Download The DNA repair helicase UvrD is essential for replication

Genomic Rescue:
Restarting failed replication forks
Part II
Andrew Pierce
Microbiology, Immunology and Molecular Genetics
University of Kentucky
MI/BCH/BIO 615
The DNA repair helicase UvrD is essential for replication
fork reversal in replication mutants.
Flores MJ, Bidnenko V, Michel B.
EMBO Rep. 2004 Oct;5(10):983-8.
Epub 2004 Sep 17.
Replication Restart Model
fork stalls
nascent
strands
anneal
linearized DNA
isomerization
HJ resolution
branch migration
find  site?
recombination
HJ resolution
exonuclease
no  site?
degradation
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?
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
recBCts
active at 30C
inactive at 37C and 42C
inactivate RecBC so
linearized DNA isn't
immediately degraded
UvrD
does
this
permissive
semi-permissive
non-permissive
for dnaN
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
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
UvrD leads to
linearization
RuvAB required
(as in 1998 paper)
no NER
no MMR
UvrD repair functions not required for UvrD-mediated linearization
Replication Restart Model
hurt replicative helicase?
reversal requires RecA
hurt DNA polymerase?
reversal requires UvrD
Situational repair of replication forks: roles of RecG
and RecA proteins.
Robu ME, Inman RB, Cox MM.
J Biol Chem. 2004 Mar 19;279(12):10973-81.
Epub 2003 Dec 29.
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?
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)
Developing a model system to study the mechanics of fork regression
"template switch"
model of repair
RecG
substrates
5' end
3' end
ssDNA
in circle
ssDNA
on tail
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
Properties of RecG protein-mediated fork regression in vitro
(no reaction reversal seen)
molar excess RecG over substrate
time course at
5x molar excess RecG
RecG can work fast
(120 - 240 bp/s)
compared to RecA
(6 bp/s)
based on minimum
time to product
free Mg is inhibitory
(d)ATP hydrolysis required
RecG helicase processivity
challenge with small
fork competitor
RecG can be competed away
therefore not that processive
same result with RecG prebound to substrate
therefore issue is RecG
processivity, rather than
RecG initial binding
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)
Effects of RecG on RecA reactions and of RecA on RecG reactions
RecA
conditions
very high RecG inhibits RecA
RecG doesn't like
RecA conditions
RecA and RecG don't really affect with each other
RecA slightly
stimulates RecG
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?