Function and Evolutionary Origins of the RAG Endonuclease

RAG 核酸内切酶的功能和进化起源

• 批准号: 10460993
• 负责人: David G. Schatz
• 金额: $ 52.53万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-11 至 2023-09-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460993
• 关键词: Adaptive Immune System; Address; Adherence; Antigen Receptors; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biophysics; Catalytic Domain; Cells; Chimeric Proteins; Complex; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; DNA; DNA Binding; DNA Binding Domain; DNA Repair; DNA Repair Gene; DNA Transposable Elements; Dangerousness; Data Set; Defect; Development; Enzymes; Event; Evolution; Failure; Family; Genetic Recombination; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Heart; Hematopoietic Neoplasms; High-Throughput Nucleotide Sequencing; Human Chromosomes; Human Genome; Immune system; Immunity; In Vitro; Jaw; Lymphocyte; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Methods; Modeling; Molecular Conformation; Mus; Mutant Strains Mice; Peptide Signal Sequences; Process; Protein Biochemistry; Proteins; Receptor Gene; Regulation; Resolution; Roentgen Rays; Source; Structure; Synapses; System; Transposase; V(D)J Recombination; Vertebrates; Work; X-Ray Crystallography; adaptive immunity; biophysical properties; endonuclease; experimental study; fascinate; in vitro activity; in vivo; insight; lens; leukemia/lymphoma; malignant lymphocyte; mammalian genome; molecular modeling; mutant; novel; prevent; recombinase; single molecule; stem; tool; tumorigenesis; unpublished works

! SUMMARY The RAG1/RAG2 recombinase, which initiates V(D)J recombination, is a defining feature of jawed vertebrate adaptive immunity and is thought to have evolved from a transposable element. Key aspects of RAG biochemistry and in vivo regulation are not understood, leaving large gaps in our understanding of the mechanisms by which RAG contributes to genome instability and the development of cancer. The Transib and ProtoRAG transposons, which encode RAG-like transposases, provide an entirely new "toolbox" with which to fill these gaps. In unpublished work, we have: i) determined the structure of ProtoRAG-DNA complexes by cryo-electron microscopy (EM); ii) obtained crystals of Transib transposase that diffract x-rays to ~3Å resolution; iii) identified a key component of the mechanism that directs coordinated (coupled) DNA cleavage by RAG; and iv) discovered two mechanisms that suppress RAG-mediated transposition in vivo. We will use these novel tools and findings to accomplish our central objective: to determine the biochemical, structural, and regulatory mechanisms that have evolved to orchestrate RAG function and to ascertain the biological consequences of failures of these mechanisms. Our proposal is organized around three core questions. First, what mechanisms explain coupled cleavage by RAG and why do those mechanisms break down? Second, how do the different modules within RAG work together to determine activity? And third, what protects the genome from RAG-mediated transposition and what are the consequences when those mechanisms fail? These questions are addressed in two interwoven aims: Aim 1: Determine the underpinnings of DNA recognition and coupled cleavage by RAG and RAG- family transposases. ProtoRAG transposase binds and cleaves DNA in a manner with striking similarities to improperly regulated cleavage by RAG. Using novel RAG-ProtoRAG chimeric proteins, biochemistry, single molecule biophysics, and cryo-EM and x-ray crystallography, we will determine how DNA binding domains, DNA bending, complex stability, and conformational changes contribute to coordinated vs. uncoordinated cleavage in synaptic complexes formed by RAG and RAG-like transposases. Aim 2: Determine the regulation, targeting, and biological consequences of transposition into the mammalian genome by RAG. Building on our discovery of RAG mutants that uncouple DNA cleavage or activate transposition in vivo, we will use a suite of in vitro and in vivo transposition, cleavage, and high- throughput sequencing assays in normal and DNA repair-deficient cells to quantitate and map transposition mediated by intact and mutant RAG enzymes. In addition, we will generate and analyze RAG-mutant mice with regulatory defects in DNA cleavage and transposition. Together, our results will reveal how DNA repair factors and RAG catalytic and regulatory modules have evolved to protect genome stability and shield developing lymphocytes from malignant transformation during the process of V(D)J recombination.

！ 概括 RAG1/RAG2 重组酶可启动 V(D)J 重组，是有颌病毒的一个决定性特征 脊椎动物适应性免疫，被认为是从转座元件进化而来的。 RAG 的生物化学和体内调节尚不清楚，这使得我们对 RAG 的理解存在很大差距 RAG 导致基因组不稳定和癌症发展的机制。 和 ProtoRAG 转座子，编码 RAG 样转座酶，提供了一个全新的“工具箱” 为了填补这些空白，我们在未发表的工作中： i) 确定了 ProtoRAG-DNA 的结构。 通过冷冻电子显微镜 (EM) 观察复合物；ii) 获得可衍射 X 射线的 Transib 转座酶晶体 达到 ~3Å 分辨率；iii) 确定了指导协调（耦合）DNA 的机制的关键组成部分 RAG 裂解；和 iv) 发现了两种抑制体内 RAG 介导的转座的机制。 我们将使用这些新颖的工具和发现来实现我们的中心目标：确定 已进化来协调 RAG 功能的生化、结构和调节机制 并确定这些机制失效的生物学后果。 首先，什么机制可以解释 RAG 的耦合裂解以及原因。 第二，RAG 中的不同模块如何协同工作？ 第三，什么可以保护基因组免受 RAG 介导的转座影响？ 当这些机制失败时会产生什么后果？这些问题通过两个相互交织的目标来解决： 目标 1：确定 RAG 和 RAG-DNA 识别和偶联切割的基础 ProtoRAG 转座酶家族的转座酶以惊人相似的方式结合和切割 DNA。 使用新型 RAG-ProtoRAG 嵌合蛋白、生物化学、 单分子生物物理学、冷冻电镜和 X 射线晶体学，我们将确定 DNA 结合的方式 结构域、DNA 弯曲、复合体稳定性和构象变化有助于协调与非协调。 RAG 和 RAG 样转座酶形成的突触复合体中的不协调切割。 目标 2：确定转座的调控、靶向和生物学后果 基于我们发现的 RAG 突变体，RAG 可以解开 DNA 裂解或 为了激活体内转座，我们将使用一套体外和体内转座、裂解和高 在正常细胞和 DNA 修复缺陷细胞中进行通量测序分析，以定量和定位转座 由完整和突变的 RAG 酶介导此外，我们将生成并分析 RAG 突变小鼠。 与 DNA 切割和转座的调控缺陷一起，我们的结果将揭示 DNA 修复的方式。 因子和 RAG 催化和调节模块已经进化，以保护基因组稳定性和屏蔽 在V(D)J重组过程中，淋巴细胞发生恶性转化。