Genomic Rescue: Restarting failed replication forks Part II

1. Genomic Rescue: Restarting failed replication forks Part II Andrew Pierce Microbiology, Immunology and Molecular Genetics University of Kentucky MI/BCH/BIO 615

2. 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.

3. nascent strands anneal linearized DNA HJ resolution exonuclease branch migration find  site? recombination no  site? degradation HJ resolution Replication Restart Model fork stalls isomerization

4. 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?

5. 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

6. 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

7. 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

8. UvrD leads to linearization RuvAB required (as in 1998 paper) no NER no MMR UvrD repair functions not required for UvrD-mediated linearization

9. Replication Restart Model hurt replicative helicase? reversal requires RecA hurt DNA polymerase? reversal requires UvrD

10. 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.

11. 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?

12. 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)

13. 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

14. 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

15. 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

16. 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

17. 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)

18. 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

19. 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?