Mechanism and Fidelity of RAG mediated DNA recombination

RAG介导的DNA重组的机制和保真度

• 批准号: 10404048
• 负责人: Eli Rothenberg
• 金额: $ 53.39万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-10 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10404048
• 关键词: Address; Affect; Antigen Receptors; B-Lymphocytes; Binding; Biochemical; Biological; Biological Assay; Cell Line; Cell Nucleus; Cells; Chromatin; Chromosomal translocation; Chromosome Deletion; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Ligation; DNA Repair; Defect; Disease; Double Strand Break Repair; Event; Functional disorder; Genetic Recombination; Genomic Instability; Glare; Goals; HMGB1 gene; Hand; Heart; Human; IgK; Image; Immune; Immune system; Immunologic Deficiency Syndromes; In Vitro; Individual; Ionizing radiation; Kinetics; Knowledge; Lead; Malignant Neoplasms; Mammalian Cell; Mediating; Methods; Molecular; Monitor; Mutation; Nonhomologous DNA End Joining; Nuclear; Nucleic Acid Regulatory Sequences; Pathway interactions; Peptide Signal Sequences; Phase; Process; Proteins; Radiation Tolerance; Reaction; Regulation; Research; Resolution; Series; Severe Combined Immunodeficiency; Site; System; Techniques; Therapeutic; Time; Transact; V(D)J Recombination; adaptive immunity; base; experimental study; in vivo; innovation; innovative technologies; insight; mutant; nanoscale; protein complex; reconstitution; recruit; repaired; response; single molecule; spatiotemporal; virtual

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.

V(D)J 重组是抗原受体多样性和适应性免疫的核心。 RAG 复合体 (RAG)，包括 RAG1、RAG2 和 HMGB1，通过结合来启动这一关键过程 重组信号序列 (RSS) 并产生 DNA 双链断裂 (DSB)。这 由此产生的断裂通过非同源末端连接（NHEJ）途径修复，这是主要的途径 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相关，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) 灵敏度。 尽管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。我们对此过程的了解中两个特别明显的差距是：1）步骤是什么 和 RAG-NHEJ 因子相互作用介导这一过程？ 2) RAG 和 NHEJ 怎么样 在“重组中心”中组织和调节（失调）的复合体，其中 V(D)J 重组发生在体内吗？对这些问题的研究受到限制 传统生物化学、结构和细胞生物学方法固有的局限性，现在可以解决 通过高分辨率单分子方法克服。 在此应用中，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和 化验。所提出的研究得到了关键初步实验的支持，包括应用 体外实时监测 RAG-NHEJ 交接过程的单分子测定及其利用 V(D)J 重组过程中重组复合物的超分辨率成像 细胞。