Function and Evolutionary Origins of the RAG Endonuclease

总结 RAG重组酶是一种驯化的转座酶，其启动V（D）J重组并促进V（D）J重组。 基因组的不稳定性。为了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，我们采取了一种独特的方法，融合了进化 生物化学和结构生物学。根据祖先RAG样（RAGL）转座酶的结构， 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” （“偶联”）切割，一种新的DNA结合模块，破坏适当的靶位点选择，和四个 RAG中的进化适应共同提供了强大的，多层次的保护，防止转座。 这些进展帮助我们建立了目前RAG进化起源的范式，并支持了“DNA 限制”模型来解释RAG定位中的错误。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”，我们将继续我们的中心目标： 了解确保RAG在适当协调的情况下削减适当目标的机制， （“偶联”）方式以及防止灾难性插入诱变的机制， 转座到基因组中。为达致这个目标，我们会致力达致以下目标： 目标1。确定RAGL→RAG转换的进化，结构和生化基础。 我们将系统地剖析“缺失环节”RAGL转座酶的活性和结构， 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， 一套体内切割和转座测定、冷冻电子显微镜和嵌合RAG酶 被工程化以具有仔细扰动的DNA结合和切割活性。 目标二。确定RAG 2在体内抑制RAG介导的转座的机制。 RAG 2和令人惊讶的“缺失的环节”RAG 2L蛋白具有酸性铰链结构域， 抑制转座，这使我们提出RAG 2L在进化早期作为“抗毒素”出现， 抑制RAG 1 L（转座酶“毒素”）的遗传毒性潜力。我们会检测出 以及介导酸性铰链和第二抑制区域的抑制活性的机制 在RAG 2中，LF 2F 3环，使用一系列生化重建和邻近标记方法。 目标3：确定RAG过度活化/失调的生物学和基因组后果， 细胞和小鼠。这一目标的目的是将机械理解与生物结果联系起来。使用in 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适应来抑制RAG介导的从基因组中的一个位点的转座 另一个？ii）对于基因组、淋巴发育和肿瘤发生， 释放RAG介导的转座或解偶联和错误的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?