Mechanism of Damage Tolerance by Nonhomologous End Joining

非同源末端连接的损伤容限机制

• 批准号: 9258650
• 负责人: Michael Patrick Conlin
• 金额: $ 2.95万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9258650
• 关键词: Address; Affect; Aging; Biological; Cancer Etiology; Catalytic Domain; Cell Death; Cell Line; Cell Respiration; Cells; Cellular Assay; Chromosome abnormality; Complex; DNA; DNA Damage; DNA lesion; DNA polymerase mu; DNA-Directed DNA Polymerase; Data; Defect; Deoxyribonucleotides; Development; Double Strand Break Repair; Effectiveness; Employee Strikes; Employment; Genome; Genomic Instability; Immune system; Immunologic Deficiency Syndromes; In Vitro; Ionizing radiation; Knowledge; LIG4 gene; Lead; Ligase; Ligation; Mammals; Measures; Meiosis; Metabolism; Methods; Mutation; Nucleotides; Organism; Oxides; Pathway interactions; Predisposition; Process; RNA; Radiation Tolerance; Reaction; Research; Ribonucleotides; Role; Safety; Shapes; Site; Specificity; Structure; Systems Development; Therapeutic Agents; Time; Variant; cancer cell; cancer therapy; cell injury; chemotherapeutic agent; cytotoxicity; experimental study; improved; insight; killings; mutant; nervous system disorder; radiosensitive; reconstitution; repaired; response; tumor

Project Summary / Abstract Double strand breaks (DSBs) are lethal DNA lesions that arise during meiosis, immune system development, and cancer therapy. Ionizing radiation (IR) and some chemotherapeutics kill tumors by inducing DSBs, which can have associated end damage that blocks repair. Repair of damaged ends is important because it shapes the efficiency and safety of cancer therapy. However, previous DSB repair research has focused on undamaged breaks. The proposed research will determine the mechanisms of damaged end repair by nonhomologous end joining (NHEJ), the predominant DSB repair pathway in mammals. The ultimate step in NHEJ is the ligation of ends by DNA Ligase IV (Lig4); I have shown that it is more likely than other pathways to join damaged ends. Lig4 functions only in the context of the NHEJ complex so it is unclear whether damage tolerance is intrinsic to the ligase. I hypothesize that Lig4 is a specialized damaged end ligase, and this activity is important for the response to cancer therapy. I am creating variants of Lig4 that separate function in vitro: they efficiently repair undamaged, but not damaged breaks. I will generate cell lines expressing these Lig4 variants and measure repair of damaged DSB ends. I will expose these cell lines to chemotherapeutic agents and ionizing radiation to determine if Lig4 damaged end repair is important for the response to cancer therapy. I will determine whether NHEJ capacity on damaged ends corresponds to radiosensitivity and chemoresistance in cells. These results will show whether Lig4 damaged end repair is a potentially druggable target to improve existing cancer therapies. Some damaged ends must be processed prior to repair. One processing factor is DNA polymerase mu, which fills in gaps at DSB ends. Surprisingly, pol mu preferentially adds ribonucleotides (RNA), rather than deoxyribonucleotides (DNA), to DNA ends. Preliminary data suggests that these ribonucleotides stimulate Lig4 activity, but it is unclear when and why this happens. I hypothesize that ribonucleotides added by pol mu facilitate Lig4 tolerance of damaged ends. I will identify when ribonucleotides are important for damaged end repair. Then, I will generate variants of Lig4 that abolish its stimulation by ribonucleotides. My results will reveal the biological significance of this startling repair phenomenon which introduces RNA into the genome. Many cancer therapies rely on damaged double strand breaks to kill cancer cells with specificity. The proposed research will provide mechanistic insight into the poorly understood process of damaged DSB repair. We will also identify requirements for damaged DSB repair that can be targeted to improve the effectiveness of cancer therapies.

项目总结/摘要 双链断裂（DSB）是在减数分裂、免疫系统 发展和癌症治疗。电离辐射（IR）和一些化疗药物通过诱导肿瘤细胞凋亡来杀死肿瘤细胞。 DSB，可能具有阻止修复的相关末端损坏。修复受损的末端很重要 因为它决定了癌症治疗的效率和安全性。然而，以前的DSB修复研究 专注于未损坏的裂缝。这项研究将确定受损末端修复的机制 通过非同源末端连接（NHEJ），哺乳动物中主要的DSB修复途径。 NHEJ的最终步骤是通过DNA连接酶IV（Lig 4）连接末端;我已经证明， 比其他途径更有可能连接受损的末端。Lig 4仅在NHEJ复合物的背景下起作用，因此其 目前还不清楚损伤耐受性是否是连接酶固有的。我假设Lig 4是一种特殊的受损的 末端连接酶，并且这种活性对于癌症治疗的反应是重要的。我正在创建Lig 4的变体， 在体外单独的功能：它们有效地修复未受损的，但不是受损的断裂。我会制造细胞系 表达这些Lig 4变体并测量受损DSB末端的修复。我会将这些细胞系 化学治疗剂和电离辐射，以确定Lig 4损伤的末端修复是否对肿瘤的发生重要。 对癌症治疗的反应。我将确定受损端的NHEJ容量是否与 细胞的放射敏感性和化学抗性。这些结果将显示Lig 4受损末端修复是否是一个重要因素。 潜在的药物靶点，以改善现有的癌症治疗。 一些损坏的端部必须在修理之前进行处理。一个加工因子是DNA聚合酶μ， 其填充DSB末端处的间隙。令人惊讶的是，pol mu优先添加核糖核苷酸（RNA），而不是核糖核苷酸。 脱氧核糖核苷酸（DNA），至DNA末端。初步数据表明，这些核糖核苷酸刺激Lig 4 活动，但尚不清楚何时以及为什么会发生这种情况。我假设由聚合物μ添加的核糖核苷酸 促进受损末端的Lig 4耐受性。我将确定何时核糖核苷酸对受损末端 修复.然后，我将产生Lig 4的变体，这些变体消除了核糖核苷酸对Lig 4的刺激。 我的结果将揭示 这种将RNA引入基因组的惊人修复现象的生物学意义。 许多癌症疗法依赖于受损的双链断裂来特异性地杀死癌细胞。的 拟议的研究将提供深入了解受损DSB修复过程的机制。 我们还将确定受损DSB修复的要求，这些要求可以有针对性地提高 癌症治疗