This single strand starts pairing with the
homologous region as in lower duplex of DNA
molecule (b).
• The short unpaired strand produced in step
(b) is degraded when the transfer of
nucleotide sequence is completed.
• The results are observed (in the next cycle)
when DNA replication has separated the two
non-matching strands (c). 12
3-Site-Specific Recombination
• Site specific recombination alters the relative position of nucleotide
sequences in chromosome. The base pairing reaction depends on protein
mediated recognition of the two DNA sequences that will combine. Very
long homologous sequence is not required.
• Unlike general recombination, site specific recombination is guided by a
recombination enzyme that recognises specific nucleotide sequences
present on one of both recombining DNA molecules. Base pairing is not
involved, however, if occurs the heteroduplex joint is only a few base pair
long.
• It was first discovered in phage λ by which its genome moves into and out
of the E. coli chromosome. After penetration phage encoded an enzyme,
lambda integrase which catalyses the recombination process (Fig. 8.28).
• Lambda integrase binds to a specific attachment site of DNA sequence on
each chromosome.It makes cuts and breaks a short homologous DNA
sequences. The integrase switches the partner strands and rejoins them to
form a heteroduplex joint of 7 bp long. The integrase resembles a DNA
topoisomerase in rejoining the strands which have previously been
broken.
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• Site specific
recombination is of the
following two types:
• (a) Conservative site-
specific recombination:
• Production of a very short
heteroduplex by requiring
some DNA sequence that
is the same on the two
DNA molecules is known
as conservative site-
specific recombination.
The detail procedure is
described in Fig. 8.28.
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• (b) Trans-positional site-specific recombination:
• There is another type of recombination system known as trans-positional
site-specific (TSS) recombination. The TSS recombination does not produce
heteroduplex and requires no specific sequences on the largest DNA.
• There are several mobile DNA sequences including many viruses and
transposable elements that encode integrates. The enzyme integrates by
involving a mechanism different from phage λ insert its DNA into a
chromosome. Each enzyme of integrates recognises a specific DNA sequence
like phage λ.
• K. Mizuuchi (1992a) reviewed the mechanism of trans-positional
recombination based on the studies of bacteriophage Mu and the other
elements. The enzyme integrase was first purified from Mu.
• Similar to integrase of phage λ, the Mu integrase also carries out of its
cutting and rejoining reactions without requirement of ATP. Also they do not
require a specific DNA sequence in the target chromosome and do not form
a joint of heteroduplex.
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• Different steps of TSS recombinational
events are shown in Fig. 8.29. The
integrase makes a cut in one strand at
each end of the viral DNA sequences, and
exposes the 3′-OH group that protrudes
out. Therefore, each of these 3′-OH ends
directly invades a phosphodiester bond
on opposite strands of a randomly
selected site on a target chromosome.
• This facilitates to insert the viral DNA
sequence into the target chromosome,
leaving two short single stranded gaps on
each side of recombinational DNA
molecule.
• These gaps are filled in later on by DNA
repair process (i.e. DNA polymerase) to
complete the recombination process. This
mechanism results in formation of short
duplication (short repeats of about 3 to
12 nucleotide long) of the adjacent target
DNA sequence. Formation of short Fig. 8.29 : Mechanism of trans-
repeats is the hall-marks of a TSS positional site-specific recombination;
recombination. SDR, short direct repeats of target
DNA sequence. 16
