Single-molecule organization and kinetics of the NHEJ repair machinery inside the cell

细胞内 NHEJ 修复机制的单分子组织和动力学

• 批准号: 9911161
• 负责人: Maria Benitez-Jones
• 金额: $ 4.55万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911161
• 关键词: Address; Affect; Behavior; Binding Proteins; Biological; Biological Assay; Cell Line; Cells; Chemicals; Chromatin; Complex; DNA Damage; DNA Double Strand Break; DNA Repair Pathway; DNA lesion; Dependence; Diffusion; Disease; Double Strand Break Repair; Environment; Event; Excision; Future; Goals; Higher Order Chromatin Structure; Human; Individual; Ionizing radiation; Kinetics; Knowledge; Lead; Malignant Neoplasms; Mammalian Cell; Measurement; Methods; Microscopy; Molecular; Monitor; Mutation; Nature; Nonhomologous DNA End Joining; Optics; Pathway interactions; Pharmaceutical Preparations; Phase; Process; Proteins; Repair Complex; Research; Role; Severe Combined Immunodeficiency; Signal Transduction; Site; Syndrome; Techniques; Testing; Therapeutic; Time; base; cytotoxic; experimental study; inhibitor/antagonist; innovation; live cell imaging; macromolecular assembly; microscopic imaging; molecular dynamics; nanoscale; novel; reconstruction; recruit; repaired; response; single molecule; spatiotemporal; temporal measurement; therapy development; virtual

PROJECT SUMMARY DNA double-stranded breaks (DSBs) are the most toxic DNA lesions, and in mammalian cells they are predominantly repaired via the non-homologous end-joining (NHEJ) pathway. Upon formation of a DSB, p-53 binding protein-1 (53BP1), a chromatin modulator, forms higher order structures (foci) and which act as platforms to recruit DSB repair proteins to damaged chromatin and promote NHEJ by blocking end resection. Several human syndromes including severe combined immunodeficiency, and an increased sensitivity to ionizing radiation (IR) and chemotherapeutic drugs result from mutations in NHEJ proteins. Additionally, deficiencies in NHEJ may also result in mutagenic alternative end-joining (a-EJ) DSB repair, which is established in some forms of cancer. Despite much progress in the field, the organization and kinetics of NHEJ and a-EJ factors remain undefined inside the cell. Furthermore, fundamental mechanisms of repair foci and their effects on NHEJ remain poorly understood. These gaps are due to inherent limitations of conventional ensemble methods. Here, I propose to resolve these knowledge gaps by defining the molecular mechanism of the human NHEJ repair process at the level of individual 53BP1 foci via single molecule techniques such as single-molecule tracking (SMT) and stochastic optical reconstruction microscopy (STORM). These approaches will provide novel information that may be applicable to future therapy development of PARP inhibitors and NHEJ-related diseases such as XLF deficiency syndrome. The aims of the proposal are to (1) establish the kinetics and organization of NHEJ factors within individual 53BP1 foci, and (2) determine the role of 53BP1 foci in regulating the kinetics and organization of the DSB repair machinery.

项目摘要 DNA双链断裂（DSB）是最有毒的DNA病变，在哺乳动物细胞中 它们主要是通过非同源末端连接（NHEJ）途径来修复的。之上 DSB，P-53结合蛋白-1（53BP1）的形成，一种染色质调节剂，形成高阶 结构（FOCI），并且是募集DSB修复蛋白损坏的染色质的平台 并通过阻止终端切除来促进NHEJ。包括严重的几种人类综合征 联合免疫缺陷，对电离辐射（IR）的敏感性提高和 化学治疗药物是由NHEJ蛋白突变引起的。此外，缺陷 NHEJ也可能导致诱变的替代末端连接（A-EJ）DSB修复，该修复已建立 在某些形式的癌症中。尽管该领域取得了很多进展，但组织和动力学 NHEJ和A-EJ因子在细胞内仍然不确定。此外，基本机制 维修灶及其对NHEJ的影响仍然很少了解。这些差距是由于固有的 常规合奏方法的局限性。在这里，我建议解决这些知识差距 通过在个体水平上定义人NHEJ修复过程的分子机制 通过单分子技术（例如单分子跟踪（SMT）和随机）的53BP1灶 光学重建显微镜（Storm）。这些方法将提供新颖的信息 这可能适用于将来的PARP抑制剂和与NHEJ相关的治疗的开发 XLF缺乏综合征等疾病。该提案的目的是（1）建立 单个53BP1焦点内NHEJ因素的动力学和组织，（2）确定角色 在调节DSB维修机械的动力学和组织方面的53BP1焦点。