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mammalian genome; molecular modeling; mutant; novel; prevent; recombinase; single molecule; stem; tool; tumorigenesis; unpublished works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 In unpublished work, we have: i) determined the structure of ProtoRAG-DNA complexes by cryo-electron microscopy (EM); ii) obtained crystals of Transib transposase that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.

呢 概括 启动V（d）J重组的RAG1/RAG2重组酶是JAWED的一个定义特征 脊椎动物自适应免疫，被认为是从转座元素演变而来的。关键方面 破布生物化学和体内调节尚不理解，在我们对 破布有助于基因组不稳定性和癌症发展的机制。 Transib 编码类似抹布的转座酶的Protorag Transoss，提供了一个全新的“工具箱” 要填补这些空白。在未发表的工作中，我们有：i）确定protorag-DNA的结构 冷冻电子显微镜（EM）的复合物； ii）获得了衍射X射线的Transib转座酶的晶体 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 抹布的乳沟； iv）发现了两种抑制体内破布介导的换位的机制。 我们将使用这些新颖的工具和发现来实现我们的核心目标：确定 进化为编排抹布功能的生化，结构和调节机制 并确定这些机制失败的生物学后果。我们的建议是 围绕三个核心问题组织。首先，哪些机制解释了抹布的裂解以及为什么 这些机制会崩溃吗？第二，抹布中的不同模块如何共同起作用 确定活动？第三，保护基因组免受抹布介导的换位的原因以及什么 这些机制失败时的后果？这些问题在两个交织的目标中解决了： AIM 1：确定DNA识别的基础和抹布的裂解的基础 家庭转座酶。 Protorag转座酶以惊人的相似性结合和切割DNA 通过抹布不当调节裂解。使用新型的rag-protorag嵌合蛋白，生物化学， 单分子生物物理学以及冷冻EM和X射线晶体学，我们将确定DNA如何结合 域，DNA弯曲，复杂的稳定性和构象变化有助于协调VS。 由抹布和抹布的转座酶形成的突触复合物中的不协调裂解。 AIM 2：确定转置向该的调节，靶向和生物学后果 RAG的哺乳动物基因组。建立在我们发现脱离DNA裂解或 激活体内换位，我们将使用一套体外和体内转座，裂解和高 - 正常和DNA修复细胞中的吞吐量测序测定，以定量和映射转座 由完整和突变的抹布酶介导。此外，我们将生成和分析抹布突变小鼠 DNA裂解和换位中的调节缺陷。一起，我们的结果将揭示DNA如何修复 因素和RAG催化和调节模块已进化以保护基因组稳定性和盾牌 在V（d）J重组过程中从恶性转化中产生淋巴细胞。