Roles and Regulations of Separase in Maintenance of Genome Integrity

突变、染色体结构和数量畸变对基因组完整性构成威胁，是DNA损伤或染色体错分离的结果。细胞进化出了有效的DNA损伤修复机制和姐妹染色单体分离的多层调控机制。分离酶在这两个过程中都处于中心位置。通过切割内聚蛋白的Scc1亚基，介导姐妹染色单体配对的分子环，它触发了所有真核生物的后期。酵母分离酶在DNA双链断裂（DSBs）的修复过程中也起作用。分离酶活动需要严格控制，以防止过早失去内聚性。脊椎动物分离酶通过与securin或Cdk1-cyclin B1的关联保持失活，直到中期到后期转变，这时E3，即后期促进复合物或环体（APC/C），介导泛素依赖性的securin和cyclin B1的破坏。APC/C由纺锤体组装检查点（SAC）控制，它催化APC/C激活子Cdc20的mad2依赖性抑制，直到所有染色体都达到适当的双极附着。缺乏securin和Cdk1-cyclin b1依赖性分离酶调控的小鼠细胞仍然存活，这表明这种重要蛋白酶必须存在额外的调控机制。根据文献和自己的初步数据，APC/C后期可能存在以下三种独立的调控，本文将对其进行研究：1)人分离酶含有一个功能性的核输出信号，该信号在RanGTP存在时与输出受体CRM1结合。鉴于内聚蛋白和RCC1 （Ran’s GEF）都是染色体结合的，我们将检测CRM1是否结合并调节有丝分裂染色体上的分离酶。2)根据小鼠遗传学，我们将评估SAC是否可能调节蛋白磷酸酶，而蛋白磷酸酶反过来调节分离酶。3)在有丝分裂前期，Sgo1保护着丝粒内聚蛋白不受蛋白水解的影响。体细胞表达第二个shugoshin， Sgo2，但其在有丝分裂中的作用仍然是谜。我们认为Sgo2是人类分离酶的相互作用物和抑制剂，它对着丝粒Scc1的保护功能一直没有被检测到，因为Sgo2通常在分离酶从securin中解放出来之前就失活了。此外，我们将测试一种有吸引力的可能性，即只有sac依赖的Mad2与Sgo2结合，使后者成为分离酶的抑制剂。在另一个子项目中，我们将寻求初步证据，表明人分离酶对dsb修复的重要功能。特别是，它将澄清分离酶是否需要用于非同源末端连接或同源定向修复，以及它是否在损伤位点局部切割Scc1以实现修复。最后，我们将验证我们的工作假设，即分级作用的翻译后修饰直接将分离酶从细胞招募到核质和dsb上的途径。

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.