Roles and Regulations of Separase in Maintenance of Genome Integrity

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Mutations and structural and numerical chromosomal aberrations pose a threat to genome integrity and result from DNA damage or chromosome missegregation. Cells have evolved efficient mechanisms of DNA damage repair and multi-layered regulations of sister chromatid separation. Separase takes center stage in both processes. By cleaving the Scc1 subunit of cohesin, the molecular rings that mediate sister chromatid pairing, it triggers all eukaryotic anaphases. Yeast separase functions also during the repair of DNA double strand breaks (DSBs). Separase activity needs tight control to prevent premature loss of cohesion. Vertebrate separase is kept inactive by association with securin or Cdk1-cyclin B1 until the metaphase-to-anaphase transition, when an E3, the anaphase promoting complex or cyclosome (APC/C), mediates the ubiquitin-dependent destruction of securin and cyclin B1. The APC/C is controllled by the spindle assembly checkpoint (SAC), which catalyses the Mad2-dependent inhibition of the APC/C activator Cdc20 until all chromosomes have achieved proper bipolar attachment. Murine cells that lack securin and Cdk1-cyclin B1-dependent regulation of separase are nevertheless viable, which demonstrates that additional regulatory mechanisms must exist for this important protease. Based on the literature and own preliminary data the following three APC/C independent regulations of anaphase might exist and will be investigated here: 1) Human separase contains a functional nuclear export signal, which is bound by the export receptor CRM1 in the presence of RanGTP. Given that both cohesin and RCC1, Ran's GEF, are chromosomally bound, it will be tested whether CRM1 binds and regulates separase at mitotic chromosomes. 2) As implied by mouse genetics, we will assess whether the SAC might regulate a protein phosphatase, which, in turn, regulates separase. 3) Sgo1 protects centromeric cohesin from proteolysis-independent displacement in mitotic prophase. Somatic cells express a second shugoshin, Sgo2, but its role in mitosis remains enigmatic. We propose that Sgo2 is an interactor and inhibitor of human separase and that its protective function of centromeric Scc1 has evaded detection because Sgo2 is usually inactivated prior to liberation of separase from securin. Furthermore, we will test the attractive possibility that it is only the SAC-dependent binding of Mad2 to Sgo2, which turns the latter into an inhibitor of separase.In another sub-project we will pursue preliminary evidence indicating an important function of human separase for the repair of DSBs. In particular, it will be clarified whether separase is required for non-homologous end joining or homology directed repair and whether it locally cleaves Scc1 at sites of damage to enable repair. Finally, we will test our working hypothesis that hierarchically acting post-translational modifications direct separase's route of recruitment from the cyto- into the nucleoplasm and onto DSBs.