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mammalian genome; molecular modeling; mutant; novel; prevent; recombinase; single molecule; stem; tool; tumorigenesis; unpublished works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 In unpublished work, we have: i) determined the structure of ProtoRAG-DNA complexes by cryo-electron microscopy (EM); ii) obtained crystals of Transib transposase that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 脊椎动物的适应性免疫，被认为是从转座元素进化而来的。 抹布生物化学和体内调节尚不清楚 破布有助于基因组不稳定性和癌症发展的机制 编码类似抹布的转子座的Protorag转座子，提供反对新的“工具箱” 在未发表的工作中填补空白，我们有：i） 通过冷冻电子显微镜（EM）进行的复合物； 〜3Å分辨率； 抹布的裂解； 我们将使用这些工具和发现来实现我们的中心目标：确定 进化为编排抹布功能的生化，结构和规律性机制 并确定这些机制失败的生物学后果。 首先组织了三个核心探索主义者。 这些机制是否分解？ 确定活动？ 这些机制在两个交织的AM中解决了这些机制的问题。 AIM 1：确定DNA识别的基础，并通过抹布和碎布进行裂解 protorag转座酶以惊人的相似性结合和切割DNA 使用新颖的碎布嵌合蛋白，生物化学的裂解不当 单分子生物物理学和冷冻EMS X射线晶体学，我们将确定DNA如何结合 域，DNA弯曲，复杂的稳定性和固定变化有助于协调VS。 由抹布和抹布样的transposess形成的突触复合物中的不协调的分裂。 AIM 2：确定转运到该的调节，靶向和生物学后果 哺乳动物的基因组，基于我们发现的抹布杂物， 激活体内换位，我们将使用一套体外和体内转座，裂解和高 - 在正常和DNA修复细胞中的吞吐量测序测定，以定量和映射转座 通过完整和突变的抹布酶介导。 随着DNA裂解和运输的调节缺陷，我们的结果将如何修复 因素和破布催化和调节模块已进化以保护基因组稳定性和屏蔽 在V（d）J重组过程中从恶性转化中产生淋巴细胞。