Simon Boulton

Novel factors critical for sensing and repair of DNA damage in S-phase

Evidence suggests that the failure to correctly repair DNA lesions in S-phase is the major threat to genome stability in normal cells. Indeed, DNA damage encountered during S-phase frequently results in replication fork collapse.

We are particularly interested in how lesion repair is coordinated with DNA replication and how this is regulated by the S-phase checkpoint, the apparatus that senses DNA damage. DNA Inter-strand DNA crosslink's (ICLs) present a major obstacle to DNA replication and must be repaired or bypassed to allow fork progression. Of particular interest to the lab is the emergence of the Fanconi anemia (FA) pathway as a central player in facilitating DNA repair at blocked replication forks.

FA is an autosomal recessive disorder associated with bone marrow failure, congenital abnormalities and cancer predisposition. Although cells derived from FA patients exhibit profound sensitivity to ICL-inducing agents suggesting that FA proteins are important for ICL repair the precise mechanism through which the FA pathway promotes ICL repair remains unclear.

We are employing C. elegans genetics, mammalian cell culture work and biochemistry to examine the interplay between the FA and homologous recombination repair pathways in ICL repair and how these processes are tightly regulated by the S-phase checkpoint.

A PhD position is available in the lab to work on one of two projects:

1. We have recently identified HCLK2, a novel S-phase checkpoint gene in C. elegans and human cells that is required for stability of the replication fork under conditions of replication stress. HCLK2 and its interacting partner ATR are also essential for the activation of the FA pathway and subsequent repair of replication blocking lesions, including ICLs. Biochemical approaches in mammalian cells have identified a number of new HCLK2 interacting factors.
   
   The project will focus on the characterization of the new associated factors using biochemistry and knockdown approaches in mammalian cells.



2. Yeast Srs2 and E. coli UvrD are related helicases that play critical roles in suppressing aberrant recombination arising during DNA replication. We have recently identified spar-1 as the likely analogue of Srs2 in complex eukaryotes.
   
   spar-1 mutants exhibit many of the hallmarks of Srs2 including synthetic lethality in combination with him-6 (BLM) mutants, hyper-recombination, an inability to deal with aberrant recombination during S-phase and meiosis and sensitivity to DNA damaging agents. The human homolog of SPAR-1 is also over-expressed in tumors and its disruption in cells confers genomic instability, sensitivity to DNA damaging agents and increased recombination frequency.
   
   A number of projects are available to further characterize the role of spar-1 in suppressing aberrant recombination that will employ C. elegans genetics, cell culture work and biochemistry.

References

1. Collis SJ, Barber LJ, Ward JD, Martin JS, Boulton SJ. CeFANCD2 responds to replication stress and functions in interstrand cross-link repair. DNA Repair (Amst) 2006; 5(11): 1398-1406.
2. Collis SJ, Barber LJ, Clark AJ, Martin JS, Ward JD, Boulton SJ. HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability. Nat Cell Biol 2007; 9(4): 391-401.
3. Ward JD, Barber LJ, Petalcorin MI, Yanowitz J, Boulton SJ. Replication blocking lesions present a unique substrate for homologous recombination. EMBO J 2007; 26(14): 3384-3396.