Genomic Rescue:Restarting failed replication forks

Part II

MI/BCH/BIO 615

Andrew PierceMicrobiology, Immunology and Molecular Genetics

University of Kentucky

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.

fork stalls

isomerization

nascent strands anneal

branch migrationexonuclease

linearized DNA

HJ resolution

find site?recombination no site?

degradationHJ resolution

Replication Restart Model

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 reversalWhat reverses these guys?

• 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

UvrD

dnaNtsinactivate -clamp at high temperature

permissive

semi-permissive

non-permissiveUvrD does this

for dnaNrecBCtsactive at 30Cinactive at 37C and 42C

inactivate RecBC so linearized DNA isn't immediately degraded

add back UvrD

extra RuvABC doesn't matter

UvrD is responsible for chromosome linearization with stalled DNA polymerase

some DnaN activity required for fork reversal

removal of UvrD gives same linearization in stalled and unstalled strains

UvrD is responsible for chromosome linearization with stalled DNA polymerasebut NOT with hurt replicative helicase

dnaBts recBCts

DnaE is part of polymerase catalytic unit(no RecA required for fork reversal)

DnaB is replicative helicase(RecA is required for fork reversal)

no NER no MMR

UvrD repair functions not required for UvrD-mediated linearization

UvrD leads to linearization

RuvAB required(as in 1998 paper)

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 PriAso… 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

RecGsubstrates

5' end

3' end

ssDNAin circle

ssDNAon 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

molar excess RecG over substrate

time course at5x molar excess RecG

(no reaction reversal seen)

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 pre-bound to substratetherefore 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 and RecG don't really affect with each other

RecAconditions

very high RecG inhibits RecA RecA slightly stimulates RecGRecG doesn't like

RecA conditions

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?