Genomic Rescue:

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Genomic Rescue Restarting failed replication
forks Part II
Andrew Pierce Microbiology, Immunology and
Molecular Genetics University of Kentucky
MI/BCH/BIO 615
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The DNA repair helicase UvrD is essential for
replication fork reversal in replication
mutants. Flores MJ, Bidnenko V, Michel B. EMBO
Rep. 2004 Oct5(10)983-8. Epub 2004 Sep 17.
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Replication Restart Model
fork stalls
isomerization
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Some Background (papers from 2000, 2001, 2002)
how nascent strands anneal depends on how fork
was blocked blocked by defective replicative
helicase (DnaB)? require RecA for fork
reversal blocked by defective polymerase? HolD
(clamp loader) DnaN (?-clamp
processivity) DnaE (polymerase catalytic subunit)
no RecA required for fork reversal What reverses
these guys?
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UvrD

• very active, very abundant dimeric helicase
• translocates 3' to 5'
• can unwind from nicks or blunt ends if at high
  concentration
• can unwind DNA/DNA and RNA/DNA duplexes
• required for nucleotide excision repair
• required for mismatch repair
• involved in RecFOR-mediated recombination (gaps)
• can act as an anti-recombinase (like yeast Srs2)
• uvrD increases recombination 5x to 10x
• rep uvrD double mutant is lethal
• lethality suppressed by inactivation of RecFOR

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permissive
recBCts active at 30C inactive at 37C and
42C inactivate RecBC so linearized DNA isn't
immediately degraded
for dnaN
semi-permissive
non-permissive
UvrD does this
dnaNts inactivate ?-clamp at high temperature
some DnaN activity required for fork
reversal removal of UvrD gives same
linearization in stalled and unstalled strains
add back UvrD
extra RuvABC doesn't matter
UvrD is responsible for chromosome linearization
with stalled DNA polymerase
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DnaE is part of polymerase catalytic unit (no
RecA required for fork reversal)
DnaB is replicative helicase (RecA is required
for fork reversal)
dnaBts recBCts
UvrD is responsible for chromosome linearization
with stalled DNA polymerase but NOT with hurt
replicative helicase
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UvrD leads to linearization
RuvAB required (as in 1998 paper)
no NER
no MMR
UvrD repair functions not required for
UvrD-mediated linearization
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Replication Restart Model
hurt replicative helicase? reversal requires RecA
hurt DNA polymerase? reversal requires UvrD
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Situational repair of replication forks roles of
RecG and RecA proteins. Robu ME, Inman RB, Cox
MM. J Biol Chem. 2004 Mar 19279(12)10973-81. Ep
ub 2003 Dec 29.
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Some Background
Blocks to leading strand synthesis allow
decoupled lagging strand synthesis to continue
for 1kbp Result is a long single-stranded gap
on the leading strand side of the fork and the
5'-PO4 ended lagging strand "priming" the other
side of the fork The 5'-PO4 ended "priming"
strand is NOT a substrate for PriA so for what
is this a substrate?
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RecG

• monomeric protein
• binds to "flayed duplex (three-armed) DNA
  structures
• prefers at least two of the three arms to be
  double-standed
• can branch-migrate Holliday junctions
• translocates on double-stranded DNA arm
• uses "wedge domain" to strip off annealed
  strands
• recG strains have complex phenotypes
• involved in supression of UV sensitivity of
  ruvABC mutants
• can bind and unravel D-loops in
  anti-recombinagenic manner
• can also bind and unravel R-loops (suppresses
  replication of plasmids)

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Developing a model system to study the mechanics
of fork regression
"template switch" model of repair
RecG substrates
ssDNA on tail
5' end
3' end
ssDNA in circle
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Electron micrograph of substrates and products of
RecG-promoted MM reaction
ssDNA in the linear piece is product (over 7kbp
processed)
ssDNA in the circular piece is substrate
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Properties of RecG protein-mediated fork
regression in vitro
(no reaction reversal seen)
molar excess RecG over substrate
RecG can work fast (120 - 240 bp/s) compared to
RecA (6 bp/s) based on minimum time to product
time course at 5x molar excess RecG
free Mg is inhibitory (d)ATP hydrolysis required
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RecG helicase processivity
challenge with small fork competitor
RecG can be competed away therefore not that
processive
same result with RecG pre-bound to
substrate therefore issue is RecG processivity,
rather than RecG initial binding
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Time course experiments of RecG-promoted MM
reactions
More RecG allows faster rebinding to substrate
after dissociation due to low processivity (time
to first detectable product)
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Effects of RecG on RecA reactions and of RecA on
RecG reactions
RecA conditions
very high RecG inhibits RecA
RecA slightly stimulates RecG
RecG doesn't like RecA conditions
RecA and RecG don't really affect with each other
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Conclusions and Questions

• RecG can regress forks quickly and extensively,
  but not processively
• RecG and RecA likely act independently of each
  other
• RecG doesn't like free Mg
• because free cations freeze Holliday junction
  geometry?
• RecG can work on fully duplex 3-stranded
  structures, but RecA cannot
• (since RecA requires ssDNA for nucleation)
• Why is the RecG reaction unidirectional?
• (How does it know which way to rebind?)
• Why is RecG in an operon with components of the
  stringent response?