Download Homologous Recombination DNA break repair by homologous

Homologous Recombination
• repair of dsDNA damage
• recombination between homologous chromosomes
STEP 1 create ssDNA with free 3’OH
ssW1 dsC2-W2
STEP 2 find homology by strand exchange:
STEP 3 extend region of strand exchange
beyond initial homology
STEP 4 resolve junction of dsDNAs to
reestablish 2 separate chromosomes
DNA break repair by
homologous recombination
This requires specialized factors:
a protein helps the ssDNA
region
to find the homologous dsDNA
in order to trade base-pairing.
ssW2 dsC2-W1
STEP 1: Create ssDNA with free 3’ OH
Eukaryotes typically load a 5’-3’ exonuclease at a dsDNA break.
Also possible to nick DNA then load a helicase:
5’
3’
3’
5’
3’
5’
3’
5’
In E. coli, homologous recombination is induced by RecBCD
RecB and RecD are helicases with opposite polarity.
They load as a complex with each other and RecC at a break.
Rec B is also a nuclease; it cuts both single strands generated by
the helicases UNTIL it encounters (running in the right polarity)
the ‘chi’ site, at which point it leaves the strand with the free
3’ OH alone and continues to degrade the other strand.
STEP 2: Strand exchange to find homology
E. coli uses RecA
The ssDNA in a RecA filament threads past dsDNA, with base flipping from
the dsDNA acting to sample homology with the ssDNA.
Binding to RecA induces underwinding of the DNA, which encourages bases
to flip back and forth between the two possible partner strands WITHOUT
additional input of energy.
Model for DNA strand exchange
mediated by RecA. A three-strand
reaction is shown. (a) RecA protein forms
a filament on the single-stranded DNA.
(b) A homologous duplex incorporates
into this complex. (c) One of the strands in
the duplex is transferred to the single
strand originally bound in the filament.
The other strand of the duplex is
displaced.
Important features of RecA:
• A monomer binds ~3 nt or bp
• Cooperative filament assembly 5’-3’
• Prefers to form filament on ssDNA,
but once formed, the filament will
take up dsDNA at a second site
• Filament has 18.6 bp DNA/turn:
bp are de-stabilized and can rapidly
exchange between two bound DNAs
• Bound ATP increases RecA DNA affinity,
ATP hydrolysis decreases affinity for DNA
Human cells have the RecA-like
protein Rad51; Rad51 needs extra help
STEP 2 Products
Each ssDNA strand exchanged
generates a Holliday junction.
Several series of steps are possible,
so ONLY consider
the model junction below.
**Stable strand
exchange by RecA
requires >50 bp
of PERFECT homology
C1
W1
W2
C2
3’
5’
5’
3’
3’
3’
STEP 3: Extend region of strand exchange
“Branch Migration” of the Holliday junction
C1
W1
W2
C2
3’
5’
5’
3’
C1
W1
W2
C2
heteroduplex
C1
W1
OR
heteroduplex
W2
C2
The Holliday junction is held in square-planar
configuration by a sandwiching octamer of RuvA
C1
W1
C1
W2
parental
W2
C2
heteroduplex
W2-C1 heteroduplex
W1
C2
parental
W2
C2
C1
W1
parental
C2-W1 heteroduplex
RuvA (in green) maintains Holliday
junction geometry, recruits RuvB
RuvB (in white) is a hexameric helicase;
it extends the heteroduplex
parental
W2
C2
**RuvB is ATP-powered:
heteroduplex formation
can proceed WITHOUT
perfect homology, over
long (>1 kb) regions
heteroduplex
W2-C1
parental
C1
W1
C2-W1
heteroduplex
Holliday junction resolution: the endonuclease RuvC (E. coli)
It must nick BOTH Crick strands OR BOTH Watson strands
to separate the two duplex DNAs (different chromosomes)
W2-C1 heteroduplex
C1
W1
C1
W2
W2
C2
W1
C2
Cut at BOTH
thin OR BOTH
thick arrows
W2
C2
parental
C1
W1
parental
C2-W1 heteroduplex
(eukaryotic equivalents of RuvABC have been purified but their identity is not certain)
C1
W2
C1
W1
W2
C2
C1
W1
W2
C2
W1
C2
C1
W1
W1
C2
W2
C2
C1
W2
C1
W1
W2
C2
OR
C1
W1
W2
C2
3’
C1
W2
100% chance of some heteroduplex
50% chance of recombinant ends
(exchange of chromosome arms)
C1
W1
5’
5’
3’
W1
C2
W2
C2
Gene conversion can make heterozygous loci homozygous
(called loss of heterozygousity or LOH)
Products of HR (shown with recombinant ends, but note that
central heteroduplex is present even with parental ends):
Large-scale genome rearrangments by inappropriate HR
RuvC cleaves to give
back parental ends
RuvC cleaves to give recombinant ends:
deletion, inversion and translocation events
Homologous recombination with RECOMBINANT
ENDS that occurs between duplicated genes
(or other duplicated loci) can result in chromosome
deletion, inversion and translocation events
DELETION
INVERSION
chromosome 1
chromosome 2
TRANSLOCATION
Site-specific recombination
Protein-DNA recognition at sites with a specific sequence
The two sites ‘synapse’ then all four strands are cut in series
to exchange the original ends for recombinant ends.
Performed by a tetramer of a site-specific recombinase.
The enzyme active site tyrosine forms a covalent protein-DNA
intermediate like a topoisomerase, so the recombination
reaction is reversible with no need for DNA ligase.
Site-specific recombination
The only difference between the reactions in (A) and (B) is the
relative orientation of the two DNA sites (indicated by arrows) at
which a site-specific recombination event occurs.
Why bother with sitespecific recombination?
A surface contour model
of Cre recombinase bound
to a recombination
intermediate. The protein
has been rendered
transparent so that the
bound DNA is visible.
Lambda phage hides in the E. coli chromosome by integration:
attB (bacterium): 25 bp
attL (left)
attP (phage): 240 bp
attR (right)
Salmonella evades the immune system by changing gene expression:
gene on
(gene off)
Example of a DNA transposon
IS = “insertion sequence” for the mode of its discovery
Non-replicative (cut-and-paste) transposition. DNA-based transposons have an
inverted repeat sequence at their ends, and any DNA between them can be moved.
Transposase multimers make a blunt double-stranded cut at the edge of the inverted
repeat termini. Transposase also has a second binding site for DNA that is not
sequence-specific, which it uses to bind an insertion target site and make a staggered
double-stranded cut. Transposase bound to the transposon ends reverses its cleavage
reaction to ligate the transposon DNA to the target site ends, but a gap remains on
each side of the inserted DNA due to the staggered target site cut. Repair synthesis is
required to rejoin the broken donor chromosome and to fill in the target site gaps.
Different transposase
enzymes make different
types of staggered cuts.
Depending on order of the next steps, transposition can result in
transposon movement or transposon retention at the donor site and
insertion elsewhere as well.
If transposase nicks the donor site ends
rather than cutting both strands at once then donor 3’ ends join target
5’ ends, target 3’ ends prime replication and result in duplication of the
transposon. The resulting donor-target fusion is fixed by the activity of
a transposon-encoded site-specific recombinase or ‘resolvase’.
Importantly, even if the transposon
departs from the donor site, the
target site direct repeat is left
behind (this is mutagenic).
Antibiotic resistance genes were found in bacterial transposons,
suggesting that ‘selfish’ mobile DNA elements can carry useful genes
LTR (long terminal repeat) retrotransposons
transposase
a virally encoded integrase
enzyme pastes the virus into
the host chromosome (like
a transposase second step).
The life cycle of
an LTR retrovirus
(like HIV)
A Non-LTR
retrovirus
lifecycle
(like the LINE elements
that constitute 21%
of our genome)
Additional mutagenesis occurs from homologous
recombination between transposable elements
DELETION
INVERSION
chromosome 1
chromosome 2
TRANSLOCATION
Some examples of single-gene diseases
Manipulating the genome using endogenous
DNA repair to perform gene conversion
A B C D E F G
a b c d e f g
A B C D E F G
a b c D e f g
X ray-induced break !
Homologous
Recombination
sister chromatid !
DNA replaced with
same sequence
induced break !
Induce a dsDNA break at the
mutation site to be repaired.
donor DNA !
Provide a plasmid template
for homologous recombination
target replaced with
donor DNA sequence !
(plasmid without origin is lost)
Zinc Finger Protein (ZFP)-Nucleases
Site-specific DNA breaks could be used for
gene correction or gene disruption
DSB
+ donor DNA?
no
non-homologous end-joining
deletion?
yes
homologous recombination
gene conversion