Chapter 11
Homologous Recombination at the
Molecular Level
吳彰哲 (Chang-Jer Wu)
Department of Food Science
National Taiwan Ocean University
1
Introduction
All DNA is recombinant DNA
During meiosis homologous recombination
results in crossing over = the exchange of
genetic material between chromosomes
The frequency of crossing over between 2
genes depends on the distance between the
genes – the greater the distance the more
frequent the cross-over
Homologous recombination is essential to
cellular processes and is catalyzed by proteins
specific for this function
Homologous recombination provides genetic
variation, allows repair (replacement) of
damaged DNA and can influence gene
expression
2
DNA breaks are common and initiate
recombination
DS breaks in DNA occur
frequently and cause cell
death if not repaired
DSB can be repaired by
recombination or by NHEJ
method
In bacteria, repairing DSB
is primary role of
homologous recombination,
also promotes genetic
exchange
In eukaryotic cells, primary function
is in chromosome pairing, although
repair is important
3
Models of homologous recombination
DNA replication is semiconservative
Recombination is conservative = direct breakage and
rejoining of DNA
Recombination also often involves limited destruction and
re-synthesis of DNA
Current models of recombination include:
1. Alignment of 2 homologous DNA molecules
2. Introduction of ds breaks into DNA
3. Base-pairing between the 2 recombining molecules strand invasion forms cross structure =
Holliday junction or structure
4. Movement of Holliday junction = branch migration
5. Cleavage of Holliday junction = resolution
4
Strand invasion is a key early step in homologous
recmbination
2 dsDNA molecules
with different alleles
Initiation begins with
two nick at the same
location in 1 strands
(dsDNA breaks)
DNA near nick is
“peeled” away, freeing
strands to invade (pair)
with homologous
duplex
Strand invasion creates
a Holliday junction
5
Strand invasion is a key early step in homologous
recmbination
Holliday junction
moves along strands
= branch migration
Migration increases
the length of
exchanged DNA
Exchange of DNA
creates
heteroduplex DNA
6
Holliday model
Resolution, finishing
recombination, requires
cutting of the DNA
The location of the cuts
determines whether the
recombination results in
crossover products or patch
products
7
Holliday model
Cutting the strands that were
not broken in the initiation
reaction results in “splice” or
crossover products = regions
of the parental molecules are
covalently linked by region of
hybrid duplex - reciprocal
strand exchange
Cutting the strands that were
broken in initiation results in
“patch” product = a region of
hybrid DNA in otherwise
parental chromosomes, no reassortment of flanking genes =
non-crossover products
8
Double-strand break repair model
Current Model - Recombination is often
initiated by dsDNA breaks in DNA
Initiating event is a DSB in 1 of 2
molecules – more likely model than
Holliday which requires 2 ssDNA breaks at
same place in 2 molecules
Meiotic cells undergo double-strand
breakage 100-1000x the rate in vegetative
cells
After initiation DNA-cleaving molecules
degrade broken DNA to form ssDNA tails
terminating in 3’OH ends
9
Double-strand break repair model
ssDNA tails invade the unbroken
homologous DNA duplex
Invasion may be initially by single strand,
then 2 invading strands, strands pair with
complementary strand
Invading strands serve as 3’ primers for
new DNA synthesis
Elongation of 3’ ends using the
complementary strand as template
replaces DNA degraded by processing at
break site
10
Double-strand break repair model
Region of DNA replaced by new
synthesis is “lost”
Gene conversion = replacement of region
on one homologous strand with sequence
from homolog
2 Holliday junctions move by branch
migration, extending region of exchange
as in Holliday model, and are then
resolved by stand cleavage
11
Double-strand break repair model
Resolution involves cuts are sites “1” or “2” of both junctions
If both junctions are cut in the same way (both 1 or both 2) then
non-crossover products will be formed
If both junctions are cut in different ways (1 and 2 or 2 and 1)
then crossover products will be formed
12
Homologous recombination protein machines
13
Recombination in E. coli
All organisms have genes for products essential for recombination,
some genes are homologous, some appear to be convergence on
function
Best understood model is from E. coli, the RecBCD pathway
RecBCD enzyme processes DNA at DSB site to generate ssDNA
RecA mediates single-stranded DNA exchange
RuvA and RuvB drive branch migration
RuvC resolves Holliday junctions
14
Recombination in E. coli
RecBCD:
1. processes broken DNA to
generate ssDNA tails – the
preferred substrate for
recombination
2. loads RecA protein onto
ssDNA
3. “selects” whether DNA will
be recombined or destroyed
15
Recombination in E. coli
RecBCD is composed of 3
subunits products of the recB,
recC and recD genes
RecBCD enters DNA from
dsDNA break and moves along
the DNA
RecB and RecD are both
helicases, unwinding DNA using
energy from ATP hydrolysis
RecBCD nuclease activity
degrades DNA as it is unwound
16
Recombination in E. coli
At chi (χ) sequence nuclease
activity is altered = RecBCD no
longer cleaves the 3’ to 5’ strand,
and cleaves the other strand even
more
This asymmetrical cleavage
results in a 3’ tail terminating at
the chi sequence – ideal for
assembly of RecA and initiation
of recombination
Change in nuclease activity
appears to be inactivation or loss
of RecD subunit
3’ overhang could also be coated
with SSB (e.g. RPA), interaction
of RecBCD and RecA ensures
that RecA binds
17
Recombination in E. coli
Chi sites increase frequency of recombination at the site 10x
Chi site is a specific sequence (GCTGGTGG), but a common
sequence, in fact it is overrepresented in the E. coli genome, +1000
instead of the predicted ~80
E. coli DNA can, and do, incorporate DNA from other E. coli
This E. coli DNA, but not viral or other invading DNA, will have
chi sites, DNA without chi sites will be degraded by RecBCD
18
Recombination in E. coli
RecA binds to ssDNA and is a strand-exchange protein, catalyzing
the pairing of homologous DNA molecules
19
Recombination in E. coli
The active form of RecA is a protein-DNA
filament – huge, variable size, 100 subunits of
RecA, 300 nucleotides
RecA filament can bind 1, 2, 3 or 4 DNA
strands
DNA in strands is extended
1.5 fold
Search for regions of
homologous sequence, and
then exchange, happens
within filament
20
Recombination in E. coli
RecA binding is cooperative, growing with the addition of
subunits to the 3’ side of the first subunit - in the 5’ to 3’
direction = filament ending in a 3’ is most likely to be coated
by RecA
21
Recombination in E. coli
RecA binds to ssDNA
RecA “looks” for sequence
homology using 2 binding sites
The ssDNA is bound in the primary
binding site
dsDNA can occupy the secondary
binding site – binding is rapid, weak,
transient and sequence independent
RecA moves along dsDNA scanning
for homology – base-flipping
22
Recombination in E. coli
Matches of 15+ bp triggers strand
exchange
RecA promotes formation of stable
complex between two DNA molecules
= three-stranded joint molecule
containing several 100 bp of hybrid
DNA
The strand in the primary site is
paired with its complement in the
duplex bound in the secondary site breaking and forming hydrogen bonds
23
24
Recombination in E. coli
Strand-exchange proteins in the RecA family are present in all
organisms.
a) human Rad51
b) E. coli RecA
c) archaebacteria RadA
All with similar helical structure
25
Recombination in E. coli
After strand invasion, the 2 recombining molecules are connected
by a Holliday junction
Branch migration increases the amount of genetic material
exchanged
RuvA is a Holliday junction specific DNA-binding protein, it
binds at junction and recruits RuvB to site
RuvB is a ATP-dependent hexameric helicase, provides energy to
drive exchange of bases along branch
26
Recombination in E. coli
Completion requires that Holliday junction(s) be resolved
RuvC is resolving endonuclease in bacteria, it functions in
conjunction with RuvAB complex
RuvC recognizes DNA-RuvAB complex and nicks 2 DNA strands
with the same polarity, resulting 5’ and 3’ ends are sealed by DNA
ligase
Depending on which strands are cut the products may, or may not,
be of the crossover type.
27
Recombination in E. coli
RuvC cleaves DNA at 5’ A/T-T-T-G/C 3’ sites
Sequence is specific, but frequent, ~1/64 nucleotides
Modest sequence specificity ensures a certain amount of branch
migration before resolution
28
Homologous recombination in eukaryotes
Homologous recombination is required for DNA repair and
restarting replication forks in eukaryotes – mutants are
hypersensitive to DNA damaging agents and predisposed to
certain types of cancer
Recombination is also required for proper
chromosome pairing in meiosis and
“reshuffles” alleles on parental chromosomes
(meiotic recombination)
without recombination chromosomes
fail to align properly - nondisjunction
29
Recombination in eukaryotes
Meiotic recombination occurs when there are 4
complete, ds, DNA molecules representing each
chromosome
Dmc1-dependent recombination is preferentially
between the non-sister homologous chromotids
Mechanism is unknown, but biological rationale
= promote interhomolog connections to assist in
alignment of chromosomes for division
30
Recombination in eukaryotes
Eukaryotes (and not E. coli)
have a specific protein to
generate dsDNA breaks in DNA
to initiate recombination =
Spo11
Spo11 cuts DNA with little
sequence selectivity, but at a
specific time = when the
replicated homologous
chromosomes pair
Spo11 also cuts at specific
locations, regions of DNA not
tightly packed on nucleosomes,
e.g. promoters = hot spots for
recombination
31
Recombination in eukaryotes
Specific tyrosine on Spo11 attacks
phosphodiester backbone forming
a covalent complex between
protein and DNA
2 Spo11 subunits make staggered
cut – protein is related to
topoisomerases
5’ ends of DNA are bound to
Spo11, energy of cleaved bond is
stored bound in the protein-DNA
linkage
32
Recombination in eukaryotes
DNA ds break is processed by MRXenzyme complex to generate singlestranded regions needed for RecA-like
protein, long ssDNA terminating in 3’
OH
MRX is not homologous to RecBCD,
but is a multisubunit nuclease
Digestions occurs only on the strand
with 5’ end bound to Spo11, 5’ to 3’
resection, generating +1kb of ssDNA
33
Recombination in eukaryotes
Eukaryotes have 2 homologs of
RecA: Rad51 and Dmc1
Rad51 is expressed in cells
undergoing mitosis or meiosis,
Dmc1 is expressed only during
meiosis
Together Rad51 and Dmc1
participate in recombination, but
how they interact is not known
34
Recombination in eukaryotes
Many proteins interact in homologous recombination, likely as a
large multicomponent complexes
These recombination factories can be visualized in the cell, e.g. colocalization of Rad51 and Dmc1 during meiosis
35
Other recombination proteins in eukaryotes
Rad52 interacts with Rad51 to
promote formation of Rad51-DNA
complexes, antagonizes action of
RPA, the normal ssDNA binding
protein
The concentration of Rad52
increases after radiation exposure
Rad52-GFP foci in nuclei after
irradiation - DNA repair and
recombination centers
36
Recombination in eukaryotes
Depending on manner of resolution of junction, recombination can
happen without generating crossover products (formation of
“patch” products)
Non-crossover recombination can have genetic consequences =
gene conversion
37
Mating-type switching = gene conversion
Homologous recombination can
change the DNA sequence at a
specific chromosomal location
The budding yeast S. cerevisiae
(single-celled eukaryote) exists
as any 1 of 3 cell types, a and α
haploids and a/α diploid
a and α haploids mate to form
the diploids
a can only mate with α, α can
only mate with a, homothallism
A yeast can switch to α, and
vice versa, to allow mating
38
Mating-type switching = gene conversion
The mating-type genes encode transcriptional regulators that
control the expression of sp. target genes that define cell type
The mating-type genes that are expressed in a given cell are
found at the mating-type locus (MAT locus)
In a-cells the a1 gene is at the MAT, in α cells the α1 and α2
are at the MAT
In diploids both sets of genes are expressed
39
Mating-type switching = gene conversion
Cells can switch mating-type by recombination
In addition to the MAT locus, there is an additional copy of
both the a and α genes present, but not expressed, in the
yeast genome
Additional, silent, copies are called the HML (α) and HMR (a)
loci = silent cassettes
Silent cassette loci store genetic information that can be used
to switch the mating type
40
Mating-type switching = gene conversion
HO endonuclease initiates DSB at MAT locus
HO endonuclease expression is tightly regulated
This is a sequence-specific endonuclease, with sequence only
found at mating-type loci
Cuts form a staggered break in chromosome, no binding of
protein to DNA
41
Mating-type switching = gene conversion
5’ to 3’ resection occurs as in meiosis, a function of MRX
protein, specifically degrading 5’ ends, forming 3’ tails
Long 3’ tails associate with Rad51 and Rad52 and search for
homologous regions of the chromosome to initiate strand
invasion and genetic exchange
Mating-type switching is unidirectional, seq. information, but
not actual DNA, moves to MAT from HMR or HML, never the
reverse – the HO endonuclease cannot cleave its recognition
seq. at either HMR or HML b/c of chromatin packing
42
Mating-type switching = gene conversion
Rad51-coated 3’ ssDNA tails from MAT locus “choose” either
the HMR or HML locus for strand invasion – if MAT is a
invade HML, if α invade HMR
This unidirectional movement of information from one site to
another is a specialized case of gene conversion
43
Mating-type switching = gene conversion
Recombination pathway diverges from DSB-repair mechanism
Crossover class of recombination products is never observed
suggests recombination without Holliday junctions
Synthesis-dependent strand annealing (SDSA) model
Initiating event is DSB and strand invasion, the invading 3’ end
serves as primer for DNA synthesis
A complete replication fork is assembled at point of strand
invasion (unlike in other types of recombination)
Both leading and lagging strand synthesis occurs,
Both strands are displaced from their templates – new ds DNA is
synthesized with sequence of template site, replacing MAT
sequence
44
Mating-type switching = gene conversion
45
Consequences of mechanism of recombination
Recombination can occur between any 2 regions of DNA with
sufficient sequence similarity
None of the steps require specific sequence recognition,
although some do have sequence preferences
The committed step in recombination is strand-exchange,
which depends only on the capacity of DNA to base-pair
The freq. of recombination between any 2 markers (genes) is
generally proportional to the distance between the genes –
greater distance = more recombination
This proportionality allows genetic mapping using
recombination freq.
46
Consequences of mechanism of recombination
Distortions in genetic maps occur when regions differ in the
probability of participating in recombination
High proportion = Hot Spots – genes appear farther apart than
they are
Low proportion = Cold Spots – genes appear closer than they
are
47
Consequences of mechanism of recombination
Gene conversion is common
during “normal” homologous
recombination
Example – heterozygous Aa
individual, after DNA
replication, expected to have 4
copies of gene: A A a a
Gene conversion alters the
observed results, e.g. A a a a
This can happen through DSB
repair if gene is close to break –
invading strand copies
information from other
homolog
48
Consequences of mechanism of recombination
Gene conversion alters the
observed results, e.g. A a a a
This can happen through DSB
repair if gene is close to
break – invading strand copies
information from other
homolog
Second mechanism involves
repair of mismatches in
intermediates = A/a
heteroduplexes
Heteroduplexes may be
recognized by mismatch
repair enzymes – which
randomly “choose” which
strand to replace
49