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

细胞周期依赖性 DNA 修复途径的机制表征 - Resubmission

• 批准号: 10756874
• 负责人: Justin Wai Chung Leung
• 金额: $ 10.39万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10756874
• 关键词: Mechanistic; characterization; cell; cycle; dependent

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.

项目总结 染色质DNA损伤反应(DDR)途径是保护细胞免受基因组伤害的基本途径 不稳定，这是癌症的一个标志。DDR途径在整个细胞周期中受到严格调控，以确保 DNA修复的时空调控。细胞周期调控的染色质修饰对DNA的编排至关重要 修理。值得注意的是，H4K20甲基化是一种依赖于细胞周期的组蛋白标记，与DNA双链有关 破损(DSB)修复途径选择。新加入的未经修改的H4招募TONSL以复制损坏 染色质进行同源重组(HR)修复；而H4K20me2招募53BP1进行损伤 染色质主要在G1期促进非同源末端连接(NHEJ)。知识鸿沟 目前的模型源于H4K20me1和DSB修复途径选择的作用和调控不清楚。 识别组蛋白H4K20me阅读器有助于深入了解染色质修饰是如何执行的 细胞通过在正确的时间招募下游效应蛋白到受损的染色质来发挥功能。我们确认了 ZMYM3(锌指骨髓增殖症和智力低下，3型)，作为HR促进因子，它 与H4K20甲基化标记特异性结合。这个项目的总体目标是阐明 ZMYM3在细胞周期调控的H4K20甲基化中的机制调控作用及其如何转化为DNA 复制后染色质上双链断裂修复途径的选择。具体地说，我们建议1)确定 生化检测和遗传学研究ZMYM3和H4K20甲基化的关系2)表征 ZMYM3功能复合体对复制后受损染色质的影响；3)阐明了ZMYM3的作用机制。 ZMYM3如何调控细胞周期调控的DSB修复途径选择和。我们将重点调查 新发现的DNA修复蛋白DYNLL1/LC8及其与生理、遗传和功能的联系 在DSB修复规则中使用ZMYM3。我们的长期目标是剖析细胞如何协调DNA的细节 通过染色质修饰进行修复。这些研究准备为H4K20me1和H4K20me1如何 H4K20me2决定了在复制后染色质修复中HR和NHEJ之间的选择。它还将破译 ZMYM3如何塑造复制后的染色质表观基因组，并在受损的染色质上招募DDR蛋白。 虽然遗传性DDR缺陷易导致癌症的发生，但这种脆弱性被用于治疗。 优先杀死肿瘤细胞。因此，DNA损伤剂是一类主要的治疗剂，包括 放射治疗。由于染色质直接调节受损染色质上积累的DNA修复蛋白，因此 表观基因组是癌症治疗药物发现的一个有吸引力的靶点。这项工作综合运用了 生化、遗传学、表观遗传学和细胞学方法来剖析细胞周期的详细机制- 受调控的表观基因组对基因组完整性的维持可转化为潜在的生物标记物和药物 癌症治疗的发现。