Mechanism and Fidelity of RAG mediated DNA recombination

V（D）J重组是抗原受体多样性和获得性免疫的核心。抹布 RAG复合物（RAG）包括RAG 1、RAG 2和HMGB 1，通过结合 重组信号序列（RSS）和产生DNA双链断裂（DSB）。的 由此产生的断裂通过非同源末端连接（NHEJ）途径修复， 哺乳动物细胞中的DSB修复机制。RAG或NHEJ蛋白的突变导致V（D）J缺陷 重组导致连接错误、染色体缺失和易位，以及基因组 不稳定缺陷性V（D）J重组与一系列人类疾病相关，包括 癌症、常见免疫缺陷（CID）和严重联合免疫缺陷（SCID），以及 电离辐射（IR）敏感性。 尽管在该领域取得了很大进展，但V（D）J重组的一个特别关键的步骤- 从RAG介导的DNA切割到NHEJ介导的DNA修复的转变仍然很差 明白我们对这一过程的认识中有两个特别明显的差距：1）步骤是什么 和RAG-NHEJ因子相互作用介导这一过程？2）如何在RAG和NHEJ 复合物在“重组中心”中组织和调节（失调），其中V（D）J 在体内发生重组吗对这些问题的研究一直受到限制 传统的生物化学、结构和细胞生物学方法中固有的局限性，现在可以 通过高分辨率单分子方法克服。 在本申请中，我们建议通过定义分子水平来解决这些知识缺口。 RAG-NHEJ交接过程的机制及其功能障碍如何导致异常V（D）J 重组为了实现这一目标，我们将使用一系列创新的单分子技术， 测定。建议的研究得到了关键的初步实验的支持，包括应用 实时监测RAG-NHEJ体外传递过程的单分子测定， 在V（D）J复合过程中复合物的超分辨率成像 细胞

V(D)J recombination lies at the heart of antigen receptor diversity and adaptive immunity. The RAG complex (RAG), which includes RAG1, RAG2 and HMGB1, initiates this critical process by binding recombination signal sequences (RSSs) and creating DNA double-stranded breaks (DSBs). The resulting breaks are repaired via the non-homologous end-joining (NHEJ) pathway, the predominant DSB repair mechanism in mammalian cells. Mutations in RAG or NHEJ proteins cause defects in V(D)J recombination leading to joining errors, chromosomal deletions and translocations, and genome instability. Defective V(D)J recombination is associated with a range of human disorders including cancer, common immune deficiency (CID) and severe combined immunodeficiency (SCID), and ionizing radiation (IR) sensitivity. Despite much progress in the field, a particularly critical step of V(D)J recombination–the transition from RAG-mediated DNA cleavage to NHEJ-mediated DNA repair–remains poorly understood. Two particularly glaring gaps in our knowledge of this process are: 1) What are the steps and RAG-NHEJ factor interactions that mediate this process? and 2) How are the RAG and NHEJ complexes organized and regulated (dysregulated) in the “recombination centers” within which V(D)J recombination takes place in vivo? Research into these questions has been hampered by limitations inherent in traditional biochemical, structural, and cell biological approaches, limitations that can now be overcome by high-resolution single molecule methods. In this application, we propose to address these knowledge gaps by defining the molecular mechanism of the RAG-NHEJ handoff process and how its dysfunction leads to aberrant V(D)J recombination. To accomplish this, we will use of an array of innovative single-molecule techniques and assays. The proposed studies are supported by key preliminary experiments including the application of single-molecule assays to monitor the RAG-NHEJ handoff process in vitro in real-time, and utilization of super-resolution imaging of recombination complexes during transactions of V(D)J recombination in cells.