Insight into homologous recombination at replication blocking lesions through analysis of the C. elegans RAD-51 paralog, RFS-1

• Main Author: Ward, Jordan David
• Published: University College London (University of London) 2008
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.499038

The ability to sense and repair replication blocking DNA damage is a critical task for a cell, as evidenced by the embryonic lethality or cancer predisposition caused by mutation in any of these processes. Recent work has implicated homologous recombinational repair (HRR) as a key player in promoting genome stability in response to stalled replication forks, in addition to its well documented role of in the error-free repair of DNA double-strand breaks (DSBs). Although the five paralogs of the mammalian recombinase, Rad51, have been suggested to be general mediators of HRR, genetic evidence fails to support this hypothesis and their exact roles have remained elusive. Here it is shown that the single C. elegans RAD-51 paralog, RFS-1, plays a specific role in promoting HRR at replication forks blocked by DNA cross-linking agents or camptothecin, but is dispensable for HRR at both meiotic and ionizing radiation-induced DSBs. Strikingly, RFS-1 is also dispensable for RAD-51 loading at forks collapsed by hydroxyurea or by the absence of the S-phase checkpoint, indicating that these lesions may resemble a conventional HRR substrate, rfs-1 mutations suppress mitotic catastrophe in a him-6 topS(RNAi) mutant background and accelerate polyG/C tract deletion in dog-1 mutants, demonstrating that these genetic backgrounds drive the formation of replication blocking lesions, a notion that had been previously suggested but never validated. These data suggest that RFS-1 is not a general HRR mediator, but instead specifically promotes RAD-51 loading at replication fork barriers (RFBs). Yeast two-hybrid (Y2H) screening identified a novel RFS-1-interacting protein, RIP-1, mutants of which phenocopied rfs-1 mutants with respect to embryonic lethality, elevated apoptosis and the RAD-51 loading defect in response to replication fork blocking lesions. Use of Y2H analysis of RFS-1 fragments, followed by RFS-1 peptide arrays, fine-mapped the interaction with RAD-51 to a single 30mer and interaction with RIP-1 to two distinct 30mers. Substitution peptide array analysis of RIP-1 has further refined the interaction surfaces in the case of the RIP-1 binding peptides, two residues are critical for binding in each 30mer. These interaction studies, combined with promising attempts at purification of RFS-1 and RIP-1, open the possibility of detailed biochemical analysis of the role of these proteins in HRR at RFBs. Preliminary work has explored the potential role of RFS-1 in aging and meiosis. Although the IGF-1 pathway transcriptional factor DAF-16 and the HRR factors HIM-6 and RFS-1 impact on spontaneous mutation rate, the mechanism behind this effect on mutation rate and interrelation between these proteins, is currently unclear, rfs-1 and rip-1 mutants both have an increased percentage of male progeny, indicating an elevation in X chromosome non-disjunction during meiosis. Additionally, loss of rfs-1 suppresses RAD-51 accumulation in mus-81 him-9/xpf-l double mutants and evidence suggests that these recombination intermediates arise from meiotic DSBs as opposed to mitotic lesions. Elucidating how RFS-1, and presumably RIP-1, impact on meiotic recombination and whether this role is mechanistically distinct from a role in promoting HRR at RFBs will be an important avenue of future study.