Local activation of mammalian separase in interphase promotes double‐strand break repair and prevents oncogenic transformation

Separase halves eukaryotic chromosomes in M‐phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double‐strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology‐directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG‐repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response‐relevant ATM, PRMT1, and Mms21 represent the corresponding kinase, methyltransferase, and SUMO ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation. Synopsis Cohesin complexes hold sister chromatids together until their mitotic segregation, but also have interphase roles in the repair of DNA double strand breaks (DSBs). Such repair is now found to also require local activation of separase, the protease that cleaves cohesin during mitosis. Human separase accumulates at DSBs in G2‐ but not G1‐phase. Human separase supports error‐free homology‐directed repair (HDR) but not error‐prone non‐homologous end joining (NHEJ). DSB‐tethered separase is activated to locally cleave cohesin. Phosphorylation, arginine methylation and SUMOylation of separase are required to target it to DSBs and support DNA damage repair. SEPARASE heterozygosity weakens HDR and predisposes to Ras‐induced neoplasia in MEFs and chemically‐induced skin cancer in mice. Graphical Abstract Intricate post‐translational modifications regulate separase recruitment to DNA breaks for local cohesin cleavage and efficient homologous recombination repair.