Characterization of V(D)J cleavage and repair complexes

项目总监/首席调查员(最后、第一、中间)：Swanson，Patrick 3R56 AI055599-06A1W1 摘要 B细胞和T细胞分别由免疫球蛋白(IGs)和T细胞受体(TCRs)介导，具有独特的抗原特异性识别能力，是人类适应性免疫系统的基础。Ig和TCR基因是独一无二的，因为编码IGs和TCRs可变区的外显子必须通过体细胞DNA重排从离散的基因片段组装起来才能获得功能。这种重排过程被称为V(D)J重组，当两种称为RAG1和RAG2的蛋白质通过蛋白质-DNA相互作用与每个基因片段两侧的重组信号序列(RSS)连接两个抗原受体基因片段时，然后切割DNA，将RSS从基因编码片段中分离出来(“切割阶段”)。随后，四个DNA末端通过非同源末端连接(NHEJ)DNA修复途径进行重组、加工和重新连接，形成由两个RSS末端组成的“信号关节”和来自两个编码末端的“编码关节”(“连接阶段”)。虽然V(D)J重组的基本步骤已经被很好地理解，但许多重要的细节仍然不清楚，特别是在解理相和连接相相交的地方。例如，一些证据表明RAG蛋白在引导DNA末端转移到NHEJ机制中发挥积极作用，但对这一过程的机制理解尚未完全阐明。阐明V(D)J重组两个阶段之间的相互作用的部分困难可能是由于历史上在这些反应的生化分析中使用截短的、具有催化活性的RAG1和RAG2的“核心”形式，这可能无法完全稳定与调节因子和NHEJ因子的关联。我们已经开发了纯化全长RAG蛋白的策略，以增加它们在生化分析中的适用性，并使用这些RAG准备材料来获得证据，表明全长RAG1可能通过新的相互作用伙伴与NHEJ中涉及的Ku70/Ku80复合体相关联。为了更深入地了解控制和连接V(D)J重组切割和连接阶段的分子机制，我们建议：(I)确定RAG蛋白质的“非核心”部分如何有助于切割后复合体的稳定性，并调节在含有一对RSS的DNA底物上组装的离散RAG复合体的组成、酶活性、蛋白质-DNA接触；(Iii)表征新发现的与全长RAG1相互作用的因子，以了解它们在V(D)J重组中的生物活性和生理意义。更多地了解RAG蛋白如何启动切割并引导RAG介导的DNA断裂的修复将提高我们对V(D)J重组受损和异常重组的机制的理解，V(D)J重组被怀疑分别是某些形式的免疫缺陷和淋巴系统恶性肿瘤的基础。

Program Director/Principal Investigator (Last, First, Middle): Swanson, Patrick 3R56 AI055599-06A1W1 ABSTRACT B and T lymphocytes form the foundation of our adaptive immune system and have the unique capacity for antigen-specific recognition mediated by immunoglobulins (Igs) and T cell receptors (TCRs), respectively. The Ig and TCR genes are unique because the exons encoding the variable domains of Igs and TCRs must be assembled from discrete gene segments by somatic DNA rearrangement to gain functionality. This rearrangement process, called V(D)J recombination, is initiated when two proteins, called RAG1 and RAG2, bridge two antigen receptor gene segments through protein-DNA interactions with a recombination signal sequence (RSS) that flanks each gene segment, and then cleaves the DNA to separate the RSS from the gene coding segment (the "cleavage phase"). Subsequently, the four DNA ends are reorganized, processed, and rejoined via the non-homologous end-joining (NHEJ) DNA repair pathway to form a "signal joint" from two RSS ends and a "coding joint" from two coding ends (the "joining phase"). While the basic steps of V(D)J recombination are fairly well understood, many important details remain unclear, particularly where the cleavage and joining phases intersect. For example, some evidence suggests that the RAG proteins play an active role in guiding the transfer of DNA ends to the NHEJ machinery, but a mechanistic understanding of this process has not been fully elucidated. Part of the difficulty in elucidating the interplay between the two phases of V(D)J recombination may be due to the historical use of truncated, catalytically active "core" forms of RAG1 and RAG2 in biochemical assays for these reactions, which may be unable to fully stabilize association with regulatory and NHEJ factors. We have developed strategies to purify full-length RAG proteins that increase their suitability for biochemical analysis, and used these RAG preparations to obtain evidence that full-length RAG1 associates with the Ku70/Ku80 complex involved in NHEJ, possibly through novel interaction partners. To gain additional insight into molecular mechanisms that control and connect the cleavage and joining phases of V(D)J recombination, we propose to: (i) determine how the "non-core" portions of the RAG proteins contribute to post-cleavage complex stability and modulate the composition, enzymatic activity, protein-DNA contacts in discrete RAG complexes assembled on DNA substrates containing a pair of RSSs; (iii) characterize newly identified factors found to interact with full-length RAG1 for their biological activity and physiological importance in V(D)J recombination. Greater knowledge of how the RAG proteins initiate cleavage and guide the repair the RAG-mediated DNA breaks will improve our understanding of the mechanisms contributing to impaired and aberrant V(D)J recombination that is suspected to underlie certain forms of immunodeficiency and lymphoid malignancy, respectively.