BIOCHEMISTRY AND REGULATION OF V(D)J RECOMBINATION

描述（改编自研究者摘要）：V（D）J研究中心特定 重组是建立免疫多样性的过程， 通过介导抗原受体基因的体细胞组装 片段 催化V（D）J反应的组分内的突变 导致严重的免疫缺陷。 其中两个组件， 重组激活蛋白Rag-1和Rag-2是淋巴细胞中的关键蛋白。 启动催化过程的特定活动。 在 此外，已经鉴定出几种普遍存在的DNA修复活性 也参与了V（D）J程序。 的长期目标 本申请旨在阐明V（D）J重组的生物化学。 具体目标是：目标1将解决结构领域和 Rag-1和Rag-2的生物化学性质。 目标2将详细分析 组装和反应动力学的各个组成部分， 所需的，以便形成V（D）J重组能力复合物。 研究 目标1和2将基于常规生物化学技术， 现代技术使用生物传感器仪器， 在真实的时间内大分子之间的相互作用。 纯化的Rag-1、Rag-2和普遍存在的 将测定DNA修复活性的DNA结合， 蛋白质/蛋白质相互作用的活动，使用足迹，交联和 生物传感器测定。 此外，已确定的域的作用将是 用遗传学方法在体内进行测试。 Rag-1的DNA结合结构域 将被结晶化以理解其结合方式。 目标3将 解决Rag-1/Rag-2的催化结构域以及Rag复合物如何切割 DNA的特异性 这些实验将采用遗传分析， 使用纯化的Rag-1的化学修饰和质谱技术 和Rag-2蛋白。 V（D）J的生物化学及调控研究 重组对于我们理解 免疫系统的建立，以及人类免疫缺陷如何 由V（D）J重组的随机失活产生 机械. 此外，V（D）J重组已经被牵连在 肿瘤发生和某些癌基因的易位。 研究 在这个建议中描述的可以帮助我们设计试剂和实验 用于诊断人类许多衰弱或致命疾病的协议 淋巴生成系统，例如严重联合免疫缺陷的形式， 白血病和淋巴瘤。 这些疾病的一个子集可以通过以下方法治疗： 基因治疗

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.