Distinct ways by which Srs2 helicase and Rrp1 translocase shape Rad51-DNA interactions in fission yeast.

Precise regulation of homologous recombination is essential for genome stability. Multiple helicases and translocases prevent toxic, unscheduled association of the Rad51 recombinase with chromatin, and their levels are frequently dysregulated in cancer. It remains unclear how anti-recombinogenic enzymes act on distinct pools of Rad51. Using Schizosaccharomyces pombe, we show that the Srs2 helicase and Rrp1 translocase may target distinct Rad51 species through recombinase substrate specificity and activity control. Genetic analyses support a model in which Srs2 may preferentially regulate Rad52-dependent Rad51 nucleofilaments associated with single-stranded DNA (ssDNA), whereas Rrp1 may preferentially regulate a distinct Rad51 population associated with double-stranded DNA. Accordingly, Rrp1 can alleviate the toxicity caused by Rad51 overproduction, a function not shared by Srs2. Unexpectedly, both loss and overproduction of Srs2 promote Rad51 accumulation, suggesting that excessive helicase levels may generate ssDNA substrates that promote Rad51 nucleation. Both Srs2 and Rrp1 fail to remove strand-exchange-deficient Rad51 from DNA. Both enzymes restrict Rad51 occupancy at centromeric regions, whereas only Srs2 acts within ribosomal DNA (rDNA) repeats, revealing locus-specific regulation. Our findings support a model in which anti-recombinogenic enzymes differentially regulate Rad51 according to its DNA substrate, thereby contributing to a regulatory system that balances Rad51 functions during DNA repair across different genomic regions.