Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCF^Fbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated. Homologous recombination is required for repairing DNA double-strand breaks (DSBs), which are induced by exogenous factors such as DNA damaging agents or by endogenous factors such as collapse of DNA replication fork in mitotic cells. If improperly processed, DSBs could lead to chromosome rearrangement, cell death, or tumorigenesis in mammals, and thus HR is strictly controlled at several steps, including Rad51 recombinase-driven DNA strand exchange reaction. Specifically, DNA helicases have been shown to be important for suppression of inappropriate recombination events. In this study, we analyzed one such DNA helicase, fission yeast Fbh1. We used an in vivo single-DSB repair assay to show that Fbh1 suppresses crossover formation between homologous chromosomes. Next, we obtained in vitro evidence that Fbh1 acts as an inhibitor of the strand-exchange reaction in the absence of Swi5-Sfr1, but stimulates the reaction after it starts. Furthermore, we found that SCF^Fbh1 has ubiquitin-ligase activity toward Rad51 in vitro and that Fbh1 regulates the protein level of Rad51 in the stationary phase. These results suggest Fbh1 regulates Rad51-mediated homologous recombination by its seemingly-unrelated two activities, DNA helicase/translocase and ubiquitin ligase.

Significance: In DNA homologous recombination, recombinase-coated single-stranded DNA filament formation is the first committed step and is subject to tight regulation. Stabilization of nucleoprotein filament by accessory proteins can be achieved by enhancing filament formation, reducing filament disassembly, or both. However, the mechanism of regulation is not understood by conventional biochemical methods. This is a study of the mechanism of how accessory proteins stimulate filament assembly by applying single-molecule methods that allow us to monitor the binding of Rad51 on DNA in mouse and fission yeast. Our results show that the Swi5–Sfr1 complex demonstrates the evolutionarily conserved stimulation of Rad51 filament assembly by stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the reduction of Rad51 dissociation.

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1–C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP. Significance A DNA double-strand break (DSB) can be repaired accurately by homologous recombination. The Mre11-Rad50-Nbs1 (MRN) complex is responsible for initiating homologous recombination by degrading 5′-ended DNA strand, where its activation by the Ctp1 cofactor plays a pivotal role. Here, by using purified fission yeast proteins, we show that two major elements comprise MRN activation. First, phosphorylation of Ctp1 promotes the physical interaction between MRN and Ctp1. Second, the C terminus of Ctp1 activates nucleolytic processing of DSB ends. In the latter case, a small peptide comprising only 15 amino acids from the Ctp1 C terminus is sufficient to activate MRN. Our results elucidate the core elements underlying MRN activation by Ctp1.