dna break repair by homologous recombination homologous recombination · 2007-01-01 · homologous...
TRANSCRIPT
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• repair of dsDNA damage
• recombination between homologous chromosomes
STEP 1 create ssDNA with free 3’OH
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
ssW1 dsC2-W2
ssW2 dsC2-W1
Homologous Recombination
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.
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STEP 1: Create ssDNA with free 3’ OH
3’
5’
5’
3’
3’
5’
5’
3’
Eukaryotes typically load a 5’-3’ exonuclease at a dsDNA break.
Also possible to nick DNA then load a helicase:
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.
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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.
STEP 2: Strand exchange to find homology
E. coli uses RecA
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
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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.
C1
W1
W2
C2
3’5’
3’5’
3’
3’**Stable strand
exchange by RecA
requires >50 bp
of PERFECT homology
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STEP 3: Extend region of strand exchange
“Branch Migration” of the Holliday junction
C1
W1
W2
C2
5’
5’
3’
3’
C1
W1
W2
C2
C1
W1
W2
C2
OR
heteroduplex
heteroduplex
The Holliday junction is held in square-planar
configuration by a sandwiching octamer of RuvA
C1
W1
W2
C2
W1
C2
C1
W2 W2-C1
C2-W1
W2
C2
C1
W1
heteroduplex
heteroduplex
parental
parental
parental heteroduplex
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W2-C1
C2-W1
W2
C2C1
W1
heteroduplex
heteroduplex
parental parental
RuvA (in green) maintains Holliday
junction geometry, recruits RuvB
RuvB (in white) is a hexameric helicase;
it extends the heteroduplex
**RuvB is ATP-powered:
heteroduplex formation
can proceed WITHOUT
perfect homology, over
long (>1 kb) regions
Holliday junction resolution: the endonuclease RuvC (E. coli)It must nick BOTH Crick strands OR BOTH Watson strandsto separate the two duplex DNAs (different chromosomes)
C1
W1
W2
C2
W1
C2
C1
W2
W2-C1
C2-W1
W2
C2
C1
W1
heteroduplex
heteroduplex
parental parental
Cut at BOTH
thin OR BOTH
thick arrows
(eukaryotic equivalents of RuvABC have been purified but their identity is not certain)
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C1
W1
W2
C2
C1
W1
W2
C2
C1
W2
W1
C2
W2
C2
C1
W1
C1
W2
C1
W1
W2
C2
5’
5’
3’
3’C1
W1
W2
C2
C1
W2
W1
C2
C1
W1
W2
C2W1
C2
OR
100% chance of some heteroduplex
50% chance of recombinant ends
(exchange of chromosome arms)
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):
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RuvC cleaves to giveback parental ends
RuvC cleaves to give recombinant ends:deletion, inversion and translocation events
Large-scale genome rearrangments by inappropriate HR
Homologous recombination with RECOMBINANTENDS that occurs between duplicated genes
(or other duplicated loci) can result in chromosomedeletion, inversion and translocation events
chromosome 1
chromosome 2
DELETION
INVERSION
TRANSLOCATION
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Protein-DNA recognition at sites with a specific sequenceThe 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
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.
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Why bother with site-
specific recombination?
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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.
attB (bacterium): 25 bp
attL (left) attR (right)attP (phage): 240 bp
gene on (gene off)
Salmonella evades the immune system by changing gene expression:
Lambda phage hides in the E. coli chromosome by integration:
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Example of a DNA transposon
IS = “insertion sequence” for the mode of its discovery
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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.
Importantly, even if the transposon
departs from the donor site, the
target site direct repeat is left
behind (this is mutagenic).
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’.
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Antibiotic resistance genes were found in bacterial transposons,
suggesting that ‘selfish’ mobile DNA elements can carry useful genes
LTR (long terminal repeat) retrotransposons
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a virally encoded integrase
enzyme pastes the virus into
the host chromosome (like
a transposase second step).
transposase
The life cycle of
an LTR retrovirus
(like HIV)
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A Non-LTR
retrovirus
lifecycle
(like the LINE elements
that constitute 21%
of our genome)
chromosome 1
chromosome 2
DELETION
INVERSION
TRANSLOCATION
Additional mutagenesis occurs from homologous
recombination between transposable elements
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Some examples of single-gene diseases
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
Manipulating the genome using endogenous
DNA repair to perform gene conversion
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X ray-induced break !
sister chromatid !
DNA replaced with same sequence
Homologous
Recombination
target replaced withdonor DNA sequence !
induced break !
donor DNA !
(plasmid without origin is lost)
Provide a plasmid template
for homologous recombination
Induce a dsDNA break at the
mutation site to be repaired.
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Zinc Finger Protein (ZFP)-Nucleases
DSB
+ donor DNA? no yes
non-homologous end-joining homologous recombination
deletion? gene conversion
Site-specific DNA breaks could be used for
gene correction or gene disruption