Unraveling unexpected functions of Separase in interphase: The trigger protease of mitotic chromosome segregation as a decision maker in the DNA damage response

揭示分离酶在间期的意外功能：有丝分裂染色体分离的触发蛋白酶作为 DNA 损伤反应的决策者

• 批准号: 501106432
• 负责人: Professor Dr. Olaf Stemmann
• 金额: --
• 依托单位: Lehrstuhl für Genetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/501106432?language=en
• 关键词: Unraveling; unexpected; functions; Separase; interphase

DNA double-strand breaks (DSBs) are constantly threatening genome integrity and cellular health and must be repaired to prevent death, oncogenic transformation, or ageing of cells. A cell that sustains DSBs has to decide whether it sacrifices itself by apoptosis (programmed cell death) in order to protect the health of the organism or whether it tries to repair the damage. In the latter case, the cell then has to choose between two major repair pathways, the fast but potentially mutagenic NHEJ (non-homologous end joining) or the slower but error-free HR (homologous recombination). Studying separase, which triggers chromosome segregation in all eukaryotic M-phases, we unexpectedly found that this essential protease also foresees important functions in interphase of the cell cycle. Our preliminary data indicate that separase is involved in both abovementioned decisions of the DNA damage response (DDR): Apoptosis or repair and, if repair, NHEJ or HR? More specifically, we found that separase is recruited to and activated at DSBs, where it cleaves anti-apoptotic Bcl-xL and NHEJ-promoting 53BP1. These cleavages require substrate phosphorylations by the DDR kinase ATM and - depending on the DSB load - favor apoptosis or HR, respectively. In the first sub-project, we will study the mitochondrial-nuclear shuttle required to bring Bcl-xL to DSBs for cleavage and the transformed, now pro-apoptotic fragment(s) back to mitochondria. We will map phosphorylations on Bcl-xL and investigate the structural changes that they induce to enable cleavage by separase. And we will unravel the molecular mechanism, by which cleaved Bcl-xL actively induces the intrinsic pathway of apoptosis. A p53-independent branch of DSB-induced apoptosis, whose existence has long been demonstrated, nevertheless remains elusive in its molecular identity until today. We propose that the separase-Bcl-xL-axis represents this thought-after pathway. We will test this provocative model and compare separase/Bcl-xL to p53 with respect to its importance for DSB-induced apoptosis. 53BP1 is known to undergo liquid-liquid phase separation to form NHEJ compartments of clustered DSBs. Its recruitment function for other factors make 53BP1 a direct antagonist of the cancer-relevant, HR-promoting BRCA1. Therefore, in the second sub-project, we will investigate whether separase-mediated cleavage dissolves damage induced 53BP1 assemblies and how it influences the repair factor composition at DSBs. By generating cells that contain only non-cleavable 53BP1 or hypo-/hyperactive separase, we will finally determine separase's impact on repair pathway choice and the sensitivity of BRCA1-compromised cells towards a DDR-specific class of anti-cancer drugs.

DNA双链断裂（DSB）不断威胁着基因组的完整性和细胞的健康，必须进行修复以防止细胞死亡、致癌转化或衰老。维持DSB的细胞必须决定是否通过凋亡（程序性细胞死亡）来牺牲自己以保护生物体的健康，或者是否试图修复损伤。在后一种情况下，细胞必须在两种主要的修复途径之间进行选择，快速但可能致突变的NHEJ（非同源末端连接）或较慢但无错误的HR（同源重组）。研究分离酶，触发染色体分离在所有真核细胞的M期，我们意外地发现，这种必需的蛋白酶也预见到重要的功能，在细胞周期的间期。我们的初步数据表明，分离酶参与上述两个决定的DNA损伤反应（DDR）：凋亡或修复，如果修复，NHEJ或HR？更具体地说，我们发现分离酶被募集到DSB并在DSB处被激活，在DSB处它切割抗凋亡Bcl-xL和NHEJ促进53 BP 1。这些裂解需要通过DDR激酶ATM的底物磷酸化，并且-取决于DSB负载-分别有利于细胞凋亡或HR。在第一个子项目中，我们将研究将Bcl-xL带到DSB进行切割所需的线粒体-核穿梭，以及转化后的促凋亡片段回到线粒体。我们将绘制Bcl-xL上的磷酸化，并研究它们诱导的结构变化，以使分离酶裂解。我们将阐明切割的Bcl-xL主动诱导细胞凋亡的内在途径的分子机制。DSB诱导的细胞凋亡的一个p53非依赖性分支，其存在早已被证明，然而直到今天仍然难以确定其分子身份。我们认为分离酶-Bcl-xL-轴代表了这种经过思考的途径。我们将测试这个挑衅性的模型，并比较分离酶/Bcl-xL的p53的重要性，DSB诱导的细胞凋亡。已知53 BP 1经历液-液相分离以形成簇DSB的NHEJ隔室。它对其他因子的募集功能使53 BP 1成为癌症相关的、促进HR的BRCA 1的直接拮抗剂。因此，在第二个子项目中，我们将研究分离酶介导的切割是否溶解损伤诱导的53 BP 1组装体，以及它如何影响DSB的修复因子组成。通过产生仅含有不可切割的53 BP 1或低活性/高活性分离酶的细胞，我们将最终确定分离酶对修复途径选择的影响以及BRCA 1受损细胞对DDR特异性抗癌药物的敏感性。