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

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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.