Mechanistic characterization of the cell cycle-dependent DNA repair pathway- Resubmission

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PROJECT SUMMARY Chromatin-based DNA damage response (DDR) pathway is fundamental for protecting cells from genome instability, which is a hallmark of cancer. The DDR pathway is tightly regulated throughout the cell cycle to ensure spatiotemporal control of DNA repair. Cell cycle-regulated chromatin modification is crucial for orchestrating DNA repair. Notably, H4K20 methylation is a cell cycle-dependent histone mark that is involved in DNA double-strand break (DSB) repair pathway choice. Newly incorporated unmodified H4 recruits TONSL to replicated damaged chromatin to execute homologous recombination (HR) repair; whereas, H4K20me2 recruits 53BP1 to damaged chromatin predominately at G1 phase to promote non-homologous end joining (NHEJ). The knowledge gap for the current model comes from the unclear role and regulation of H4K20me1 and DSB repair pathway choice. Identifying histone H4K20me readers provides important insights into how chromatin modifications execute cellular functions by recruiting downstream effector proteins to damaged chromatin at the right time. We identified ZMYM3 (Zinc finger myeloproliferative and mental retardation, type-3), as an HR promoting factor, which specifically binds to the H4K20 methylation mark. The overall objective of this project is to elucidate the mechanistic regulatory role of ZMYM3 on cell cycle-regulated H4K20 methylation, and how it translates into DNA DSBs repair pathway choice on post-replicative chromatin. Specifically, we propose to 1) determine the connection between ZMYM3 and H4K20 methylation by biochemical assays and genetic studies; 2) characterize the ZMYM3 functional complex(es) on post-replicative damaged chromatin; and 3) elucidate the mechanism of how ZMYM3 regulates cell cycle-regulated DSB repair pathway choice and. We will focus on investigating DYNLL1/LC8, a recently characterized DNA repair protein, and its physical, genetic and functional connections with ZMYM3 in DSB repair regulation. Our long-term goal is to dissect the detail of how cells orchestrate DNA repair via chromatin modifications. These studies are poised to provide critical insights into how H4K20me1 and H4K20me2 dictate the choice between HR and NHEJ on post-replicative chromatin repair. It will also decipher how ZMYM3 shapes the post-replicative chromatin epigenome and recruits DDR proteins at damaged chromatin. Although inherited DDR defects predispose in cancer development, the vulnerability is therapeutically exploited to preferentially kill tumor cells. Thus, DNA damaging agents are a major class of therapeutic agents that include radiotherapy. Since chromatin directly regulates DNA repair proteins accrual at damaged chromatin, the epigenome is an attractive target for drug discovery for cancer treatment. This work exploits a combination of biochemical, genetic, epigenetics and cellular approaches to dissect the detailed mechanism of cell cycle- regulated epigenome on genome integrity maintenance that can translate to potential biomarkers and drug discovery for cancer treatment.