BIOCHEMISTRY AND REGULATION OF V(D)J RECOMBINATION

描述(改编自调查员摘要)：V(D)J站点特定 重组是建立免疫多样性的过程。 介导抗原受体基因体细胞组装的全套技术 细分市场。催化V(D)J反应的成分中的突变 导致严重的免疫缺陷。其中有两个组件，即 重组激活蛋白RAG-1和RAG-2是关键的淋巴组织 启动该过程催化作用的特定活动。在……里面 此外，还发现了几种普遍存在的DNA修复活动 也参与V(D)J进程的人。的长期目标 这一应用旨在阐明V(D)J重组的生物化学。 具体目标是：目标1将涉及结构性领域和 RAG-1和RAG-2的生化特性。Aim 2将详细分析 具有以下特性的各个组件的组装和反应动力学 形成V(D)J重组活性复合体所必需的。研究 在目标1和目标2中，将基于常规生化技术和 使用生物传感器仪器监测相互作用的现代技术 大分子之间的实时联系。纯化的RAG-1、RAG-2和无处不在 DNA修复活动将被检测其DNA结合和 蛋白质/蛋白质相互作用活动使用足迹、交联和 生物传感器分析。此外，已确定的域的作用将是 使用遗传方法在体内进行了测试。RAG-1的DNA结合区 将被结晶以了解其结合模式。目标3将 阐述RAG-1/RAG-2的催化结构域以及RAG复合体如何裂解 具有特异性的DNA。这些实验将使用基因分析， 利用纯化的RAG-1进行化学修饰和质谱学技术 和RAG-2蛋白。V(D)J的生化及其调控研究 重组对于我们理解 免疫系统的建立以及人类免疫缺陷的程度 由V(D)J复合的随机失活产生 机械设备。此外，V(D)J重组被认为与 癌的发生和某些癌基因的移位。这些研究 这份提案中所描述的可能有助于我们设计试剂和实验 诊断人类许多衰弱或致命疾病的方案 淋巴生成系统，如各种形式的严重联合免疫缺陷， 白血病和淋巴瘤。这些疾病的一部分可能可以通过以下方法治疗 基因疗法。

DESCRIPTION (Adapted from the investigator's abstract): V(D)J site-specific recombination is the process that establishes diversity of the immune repertoire by mediating the somatic assembly of the antigen receptor gene segments. Mutations within the components that catalyze the V(D)J reaction result in severe immunodeficiencies. Two of these components, the recombination activating proteins, Rag-1 and Rag-2, are the key lymphoid specific activities that initiate the catalysis of the process. In addition, several ubiquitous, DNA repair activities have been identified that also participate in the V(D)J process. The long-term objectives of this application are to elucidate the biochemistry of V(D)J recombination. The specific aims are: Aim 1 will address the structural domains and the biochemical properties of Rag-1 and Rag-2. Aim 2 will analyze in detail the assembly and reaction kinetics of the individual components that are required in order to form a V(D)J recombination competent complex. Studies in aims 1 and 2 will be based on conventional biochemical techniques and modern technology using a biosensor instrument that can monitor interactions between macromolecules in real time. Purified Rag-1, Rag-2 and ubiquitous DNA repair activities will be assayed for their DNA binding and protein/protein interaction activities using footprinting, cross-linking and biosensor assays. In addition, the role of the identified domains will be tested in vivo using genetic approaches. The DNA binding domain of Rag-1 will be crystallized in order to understand its mode of binding. Aim 3 will address the catalytic domain of Rag-1/Rag-2 and how the Rag complex cleaves the DNA with specificity. These experiments will employ genetic analyses, chemical modifications and mass spectroscopy techniques using purified Rag-1 and Rag-2 proteins. Studies on the biochemistry and regulation of V(D)J recombination are of profound significance for our understanding of how the immune system is established and how immunodeficiencies in humans can be generated by the stochastic inactivation of the V(D)J recombination machinery. In addition, V(D)J recombination has been implicated in oncogenesis and the translocation of certain oncogenes. The studies described in this proposal may help us design reagents and experimental protocols to diagnose many debilitating or fatal diseases of the human lymphopoietic system such as, forms of severely combined immunodeficiencies, leukemias and lymphomas. A subset of these diseases may be treatable by gene therapy.