DNA Recombination
Mechanisms
Definition of Recombination
• Breaking and rejoining of two parental DNA
molecules to produce new DNA molecules
2
Recombination
ABCDEFGHIJKLMNOPQRSTUVWXYZ
abcdefghijklmnopqrstuvwxyz
ABCDEFGhijklmnoPQRSTUVWXYZ
abcdefgHIJKLMNOpqrstuvwxyz
Types of recombination
A+ B+ C+ A+ B- C-
Homologous
or general
A- B- C- A- B+ C+
Nonhomologous A B C A B F
or illigitimate
D E F D E C
l att l integrase
l
Site-specific
att att
att E. coli
Replicative transposase
recombination, A B C A B C
transposition
Transposable element 4
Mitotic and meiotic recombination
Recombination can occur both during mitosis
and meiosis
Only meiotic recombination serves the
important role of reassorting genes
Mitotic recombination may be important for
repair of mutations in one of a pair of sister
chromatids
Meiotic recombination generates new
combinations of alleles in offspring
Each line is duplex DNA, starting at pachytene of meiosis I
A1 B2 C2 A1 B3 C1
Dad Mom
A2 B1 C4 A3 B1 C3
Finish Meiosis I
Meiosis II
A1 B2 C2 A1 B3 C1
A1 B2 C4 A3 B3 C1
A2 B1 C2 A1 B1 C3
A2 B1 C4 A3 B1 C3
Fertilization
A1 B2 C4
A3 B3 C1 Child
Benefits of recombination
• Greater variety in offspring: Generates new
combinations of alleles
• Negative selection can remove deleterious
alleles from a population without removing the
entire chromosome carrying that allele
8
Recombination between
homologous DNA sites
Recombination provides a means by which a genome can
change to generate new combinations of genes
Homologous recombination allows for the exchange of blocks
of genes between homologous chromosomes and thereby is
a mechanism for generating genetic diversity
Recombination occurs randomly between two homologous
sequences and the frequency of recombination between two
sites is proportional to the distance between the sites
Recombination mechanisms
Best studied in yeast, bacteria and phage
Recombination is mediated by the breakage
and joining of DNA strands
Holliday model for recombination
• 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.
11
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
12
flanking markers are not recombined.
The cross-strand Holliday structure is
an intermediate in recombination (part I)
The cross-strand Holliday structure is an
intermediate in recombination (part II)
Generation of a chi
intermediate
Electron micrograph
of the chi form
Double
strand
break
model
Double
strand
break
model
Branch migration and resolution of
Holliday structures depends on Ruv
proteins
RuvA and RuvB
• DNA helicase that catalyzes branch migration
• RuvA tetramer binds to HJ (each DNA
helix between subunits), forces it into square
planar conformation
• 2 copies of RuvB bind at the HJ (to RuvA and 2
of the DNA helices)
– RuvB is a hexamer ring, has helicase & ATPase
activity
• Branch migration is in the direction of recA
mediated strand-exchange
RuvA RuvB
RuvA removed for visual purposes only
Model based on EM images.
RuvC : resolvase
• Endonuclease that cuts 2 strands of HJ
• Binds to HJ as a dimer (that already has RuvA/RuvB)
• Consensus sequence: (A/T)TT (G/C)
- occurs frequently in E. coli genome
- branch migration needed to reach consensus sequence!
Initiation of recombination by the
RecBCD enzyme
24
recBCD Pathway of Homologous Recomb.
Part I: Nicking and Exchanging
1. A nick is created in one strand by recBCD at a Chi
sequence (GCTGGTGG), found every 5000 bp.
2. Unwinding of DNA containing Chi sequence by recBCD
allows binding of SSB and recA.
3. recA promotes strand invasion into homologous DNA,
displacing one strand.
4. The displaced strand base-pairs with the single strand left
behind on the other chromosome.
5. The displaced and now paired strand is nicked (by
recBCD?) to complete strand exchange.
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recBCD Pathway of Homologous Recom.
Part II: Branch Migration and Resolution
1. Nicks are sealed Holliday Junction
2. Branch migration (ruvA + ruvB)
3. Resolution of Holliday Junction (ruvC)
26
RecBCD : A Complex Enzyme
• RecBCD has:
1. Endonuclease subunits (recBC) that cut
one DNA strand close to Chi sequence.
2. DNA helicase activity (recD subunit) and
a DNA-dependent ATPase activity
– unwinds DNA to generate the 3’ SS tails
27
RecA
• 38 kDa protein that polymerizes onto SS DNA 5’-3’
• Catalyzes strand exchange, also an ATPase
• Also binds DS DNA, but not as strongly as SS
RecA binds preferentially
to SS DNA and will
catalyze invasion of a DS
DNA molecule by a SS
homologue.
Important for many types
of homologous
recombination, such as
during meoisis (in yeast).
Fig. 6.19 in Buchanan et al.
RecA Function Dissected
• 3 steps of strand exchange:
1. Pre-synapsis: recA coats single-stranded DNA
(accelerated by SSB, so get more relaxed
structure).
2. Synapsis: alignment of complementary sequences
in SS and DS DNA (paranemic or side-by-side
structure).
3. Post-synapsis or strand-exchange: SS DNA
replaces the same strand in the duplex to form a
new DS DNA (requires ATP hydrolysis).
Cre protein and other recombinases
catalyze site-specific recombination
The Cre protein is a site-specific DNA recombinase, that is, it
can catalyse the recombination of DNA between specific sites in
a DNA molecule. These sites, known as loxP sequences, contain
specific binding sites for Cre that surround a directional core
sequence where recombination can occur.
Cre recombinase proteins bind to the first and last 13 bp
regions of a lox site forming a dimer. This dimer then binds to a
dimer on another lox site to form a tetramer. Lox sites are
directional and the two sites joined by the tetramer are parallel
in orientation. The double stranded DNA is cut at both loxP
sites by the Cre protein. The strands are then rejoined with
DNA ligase in a quick and efficient process.
32
33
The mechanism of Cre-loxP site-
specific recombination
Repair by End Joining
(Recombination Repair)
DNA non-homologous end-
joining (NHEJ)
Predominant mechanism for DSB repair in
mammals.
Also exists in single-celled eukaryotes, e.g.
Saccharomyces cerevisiae
Particularly important in G0/G1
Homologous recombination Non-homologous end-joining
DSB DSB
Rad50, Mre11,
Xrs2 complex Resection
Rad52 Ku70, Ku80
DNA-PKcs
Rad50, Mre11,
Strand invasion
Xrs2 complex “Cleaning up”
of ends
Rad51; BRCA2
DNA synthesis
XRCC4/
Ligase IV
Ligation, branch migration, Ligation
Holliday junction resolution
End-joining repair of nonhomologous
DNA
DNA-dependent protein kinase
(DNA-PK)
DNA
DNA-PK
DNA-PK
ACTIVE
INACTIVE KINASE
DNA-PK has three subunits
DNA
Ku70 X
Ku80
P
Ku70 DNA-PKcs
Ku80
69 kDa DNA-PKcs
83 kDa ATP ADP
470 kDa
INACTIVE ACTIVE
Target sites: Ser/Thr-Gln
DNA-PK has three subunits
DNA
Ku70 X
Ku80
P
Ku70 DNA-PKcs
Ku80
69 kDa DNA-PKcs
83 kDa ATP ADP
470 kDa
INACTIVE ACTIVE
… and is activated by DNA DSBs!
Multiple potential roles for
Ku/DNA-PKcs in NHEJ
