STBP1013

FUNDAMENTALS OF MOLECULAR
BIOLOGY

DNA RECOMBINATION
 WHAT IS RECOMBINATION??
• process or set of processes by which
DNA molecules interact with one another
to bring about a rearrangement of the
genetic information or content in an
organism.
• In eukaryotic systems -
recombination as the process
that is responsible for crossing-
over during meiosis.

• Crossing-over has been well-

documented genetically and is
used to map the relative
locations of genes on a
chromosome
• Examples of recombination in prokaryotic
systems are
(i) integration of the bacteriophage
lambda prophage,
(ii) recombination of bacterial DNA
following conjugation between bacteria
(iii) formation of plasmid multimers
Replication of Bacteriophage- Evidence for
Recombination Events

5
Bacteriophage – Plaque assay
 Recombination in a bacterial
system
• was first demonstrated independently by Al
Hershey and Max Delbrück in 1947.
• They studied the infection of E. coli with
bacteriophage.
• If an E. coli cell was infected at the same time
with two genetically different bacteriophage,
the resulting phage population included
recombinant phage types as well as the
original parental phage types.
• The h locus determines whether the phage
can grow on a particular strain of E. coli:
• phage that are h- can infect the strain;
• phage that are h+ cannot.
• The r locus is a gene that determines whether
the phage will lyse the host cells rapidly or
slowly:
• phage that are r - will lyse the host cells
rapidly;
• phage that are r + will lyse the host cell slowly.
• In addition, two strains of E. coli were used in
the experiment:
• strain 1 supports growth of h- phage but not
h+ phage;
• strain 2 supports the growth of both phages.
• In the experiment, bacteriophages are plated
on a lawn of bacteria that consists of a
mixture of both strains of E. coli.
• If a phage can infect both strains of bacteria
(i.e. if it is h-) then the resulting plaque will be
clear.
• If the phage can infect only one of the two
strains of bacteria (i.e. if it is h+) then the
resulting plaque will be turbid because the
non-infected bacteria will be growing.
• When the experiment is performed, four types
of plaque were observed:
phenotype inferred genotype
Clear and small h-r+
Cloudy and large h+r-
Cloudy and small h+r+
Clear and large h-r-

Note:
r – phage will lyse the host cells rapidly;
r + phage will lyse the host cell slowly
phage that are h- can infect both E. coli strains;
phage that are h+ can infect only one E. coli strain.
• Most of the plaques correspond to the
parental phenotypes but a significant number
have the recombinant phenotypes.
• Most of the plaques correspond to the
parental phenotypes but a significant number
have the recombinant phenotypes.
• However, when the progeny phage were used
to reinfect E. coli so as to examine their
phenotype, a low but definite percentage of
the resulting plaques were found to contain
two different types of phage although only
one type had been expected.
• This implies that some of the progeny phage
were not genetically homogeneous.
• This observation can be explained by models
of recombination that allow for heteroduplex
forms to be generated.
• Al Hershey and Max Delbrück shared the 1969
Nobel prize in Medicine & Physiology with
Salvador Luria for their discoveries concerning
"the replication mechanism and the genetic
structure of viruses"
The Meselson - Weigle Experiment
• In the simplest sense, recombination is an
exchange of both strands between two DNA
molecules:
• Note: each line in the above cartoon figure
represents one strand of a DNA double helix.
• This representation implies that both strands
of each molecule must be broken and then
rejoined. This was first demonstrated by an
experiment performed by Matt Meselson and
Jean Weigle in 1961.
• Meselson and Weigle infected E. coli cells at
the same time with phage from two different
stocks of bacteriophage lambda.
• One stock had been prepared by growing the
bacteriophage lambda c-mi- in cells grown in
medium containing heavy isotopes of carbon
(13C) and nitrogen (15N).
• The other stock had been prepared by
growing bacteriophage lambda c+mi+ in
medium containing light isotopes of carbon
and nitrogen.
Note: each line in the above figure represents a phage
chromosome, i.e. a double helical DNA molecule.
•After infection, the progeny phage were
isolated and banded on a CsCl gradient.

•A broad band of phage particles were

found on the gradient.

•Non-recombinant phage were found, as

expected, at two well-defined densities
corresponding to the parental light and
heavy phages.

•Recombinant phage were found -

surprisingly - at all intermediate densities
between these two.
• They also followed the course of the infection
using two genetic markers, c and mi, which
were located near one end of the lambda
chromosome.
• Note: each line in the above figure represents a
phage chromosome, i.e. a double helical DNA
molecule.
• When the phenotypes of the intermediate
density phage particles were analyzed,
recombinant phage that were c-mi+ were
found near the band of "heavy" phage while
recombinant phage that were c+mi- were
found near the band of "light" non-
recombinant phage.
 3 general types of recombination
1. Homologous genetic recombination
2. Site-specific recombination
3. Illegitimate recombination
 3 general types of recombination
1. Homologous genetic recombination
2. Site-specific recombination
3. Illegitimate recombination
 1. Homologous Recombination
• Also known as general recombination or general
homologous recombination
• The exchange of genetic material between two
molecules that share a large degree of identity with one
another.
• This is the type of recombination that is required during
meiotic crossing over, for bacteriophage recombination,
for recombination following bacterial conjugation, and
during the formation of plasmid multimers
 Homologous Recombination

• Exchange of DNA sequences between

DNA molecules that contain identical or
nearly identical sequences along their
length.

• The region to be recombined is known

as homology between sequences – can be
as few as 50-100 bp or as much as the
whole chromosome
 The requirement for homologous recombination

1. Two DNA sequences with similar or almost identical

base pair sequence (homologous sequence)

2. The ability to form stable hydrogen bonds between the

bases on one strand of DNA sequence and the bases
on the complementary strand on the other DNA
sequence

3. Proteins needed to carry out recombination. These

proteins include those make two DNA sequence to
stay close to each other, enzymes that break
phosphodiester bonds (endonuclease or exonuclease)
and enzymes that rejoin phosphodiester bond (ligase).
Early Models for Homologous
Recombination
 Important protein in E. coli for homologous
recombination
 RecA – binds single-stranded DNA,
promote base pairing
 RecA binds to single stranded DNA
in the presence of ATP to form
RecA-ssDNA.
 A RecA-ssDNA filament can bind RecA
and unwind (by breaking hydrogen
bonds) another double stranded
DNA promoting base pairing of the
nucleotides in the ssDNA with RecA

nucleotides in the complementary

strand of the homologous dsDNA
 RecB, RecC and RecD – also known
as RecBCD or Exonuclease V. Has
various function
 Nuclease - nick dsDNA, ssDNA.
Created the ssDNA , first
function needed in homologous
recombination
 Helicase activity – separate the
strands of dsDNA.
 ATPase activity - hydrolyses ATP

 Other protein RecE, RecF, RecG,

RecJ, RecN, RecO, RecQ, RecR, RecT,
RusA, RuvA, RuvB, RuvC – various
functions such as exonuclease,
endonuclease, ATPase, dsDNA
helicase, binds to Holliday junction,
etc
Proposed function of RecBCD and RecA in homologous recombination in E. coli

• RecBCD enzyme binds to a blunt-end

DNA from a double-stranded DNA break.

• It unwinds the dsDNA and preferentially

degrades the 3’-terminating strand (top
strand).

• Interaction with χ (chi site) results in

attenuation of the 3’ → 5’ nuclease
activity, activate the weaker 5’ → 3’
nuclease activity, and the facilitated
loading of RecA protein onto the χ-
containing ssDNA.
Model for Homologous Recombination
• The intermediate that is formed is called a
Holliday intermediate or Holliday structure.
The shape of this intermediate in vivo is
similar to that of the greek letter chi, hence
this is also called a chi form.
• Resolve the structure.
• There are two ways in which this can happen:
• If the same strands are cleaved a second time
then the original two DNA molecules are
generated:
• If the other strands are cleaved, then
recombinant molecules are generated:
 Holliday Structure
Name after Robin Holliday
who in 1964, proposed a
model for homologous
recombination and re-
established by David
Dressler and Huntington
Potter in 1976
 Potter & Dressler's evidence for
the Holliday Model
• In 1976, David Dressler and Hunt Potter
published the results of a series of
experiments that demonstrated the validity of
the Holliday model of recombination.
• They used E. coli cells containing the colicin E1
derived plasmid, pMB9.
• This plasmid was one of the very earliest
plasmids developed for cloning in Herbert
Boyer's laboratory.
• Normally, E. coli contain about 20 copies of
this plasmid per cell.
• However, if the cells are exposed to
chloramphenicol then, although chromosomal
replication stops, plasmid replication does not
and the number of plasmid molecules
increases to 1000 copies per cell.
• With so many more copies of the plasmid in
the cell, the chances of recombination
increase as does the probability of observing a
recombination intermediate.
• When plasmid was isolated from the cells,
purified by CsCl gradient centrifugation, and
observed in the electron microscope, a
number of candidates for intermediates were
observed.
• These all had the appearance of "figure 8"
structures. However, there are 3 possible ways
such structures might arise:
I. as a double-sized circular plasmid twisted
over on itself.
II. as two interlocking circular plasmid
molecules.
III. as a genuine recombination intermediate.
• In order to distinguish between the three
possibilities, Potter and Dressler digested their
plasmid preparations with EcoRI.
• This enzyme will generate monomer sized
linear molecules from either of the first two
possible structures.
• However, it will generate unique chi-shaped
structures from the third.
• When they did this, Potter and Dressler found
that between 0.5% and 3% of the molecules
they observed were chi-shaped structures.
• The molecules were symmetrical in that the
opposite arms were identical lengths and had
identical denaturation patterns.
• Finally, they saw no such structures if they
prepared their plasmids from recA- strains of
E. coli.
• From this evidence they concluded:
• . . . the intermediates we have observed in
the electron microscope provide physical
evidence in support of the recombination
intermediate postulated by Holliday on
genetic grounds.
 3 general types of recombination
1. Homologous genetic recombination
2. Site-specific recombination
3. Illegitimate recombination
 Site-specific recombination
• This type of recombination
involves the exchange of
genetic material at very
specific sites only.
• Examples include the
integration of
bacteriophage lambda into
the host chromosome to
form the prophage
 Site-specific Recombination

• Involves a protein called

recombinase, that acts on both
participating DNA sequences at a very
specific sequence on the nucleotides.

•The sequence is often short and must

be present on both DNA segments
•Different site-specific recombinase
will recognise different sequence.

• Site specific recombination can take

place between two separate DNA
molecules as long as they have the
same specific site (intermolecular).
• It can also take place within
the same DNA molecule
(intramolecular) – the specific
site must present at least
twice in the molecule

• Described as conservatives
due:
 No nucleotide are lost
 DNA replication is not
required. Involves in
breakage and rejoining of
DNA strands .
 ATP is not required for
site specific recombination
 Site-specific Recombination: Integration of Bacteriophage 
chromosome into E. coli

The molecular event for

integration and excision
of bacteriophage .

To integrate the  genome

into the E.coli
chromosome, two specific
sites (attP and attB) and
the activities of  Int and
E. coli IHF are required
 3 general types of recombination
1. Homologous genetic recombination
2. Site-specific recombination
3. Illegitimate recombination
 Illegitimate recombination
• There are a number of other genetic
exchanges which do not fall into any of the
above classes - hence their name: illegitimate
recombination.
• Illegitimate recombination is a broad term
designating recombination events that are
independent of RecA (Michel 1999).
• Include spontaneous DNA rearrangement such
as deletions, duplications and formation of
specialized phage.
Illegitimate recombination, repeats
and SSR
• Illegitimate recombination events are frequently
associated with very small repeated sequences.
• Direct repeats are nucleotide sequences present in
multiple copies in the genome
• These repeats can be as small as 3 or 4 bp and know
as short sequence repeat (SSR).
• The frequency of Illegitimate recombination
decreases as the SSR located further apart.
• The frequency increase exponentially as the SSR get
longer. At 8 bp in length, Illegitimate recombination
can be quite frequent.
Scenarios of illegitimate recombination between close direct repeats and SSRs. Black boxes
represent start and stop codons of the original gene, grey boxes represent strict repeats, and
light grey boxes represent regions of weaker homology. Dashed lines indicate that deletions
and duplication may induce frameshifts and therefore produce ORFs of very different length.
(A and B) Duplication/deletion of the repeat and the region between occurrences. (C) The
regions of non-strict similarity become similar after conversion. (D and E) Increase/decrease in
the number of motifs of the SSR. Homologous recombination between long repeats closely
follows the scenarios of (A), (B) and (C), except that large duplications are unstable and large
deletions are strongly counter selected. Thus, conversions or reciprocal translocations are the
most frequent outcome of homologous recombination between long distant repeats.
 Homology-facilitated illegitimate
recombination

The process of branch migration frequently stops when it

encounters the end of homology between the recombining
molecules.

But sometimes, the heterologous regions are integrated.

• The process termed as "homology-facilitated
illegitimate recombination" (HFIR) to indicate
the essential role of homologous anchor
sequences.
• For example, HFIR has been shown to
mediate the transfer of genes from transgenic
tobacco plastids (trnL und ycf5) to
Acinetobacter sp. equipped with a truncated
aadA gene.