Function and Evolutionary Origins of the RAG Endonuclease

摘要 RAG重组酶是一种驯化的转座酶，它启动V(D)J重组并对 对基因组的不稳定性有重大影响。为了了解保护基因组免受危险的机制 RAG内切酶/转座酶活性，我们采取了一种独特的方法，将进化 生物学与生物化学和结构生物学。从祖传RAG样转座酶(RAGL)的结构， 我们发现了RAG的基本模块化结构，一个控制适当调节的“开-关”开关 (“偶联”)切割，一种新的DNA结合模块，它扰乱了正确的靶点选择，以及四个 RAG中的进化适应共同提供了强大的多层保护，防止移位。 这些进展帮助建立了我们目前关于RAG进化起源的范例，并支持 限制“模型来解释RAG目标定位中的错误。使用这些新颖的概念框架和我们的 最近在RAG的进化史上发现了一个关键的“缺失环节”，我们将追求我们的中心目标： 要了解确保RAG在适当的精心策划的情况下切割适当目标的机制 (“偶联”)方式以及防止灾难性插入突变的机制 转座到基因组中。为了达到这一目标，我们将追求以下目标： 目的1.确定RAGL→RAG转变的进化、结构和生化基础。 我们将系统地剖析RAGL转座酶的活性和结构，并严格地 使用体外蛋白质生物化学测试我们的DNA限制和“开-关”开关模型的预测 一套体内切割和转座分析、冷冻电子显微镜和嵌合RAG酶 经过精心设计，具有被仔细干扰的DNA结合和切割活性。 目的2.确定RAG2在体内抑制RAG介导的转座的机制。 令人惊讶的是，RAG2和“缺失环节”的RAG2L蛋白都具有一个酸性铰链结构域， 抑制转座，导致我们提出RAG2L在进化早期作为一种抗毒素出现在 抑制RAG1L(转座酶“毒素”)的遗传毒性。我们将测定蛋白质残留量 以及调节酸性铰链和第二抑制区抑制活性的机制 在RAG2中，使用一系列生化重组和邻近标记方法的LF2F3环。 目的3.确定RAG过度激活/失控的生物学和基因组后果 细胞和老鼠。这一目标的目的是将机械理解与生物学结果联系起来。使用中 体内转座试验和携带突变RAG等位基因的小鼠，我们将回答两个突出的问题： I)需要哪些RAG适配来抑制来自基因组某一位置的RAG介导的转座 对另一个人？二)对人的基因组、淋巴发育和肿瘤形成有什么影响 释放RAG介导的转位，还是解偶联和错误定位RAG的切割活性？

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?