Replication blocking lesions present a unique substrate for homologous recombination

Abstract

Homologous recombination (HR) plays a critical role in the restart of blocked replication forks, but how this is achieved remains poorly understood. We show that mutants in the single Rad51 paralog in Caenorhabditis elegans, rfs-1, permit discrimination between HR substrates generated at DNA double-strand breaks (DSBs), or following replication fork collapse from HR substrates assembled at replication fork barriers (RFBs). Unexpectedly, RFS-1 is dispensable for RAD-51 recruitment to meiotic and ionizing radiation (IR)-induced DSBs and following replication fork collapse, yet, is essential for RAD-51 recruitment to RFBs formed by DNA crosslinking agents and other replication blocking lesions. Deletion of rfs-1 also suppresses the accumulation of toxic HR intermediates in him-6; top-3 mutants and accelerates deletion formation at presumed endogenous RFBs formed by poly G/C tracts in the absence of DOG-1. These data suggest that RFS-1 is not a general mediator of HRdependent DSB repair, but acts specifically to promote HR at RFBs. HR substrates generated at conventional DSBs or following replication fork collapse are therefore intrinsically different from those produced during normal repair of blocked replication forks.

Genome Integrity Is Regulated by theCaenorhabditis elegansRad51D Homologrfs-1

“…This group contains the human proteins rad51B, rad51C, rad51D, XRCC2, and XRCC3, which form two known complexes that are implicated in DNA damage repair. Rfs-1 appears most similar to Rad51D (Ward et al 2007). Thus, rfs-1 appeared to be a likely candidate to be the gene responsible for the Him phenotype.…”

“…One of these deletions, eDf25, originally known as unc-86(e1416), removes part of the unc-86 promoter and an upstream operon with three genes ( Figure 1A). The first two genes in the operon are uncharacterized; the third gene, rfs-1, is the only member of the Rad-51 paralog group found in C. elegans (Ward et al 2007). This group contains the human proteins rad51B, rad51C, rad51D, XRCC2, and XRCC3, which form two known complexes that are implicated in DNA damage repair.…”

“…The rfs-1 mutation provides a unique opportunity to investigate the effect of such lesions on the development of a multicellular organism. The rad-51 short protein (rfs-1) gene has recently been shown to enhance the dog-1 mutant phenotype (Ward et al 2007) and to interact with the DNA damage-repair genes rad-51 and brc-1 . rfs-1 also appears to act upstream of him-6 in the HR repair of stalled replication forks and displays acute sensitivity to nitrogen mustards and cisplatin (Ward et al 2007).…”

“…The rad-51 short protein (rfs-1) gene has recently been shown to enhance the dog-1 mutant phenotype (Ward et al 2007) and to interact with the DNA damage-repair genes rad-51 and brc-1 . rfs-1 also appears to act upstream of him-6 in the HR repair of stalled replication forks and displays acute sensitivity to nitrogen mustards and cisplatin (Ward et al 2007). These agents cause massive DNA damage, leading to immediate sterility of the exposed animals, suggesting that rfs-1 is important for maintaining fertility.…”

Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells

“…Contrary to findings in G1 cells, which lack the opportunity for recombinational repair (14), in the G2 cells we found efficient loss of SMD at later times, restitution of chromosomes, the disappearance of MBN-sensitive sites, and high survival rates, all of which required Rad52 and Rad51. Although recombination repair can restore collapsed replication forks (45,46), there is little direct evidence that HR directly processes and repairs replication-blocking lesions located on ssDNA. Using a plasmid-based assay, Adar et al (47) showed that gaps opposite an abasic site or a benzo[a]pyrene-guanine adduct could be repaired by HR in mammalian cells.…”