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

项目摘要 DNA双链断裂（DSB）是最具毒性的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。后 DSB的形成，即p-53结合蛋白-1（53 BP 1），一种染色质调节剂， 结构（病灶），并作为平台招募DSB修复蛋白到受损的染色质 通过阻断末端切除促进NHEJ。几种人类综合征，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， 化学治疗药物由NHEJ蛋白的突变产生。此外， NHEJ也可能导致突变性替代性末端连接（a-EJ）DSB修复， 在某些癌症中。尽管在该领域取得了很大进展， NHEJ和a-EJ因子在细胞内仍然是不确定的。此外，基本机制 修复灶及其对NHEJ的影响仍然知之甚少。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， 通过单分子技术如单分子跟踪（SMT）和随机 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PARP抑制剂和NHEJ相关的未来治疗开发 XLF缺乏综合征等疾病。该提案的目的是（1）建立 在单个53 BP 1病灶内NHEJ因子的动力学和组织，和（2）确定作用 53 BP 1焦点在调节DSB修复机制的动力学和组织。

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.