UNIT 3

MOLECULAR
MECHANISM OF
RECOMBINATION
 INTRODUCTION
Gene is a polynucleotide chain that can control a
specific trait
Can be unit of function- cistron

Unit of mutation-mutan

Unit of recombination recon

Recombination- process of formation of new

recombinant chromosomes by combining genetic
materials from 2 organisms.
 Steps involved:
Crossing over
Gene conversion
Exchange between sister chromatids
Repair of DNA damage
 HOMOLOGOUS RECOMBINATION

Between complementary strands of 2 homologous

DNA molecules
Crossing over: genetic exchange between
chromosomes as a result of HR
Frequency of crossing over is proportional to the
physical distance between genes
Helps to get new combination and repair the prior
damages
 Steps:
Allignment of 2 DNA molecules

Introduction of breaks in DNA( single or double strand

break)
Strand invasion formation of initial short region of base
pairing
Causes the formation of a junction ie:- 2 DNA connected
through crossing over Holliday junction
Movement of holliday juction by continuous melting and
formation of base pairs branch migration
Cleavage of holliday juction - resolution
HOLLIDAY MODEL
 Pairing: align homologous duplexes

Single strand invasion:

Endonuclease nicks at corresponding regions of the

same strands of homologous chromosomes
Ends generated by the nicks invade the other,
homologous duplex
Ligase seals nicks to form a joint molecule.

(Holliday intermediate or Chi structure)

Branch migration expands heteroduplex region.

 Resolution of joint molecules

Can occur in one of two ways

The Holliday junction can be nicked in the same strands

that were initially nicked = horizontal resolution. This
results in NO recombination of flanking markers.
The Holliday junction can be nicked in the strands that
were not initially nicked = vertical resolution. This
results in RECOMBINATION of flanking markers.
Heteroduplex formation
 Vertical & horizontal resolution
A+

B+
V

B-
A-

or
1. Horizontally H
2. Vertically V

A+ B- A+ B+

A- B+ A- B-

This leaves a region of heteroduplex, and

the flanking markers have recombined.
A region of heteroduplex is left, but the
flanking markers are not recombined.
 Enzymes involved
rec A protein and recBCD endonuclease are purified
from bacterial systems
Topoisomerases

int proteins, xis protein and IHF Protein in phage

rec A and rec BCD promotes basepairing in single

strand of DNA
 Double strand break model
This model provides a better explanation for recombination
events in yeast
A double strand break precedes recombination.

HR in bacteria helps in repair of DSBs.

One DNA molecule is used preferentially as the donor of genetic

information.
A double-strand break is expanded to a gap, which is then
repaired by copying a homologous sequence
commonly results in crossover

2 holliday junctions will form

http://web.mit.edu/engelward-lab/an
imations/DSBR.html
 Steps
DSB is introduced into one of the DNA
the DSB is processed by either a helicase, a nuclease, or a
combination of both, to yield a free 3-ssDNA overhang(s)
free 3-terminal end is homologously paired with its homologue
by a RecA-like protein to form an intermediate known as a joint
molecule
pairing of the displaced complementary strands results in the
formation of characteristic intermediates known as Holliday
junctions
Holliday junction(s) is extended unidirectionally by branch
migration proteins
the Holliday junction is cleaved by a resolvase to yield
recombinant products (e.g.spliced or patched)
 Site specific

recombination
Homologous recombination is to ensure that the
genome of organism is nearly identical from
generation to generation
some recombinations alter the relative position of
nucleotide sequence in chromosome- site specific
recombination
It can be of 2 types
Conservative site specific recombination (CSSR)

Transpositional site specific recombination (TSSR)

 Conservative Site Specific
Recombination
GENERAL FEATURES:
Recombinase recognize specific sequences where
recombination occurs
It brings these specific sequences together to form a
protein- DNA complex(synaptic complex)
Within synaptic complex recombinase catalyse
cleavage and rejoing of DNA molecule either to invert
or to move a segment to a new site
One recombinase enzyme will do all these steps

Controlled process to minimise the DNA damage

 Recombinases

Cleaves all 4 strands prior to exchange

For each strand 4 molecules of reombinase is needed

Recombinase covalently linked to DNA through

phosphotyrosine/phosphoserine bond based on type of
recombinase(Serine recombinase &Tyrosine recombinase(cre-lox
recombination))
Energy released by cleavage will be compensated when the
recombinase join the molecule forming Protein-DNA
intermediate
Hence called conservative site specific recombination
Then cleaved strands will be rejoined with new molecules and
hence form a holliday intermediate.
In some cases no holliday junction is formed
Eg :CSSR is integration of phage genome in bacterial
chromosome:
During integration recombination occurs at exactly same
nucleotide sequence on phage and host DNA.
Recombination sites are ~20bp long

It have sites for the attachment of recombinase enzyme

3 different arrangements occur due to CSSR based on the

organisation of recombination sites

i. Insertion

ii. Deletion

iii. Inversion
 Prophage in lysogeny
Independent in lytic
Transition between the two involve site specific recombination
Because integrase remained bound
with DNA just like topoisomerase, the
action of lambda integrase does not
require ATP.
excisionase
 Application of CSSR
To delete unwanted transgenes like markers
To activate transgene expression/ to switch between alternate
transgenes
To fascilitate precise transgene integration
Chromosome engineering
 Transpositional recombination
Does not produce heteroduplex
Require no specific sequence
Mobile genes are involved
Jumping genes
Can move from 1 position to another on same or different
chromosome
Transposible elements encode integrase enzyme
These enzymes will recognize specific DNA
Steps:
Integrase makes a cut at 1 strand at each end of DNA-results in 3
OH
These OH groups will invade phospho diester bond on opposite
strands of randomly selectd site on target chromosome
DNA sequences insert to target and a short gap will be left
Another repair process will occur to fill the gap and complete the
process
Process result in short repeats of sequences( hall mark of TSSR)