Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans

Abstract

Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSBinducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1. Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1. We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability.KEYWORDS homologous recombination; RAD51 paralog; Shu complex; camptothecin; helq-1 D NA double-strand breaks (DSBs) are extremely cytotoxic lesions that threaten genome integrity. DSBs arise from both endogenous sources, such as replicative damage, or exogenous sources, such as ionizing radiation (IR) and chemotherapeutic agents. To ensure the maintenance of the genome, DSBs need to be repaired by high-fidelity repair pathways, the most robust of which is homologous recombination (HR), in which DNA from a sister chromatid or homologous chromosome provides a repair template. Initial processing of DSB ends by resection forms 39 singlestranded DNA (ssDNA) overhangs that are coated with the ssDNA-binding protein RPA. The exchange of RPA for the recombinase enzyme RAD51 facilitates the homology search and strand invasion of homologous DNA templates to form displacement loop structures. Subsequent stabilization of HR intermediates then requires removal of RAD51 from the doublestranded DNA to allow access to the DNA polymerization machinery. Given the central role of the RAD51 filament in HR, its assembly and disassembly are tightly regulated to ensure the fidelity of repair (Krejci et al. 2012;Jasin and Rothstein 2013;Heyer 2015).Key mediators of RAD51 filament assembly are the RAD51 paralogs. In humans, there are six RAD51 paralogs: RAD51B, RAD51C, RA...

Novel insights into RAD51 activity and regulation during homologous recombination and DNA replication

“…This novel ceRAD-51 paralogue physically associates with the only known ceRAD-51 paralogue in C. elegans, ceRFS-1, forming the first known ceRAD-51 paralogue complex in nematodes (Taylor et al 2015; Ward et al 2010). Recently, we identified ceRIP-1 to directly interact with the worm scShu2 orthologue, ceSWS-1 by Y2H and to bridge an interaction between ce-SWS-1 with ceRFS-1 by Y3H (McClendon et al 2016). Our results show that ceSWS-1 interaction with ceRIP-1 is mediated by the SWIM domain of ce-SWS-1 and the Walker B motif of ceRIP-1 (McClendon et al 2016).…”

“…Recently, we identified ceRIP-1 to directly interact with the worm scShu2 orthologue, ceSWS-1 by Y2H and to bridge an interaction between ce-SWS-1 with ceRFS-1 by Y3H (McClendon et al 2016). Our results show that ceSWS-1 interaction with ceRIP-1 is mediated by the SWIM domain of ce-SWS-1 and the Walker B motif of ceRIP-1 (McClendon et al 2016). Using CRISPR to create a null sws-1 allele, sws-1 worms exhibit defects consistent with a role in HR such as DNA damage sensitivity, increased males, increased mutation rates, and fewer mitotic ceRAD-51 foci (McClendon et al 2016).…”

CRISPR Technology Reveals RAD(51)-ical Mechanisms of Repair in Roundworms: An Educational Primer for Use with “Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans”

“…The rationale for this experiment was that helq-1 encodes a protein involved in HR repair whose absence confers synergistic lethality with either rfs-1, rip-1, or sws-1 (Ward et al 2010;Taylor et al 2015;McClendon et al 2016). By examining chromosomes in the the sws-1; helq-1 double mutant, the researchers could test the hypothesis that these genes play overlapping roles in HR repair.…”

“…McClendon et al (2016) studied the effects of DNA damage in germ cells that are collectively referred to as the germ line. Unlike somatic cells, which give rise to all of our "body" cells, germ cells are unique as they are the only cell type to undergo both mitosis and meiosis and therefore directly contribute to future generations by forming gametes (egg or sperm).…”

“…Researchers in the J. L. Yanowitz and K. A. Bernstein laboratories, both at the University of Pittsburgh, study DNA repair using model organisms. In their recently published study, McClendon et al (2016) describe the role of a newly characterized Caenorhabditis elegans gene called sws-1 that they find plays a role in HR during both mitosis and meiosis. Using a combination of several established and more recently described cellular, genetic, and molecular techniques, the discoveries of McClendon et al (2016) have direct implications for understanding how mutations in the human versions of sws-1 and related genes can predispose individuals to cancer and hinder fertility.…”