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: 10801641
• 负责人: David G. Schatz
• 金额: $ 62.48万
• 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 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10801641
• 关键词: Adaptive Immune System; Address; Alleles; Antigen Receptors; Architecture; B-Cell Development; Biochemical; Biochemistry; Biological; Biological Assay; Biology; Cell Line; Cells; Chimeric Proteins; Chromatin; Chromosome Pairing; Closure by clamp; Complex; Coupled; Cryoelectron Microscopy; DNA; DNA Binding; Dangerousness; Development; Elements; Engineering; Ensure; Enzymes; Ephrin-A5; Event; Evolution; Family; Functional disorder; Funding; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Heart; Hematopoietic Neoplasms; Human Chromosomes; Human Genome; Immune system; In Vitro; Insertional Mutagenesis; Integration Host Factors; Invertebrates; Jaw; Label; Lead; Left; Link; Lymphocyte; Lymphoid; Malignant Neoplasms; Mediating; Methods; Modeling; Molecular; Mus; Mutagenesis; Outcome; Protein Biochemistry; Proteins; Publishing; Rag1 Mouse; Receptor Gene; Recording of previous events; Regulation; Site; Structure; T-Lymphocyte; Testing; Toxin; Transposase; V(D)J Recombination; Work; adaptive immunity; antitoxin; design; endonuclease; experimental study; fascinate; genotoxicity; in vitro Model; in vivo; insight; leukemia/lymphoma; mutant; novel; prevent; recombinase; reconstitution; structural biology; tool; tumorigenesis

SUMMARY The RAG recombinase is a domesticated transposase that initiates V(D)J recombination and contributes significantly to genome instability. To understand the mechanisms that protect the genome from dangerous RAG endonuclease/transposase activity, we have taken a distinctive approach that melds evolutionary biology with biochemistry and structural biology. From structures of ancestral RAG-like (RAGL) transposases, we discovered RAG’s fundamental modular organization, an “on-off” switch that controls properly regulated (“coupled”) cleavage, a novel DNA binding module that disrupts proper target site selection, and four evolutionary adaptations in RAG that together provide powerful, multilayered protection against transposition. These advances helped establish our current paradigm for RAG’s evolutionary origins and support a “DNA confinement” model to explain errors in RAG targeting. Using these novel conceptual frameworks and our recent discovery of a critical “missing link” in RAG’s evolutionary history, we will pursue our central objective: to understand the mechanisms that ensure that RAG cuts appropriate targets in a properly orchestrated (“coupled”) manner as well as the mechanisms that protect against catastrophic insertional mutagenesis due to transposition into the genome. To achieve this objective, we will pursue the following aims: Aim 1. Determine the evolutionary, structural, and biochemical basis of the RAGL→RAG transition. We will systematically dissect the activity and structure of “missing link” RAGL transposases and rigorously test the predictions of our DNA confinement and “on-off” switch models using in vitro protein biochemistry, a suit of in vivo cleavage and transposition assays, cryo-electron microscopy, and chimeric RAG enzymes engineered to possess carefully perturbed DNA binding and cleavage activities. Aim 2. Determine the mechanisms by which RAG2 suppresses RAG-mediated transposition in vivo. RAG2 and, surprisingly, “missing link” RAG2L proteins, possess an acidic hinge domain that powerfully suppresses transposition, leading us to propose that RAG2L arose early in evolution as an “antitoxin” to suppress the genotoxic potential of RAG1L (the transposase “toxin”). We will determine the protein residues and mechanisms that mediate the suppressive activity of the acidic hinge and a second suppressive region in RAG2, the LF2F3 loop, using an array of biochemical reconstitution and proximity labeling approaches. Aim 3. Determine the biological and genomic consequences of hyperactivated/dysregulated RAG in cells and mice. The goal of this aim is to connect mechanistic understanding to biological outcome. Using in vivo transposition assays and mice harboring mutant RAG alleles, we will answer two outstanding questions: i) Which RAG adaptations are needed to suppress RAG-mediated transposition from one site in the genome to another? ii) What are the consequences for the genome, lymphoid development, and tumorigenesis, of unleashing RAG-mediated transposition or of uncoupling and mistargeting RAG’s cleavage activity?

概括 RAG 重组酶是一种驯化的转座酶，可启动 V(D)J 重组并有助于 显着影响基因组的不稳定性。了解保护基因组免受危险的机制 RAG 核酸内切酶/转座酶活性，我们采取了融合进化的独特方法 生物学与生物化学和结构生物学。从祖先 RAG 样 (RAGL) 转座酶的结构来看， 我们发现了 RAG 的基本模块化组织，一个控制适当调节的“开关”开关 （“耦合”）切割，一种新型 DNA 结合模块，可破坏正确的靶位点选择，以及四个 RAG 中的进化适应共同提供了针对转座的强大、多层保护。 这些进步帮助我们建立了当前 RAG 进化起源的范式，并支持“DNA 限制”模型来解释 RAG 靶向中的错误。使用这些新颖的概念框架和我们的 最近发现了 RAG 进化史上一个关键的“缺失环节”，我们将追求我们的中心目标： 了解确保 RAG 以适当协调的方式削减适当目标的机制 （“耦合”）方式以及防止灾难性插入突变的机制 转座到基因组中。为了实现这一目标，我们将追求以下目标： 目标 1. 确定 RAGL→RAG 转变的进化、结构和生化基础。 我们将系统剖析“缺失环节”RAGL转座酶的活性和结构，并严格 使用体外蛋白质生物化学测试我们的 DNA 限制和“开关”模型的预测， 一套体内裂解和转座测定、冷冻电子显微镜和嵌合 RAG 酶 被设计为具有精心扰乱的 DNA 结合和切割活性。 目标 2. 确定 RAG2 在体内抑制 RAG 介导的转座的机制。 RAG2 和令人惊讶的“缺失环节”RAG2L 蛋白拥有一个酸性铰链结构域，可以强有力地 抑制转座，使我们提出 RAG2L 在进化早期作为一种“抗毒素”出现 抑制 RAG1L（转座酶“毒素”）的潜在基因毒性。我们将测定蛋白质残留 以及介导酸性铰链和第二抑制区的抑制活性的机制 在 RAG2 中，LF2F3 循环，使用一系列生化重建和邻近标记方法。 目标 3. 确定 RAG 过度激活/失调的生物学和基因组后果 细胞和小鼠。这一目标的目标是将机械理解与生物学结果联系起来。使用于 体内转座试验和携带突变 RAG 等位基因的小鼠，我们将回答两个悬而未决的问题： i) 需要哪些 RAG 适应来抑制基因组中某个位点的 RAG 介导的转座 到另一个？ ii) 对基因组、淋巴发育和肿瘤发生有何影响？ 释放 RAG 介导的转座或解偶联和误定位 RAG 的裂解活性？