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。破布 包括RAG1、RAG2和HMGB1的复合体(RAG)通过结合启动这一关键过程 重组信号序列(RSS)和产生DNA双链断裂(DSB)。这个 由此产生的断裂通过非同源末端连接(NHEJ)途径修复，NHEJ是主要的 哺乳动物细胞中的DSB修复机制。RAG或NHEJ蛋白突变导致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。V(D)J重组缺陷与一系列人类疾病有关，包括 癌症、普通免疫缺陷(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 明白了。在我们对这一过程的了解中，有两个特别明显的差距：1)步骤是什么 以及调节这一过程的RAG-NHEJ因子相互作用？2)RAG和NHEJ怎么样 在V(D)J所在的“复合中心”中有组织和调节(失调)的复合体 重组在体内发生？对这些问题的研究一直受到限制。 在传统的生化、结构和细胞生物学方法中，现在可以 由高分辨率单分子方法克服。 在这个应用中，我们建议通过定义分子来解决这些知识差距 RAG-NHEJ切换过程的机制及其功能障碍如何导致V(D)J异常 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和 化验。拟议的研究得到了包括应用程序在内的关键初步实验的支持 单分子检测方法实时监测RAG-NHEJ体外移交过程，并利用 V(D)J复合过程中复合络合物的超分辨成像 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.