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

项目总结 DNA双链断裂(DSB)是毒性最大的DNA损伤，在哺乳动物细胞中 它们主要通过非同源末端连接(NHEJ)途径修复。vt.在.的基础上 染色质调节剂DSB，p-53结合蛋白-1(53BP1)的形成形成更高的顺序 结构(焦点)，作为平台将DSB修复蛋白招募到受损的染色质 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，对电离辐射(IR)和 化疗药物是由NHEJ蛋白突变产生的。此外，在以下方面存在缺陷 NHEJ也可能导致突变的替代末端连接(a-EJ)DSB修复，这是建立的 在某些形式的癌症中。尽管在这一领域取得了很大进展，但 NHEJ和a-EJ因子在细胞内仍未确定。此外，基本机制 修复焦点及其对NHEJ的影响仍然知之甚少。这些差距是由于先天的 传统系综方法的局限性。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 通过单分子跟踪(SMT)和随机等单分子技术获得53BP1焦点 光学重建显微镜(STORM)。这些方法将提供新的信息 这可能适用于未来PARP抑制剂和NHEJ相关药物的治疗开发 XLF虚证等疾病。这项建议的目的是(1)建立 NHEJ因子在单个53BP1焦点内的动力学和组织，以及(2)决定其作用 53BP1的焦点在调节DSB修复机制的动力学和组织方面。

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.