Function and Evolutionary Origins of the RAG Endonuclease

! 总结 启动V（D）J重组的RAG 1/RAG 2重组酶是颌型关节炎的一个定义性特征。 脊椎动物的适应性免疫，被认为是从转座因子进化而来的。的关键方面 RAG的生物化学和体内调控尚不清楚，在我们对RAG的理解中留下了很大的空白。 RAG导致基因组不稳定和癌症发展的机制。The Transib 和ProtoRAG转座子，编码RAG样转座酶，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ProtoRAG-DNA的结构 复合物通过冷冻电子显微镜（EM）; ii）获得晶体的Transib转座酶， ~ 3 μ m分辨率; iii）确定了指导协调（偶联）DNA的机制的关键组成部分 通过RAG切割;和iv）发现了在体内抑制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： 生物化学、结构和调节机制已经进化到协调RAG功能 并确定这些机制失效的生物学后果。我们的建议是 围绕三个核心问题展开。首先，什么机制可以解释RAG的偶联裂解，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， 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在两个相互交织的目标中得到解决： 目的1：确定RAG和RAG-DNA识别和偶联切割的基础。 家族转座酶ProtoRAG转座酶以惊人的相似性结合并切割DNA RAG的不适当调节切割。使用新的RAG-ProtoRAG嵌合蛋白，生物化学， 单分子生物物理学，冷冻电镜和X射线晶体学，我们将确定如何DNA结合 结构域、DNA弯曲、复合物稳定性和构象变化有助于协调与 在由RAG和RAG样转座酶形成的突触复合物中的不协调切割。 目的2：确定转座到细胞内的调节、靶向和生物学后果。 哺乳动物基因组RAG。基于我们发现的RAG突变体， 激活转座在体内，我们将使用一套在体外和体内转座，切割，和高- 在正常和DNA修复缺陷细胞中进行通量测序测定，以定量和绘制转座图 由完整的和突变的RAG酶介导。此外，我们将产生和分析RAG突变小鼠， 在DNA切割和转座中存在调节缺陷。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到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发展淋巴细胞。

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 In unpublished work, we have: i) determined the structure of ProtoRAG-DNA complexes by cryo-electron microscopy (EM); ii) obtained crystals of Transib transposase that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