Function and Evolutionary Origins of the RAG Endonuclease

好了！ 摘要 启动V(D)J重组的RAG1/RAG2重组酶是JAWED的一个决定性特征 脊椎动物的适应性免疫，被认为是从转座因子进化而来的。关键方面 RAG生物化学和体内调节尚不清楚，在我们对RAG生物化学和体内调控的理解中留下了很大的空白 RAG促进基因组不稳定和癌症发展的机制。《过渡期》 而编码RAG样转座酶的ProtoRAG转座子提供了一个全新的工具箱 来填补这些空白。在未发表的工作中，我们已经：i)确定了原RAG-DNA的结构 用低温电子显微镜(EM)观察配合物；II)获得衍射光的TRANSB型转座酶晶体 3)确定了指导协调(偶联)DNA的机制的一个关键组成部分 被RAG切割；以及IV)在体内发现了抑制RAG介导的转座的两种机制。 我们将使用这些新的工具和发现来实现我们的中心目标：确定 进化为协调RAG功能的生化、结构和调节机制 并确定这些机制失效的生物学后果。我们的建议是 围绕三个核心问题进行了组织。首先，是什么机制解释了RAG的偶联卵裂？为什么？ 这些机制会崩溃吗？其次，RAG中的不同模块如何协同工作以 确定活动？第三，是什么保护基因组免受RAG介导的转座？ 当这些机制失效时会产生什么后果？这些问题有两个相互交织的目标： 目的1：确定DNA识别和RAG和RAG偶联切割的基础 家族转座酶。ProtoRAG转座酶以惊人的相似性结合和切割DNA 用碎布不适当地调节乳沟。利用新型RAG-ProtoRAG嵌合蛋白，生物化学， 单分子生物物理学，以及低温电子显微镜和x射线结晶学，我们将确定DNA结合 结构域、DNA弯曲、复杂的稳定性和构象变化有助于协调与 RAG和RAG样转座酶形成的突触复合体中的非配位裂解。 目标2：确定转座到TRAN的调节、靶向和生物学后果 哺乳动物基因组的碎布。基于我们发现的破布突变体，它可以解开DNA裂解或 激活体内转座，我们将使用一套体外和体内的转座、切割和高- 正常细胞和DNA修复缺陷细胞的吞吐量测序分析以定量和定位转座 由完整和突变的RAG酶介导。此外，我们还将培育和分析RAG突变小鼠 在DNA切割和转座方面存在调控缺陷。总之，我们的研究结果将揭示DNA修复 因子和RAG催化和调节模块已经进化来保护基因组的稳定性和屏蔽 在V(D)J重组过程中发生恶性转化的淋巴细胞。

! 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 In unpublished work, we have: i) determined the structure of ProtoRAG-DNA complexes by cryo-electron microscopy (EM); ii) obtained crystals of Transib transposase that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.