Genetic Recombination

A/P Deng Lih Wen

Department of Biochemistry
bchdlw@nus.edu.sg

Readings:
- Lewin, Genes XI, Chapter 15
- Alberts et al, MBoC5, Chapter 5
 Types of genetic recombination
2 types in bacteria
• General recombination
– Require long (> 50 bp) sequence homology
– RecA-dependent

• Site-specific recombination
– Require very short (< 5 bp) sequence
homology;
– Special site recognition
– RecA-independent but require specialized
proteins
 1. General Recombination

• Genetic exchange takes place

between 2 pieces of
homologous DNA sequences
• May be intra- or inter-molecular
events
 Recombination may
results in insertion, gene amplification
and deletions
1. Insertion 3. Deletion

z y x

2. Duplication (amplification) 4. inversion

4
• Heteroduplex formation at
the site of crossover
• New recombinant DNA
molecules are produced
• Heteroduplex DNA (Hybrid
DNA from the different
parental duplex molecules);
it occurs during genetic
recombination.
 Single-strand invasion model
Recombination is initiated by a nick in one strand

RecA, RecBCD RecA Ligase

Single stranded DNA,

coated by RecA,
invades homologous Holliday
duplex junction

Holliday junction is an intermediate structure in homologous recombination, where the two duplexes of
DNA are connected by the genetic material exchanged between two of the four strands, one from each
duplex.
Double strand break model
1. Limited degradation at double-strand
break by a 5’3’ exonuclease to
create protruding single-stranded 3’
tails
2. Single-stranded DNA are recognized
by RecA protein which initiates
homology search in the other
chromosome
3. ATP-dependent strand exchange
occurs followed by DNA synthesis
and ligation
4. Branch migration of Holliday
junctions
5. Resolution by strand cutting

Branch migration describes the ability of a DNA strand partially

paired with its complement in a duplex to extend its pairing by
displacing the resident strand with which it is homologous.
 isomerization
Splice recombinant DNA results
from a Holliday junction being
resolved by cutting the non-
exchanged strands. Both strands
of DNA before the exchange point
come from one chromosome; the
DNA after the exchange point
come from the homologous
chromosome.
Patch recombinant DNA results
from a Holliday junction being
resolved by cutting the exchange
strands. The duplex is largely
unchanged, except for a DNA
sequence on one strand that came
from the homologous
chromosome. patch splice
 The Ruv complex acts on
recombinant junctions.
• RuvA
22 kD protein which binds to
RuvB and Holliday
junctions
• RuvB
37 kD helicase that catalyzes
branch migration
RuvB RuvA
• RuvC
19 kD nuclease which
resolves Holliday structures
(resolvase)
• DNA ligase
RecBCD is a helicase-
nuclease complex that
initiates the repair of
helicase
double-strand breaks
Chi (crossover hotspot instigator) sites
3’5’ & 5’3’
• ~1000 chi sites (5'- exonucleases
GCTGGTGG-3’) are present
on the E. coli chromosome
• RecBCD generates single-
stranded DNA at Chi sites
• Chi sites are “hotspots” for
general recombination
ss DNA is coated by RecA for
homologous recombination 11
 AA
TT Site in gene X
CG where blue and red
GC
allele different

Gene conversion is non-

reciprocal exchange
Only small sections of DNA or
part of a gene undergoes
gene conversion
AA

CG
CG
GC

AA
Please refer to the next slide
CG
CG 12
GC
Mismatched DNA in a heteroduplex are recognized
and removed by the DNA repair enzymes and
replaced with a copy of the complementary strand

NO GENE CONVERSION
2. Site-Specific Recombination

A. Transposons
B. Phage integration and excision
C. Cre-Loxp System
 Three major classes of transposable elements

Table 5-3 Molecular Biology of the Cell (© Garland Science 2008)

 Class I: DNA-only transposons
 Move by a CUT-AND-PASTE mechanism
 Predominately in bacteria and responsible for spread of
antibiotics resistance in bacterial strains
 Excised from one spot on a genome and inserted into
another
 Transposase inserts into target site (<20 nt) on chromosome
 Transposition is rare (~ once in 105 cell generations)
Short DNA sequence that recognised only by the transposase
encoded by that element

Figure 5-68 Molecular Biology of the Cell (© Garland Science 2008)

 Cut-and-Paste transposition
These short direct repeats
are the clues in identifying
transposon in genome
sequence)

Transposase functions as a dimer, each

monomer recognizes the same specific DNA
sequence at the ends of the transposon.

Staggered cut made on the

end of target sites

DNA pol + ligase

Figure 5-69 Molecular Biology of the Cell (© Garland Science 2008)
 Class II: Retroviral-like retrotransposons
 Some viruses use transpositional site-specific recombination
to move themselves into host chromosomes E.g.
Bacteriophage Mu, retroviruses
 Move via RNA intermediate and use a reverse transcriptase
and integrase (transposase) Once the reverse transcriptase
has produced a double
stranded DNA, specific
sequence near its two ends
can be recognised by a virus-
encoded transposase
(integrase) which then insets
the viral DNA into the
chromosome using a similar
cut-and-paste DNA only
transposons.
 Class III: Nonretroviral
retrotransposons
 Descendants of retroviral DNA (remnants of
polyA tail)
 Occur as repetitive DNA sequences (e.g. L1
element)
 Move via endonuclease-reverse
transcriptase complex
Recap for three classes
1. DNA-only transposons move by DNA breakage
and joining
2. Retroviral-like retrotransposons also move by
breakage and joining, but via an RNA intermediate
3. Non-retroviral retrotransposons move by making an
RNA copy which acts as a direct template for a DNA
target-primed reverse transcription event

Figure 5-74 Molecular Biology of the Cell (© Garland Science 2008)

 B. Phage integration and excision
Circular phage lambda DNA is converted to 
an integrated prophage by a reciprocal 
recombination between attP and attB. 

Gateway cloning system

(Integration
host factor)

Refer to Lecture 4
specialised transduction

http://blog.addgene.org/plasmids-101-gateway-cloning
C. Cre-Loxp System

• Cre is a bacteriophage P1
integrase which catalyzes
site-specific recombination
between loxP sites (34 bp
short direct repeats)

• This recombination also

works in mammalian cells
in vitro and in vivo

Cre is derived from bacteriophage P1

and is a member of the integrase
family of site specific recombinase
 Use site-specific recombination to
regulate gene expression
 Induction of recombination enzyme results in activation
of transgene expression
Cre-LoxP
system

Inactive
gene
Active
gene
Brain specific promoter
 Tissue-specific gene regulation

GENE OFF

GENE ON

Figure 5-79 Molecular Biology of the Cell (© Garland Science 2008)