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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 DNA 双链断裂 (DSB) 是哺乳动物细胞中毒性最强的 DNA 损伤它们主要通过非同源末端连接（NHEJ）途径进行修复。之上 DSB、p-53 结合蛋白-1 (53BP1)（一种染色质调节剂）的形成，形成更高阶结构（焦点），并充当招募 DSB 修复蛋白修复受损染色质的平台并通过阻断末端切除促进NHEJ。几种人类综合症，包括严重的联合免疫缺陷，以及对电离辐射 (IR) 的敏感性增加和化疗药物是 NHEJ 蛋白突变的结果。此外，还存在以下不足： NHEJ 还可能导致突变替代末端连接 (a-EJ) DSB 修复，这一点已被证实在某些形式的癌症中。尽管该领域取得了很大进展，但其组织和动力学 NHEJ 和 a-EJ 因子在细胞内仍未定义。此外，基本机制修复灶及其对 NHEJ 的影响仍知之甚少。这些差距是由于固有的传统集成方法的局限性。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类 NHEJ 修复过程的分子机制通过单分子技术（例如单分子追踪 (SMT) 和随机）进行 53BP1 焦点光学重建显微镜（STORM）。这些方法将提供新颖的信息可能适用于未来 PARP 抑制剂和 NHEJ 相关治疗的开发 XLF 缺乏综合症等疾病。该提案的目的是 (1) 建立个体 53BP1 病灶内 NHEJ 因子的动力学和组织，以及 (2) 确定作用 53BP1 焦点调节 DSB 修复机制的动力学和组织。