Characterization of V(D)J cleavage and repair complexes

项目负责人/主要研究者（最后一名、第一名、中间名）：Swanson，帕特里克3R 56 AI 055599 - 06 A1 W1 摘要 B和T淋巴细胞形成我们的适应性免疫系统的基础，并具有分别由免疫球蛋白（Ig）和T细胞受体（TCR）介导的抗原特异性识别的独特能力。IG和TCR基因是独特的，因为编码Ig和TCR可变结构域的外显子必须通过体细胞DNA重排从离散的基因片段组装以获得功能性。这种重排过程称为V（D）J重组，当两种称为RAG 1和RAG 2的蛋白质通过蛋白质-DNA相互作用与每个基因片段侧翼的重组信号序列（RSS）桥接两个抗原受体基因片段，然后切割DNA以将RSS与基因编码片段分离（“切割阶段”）时，启动。随后，四个DNA末端通过非同源末端连接（NHEJ）DNA修复途径重组、加工和重新连接，以形成来自两个RSS末端的“信号接头”和来自两个编码末端的“编码接头”（“连接阶段”）。虽然V（D）J重组的基本步骤已经相当清楚，但许多重要的细节仍然不清楚，特别是在裂解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机制中起着积极的作用，但对该过程的机制理解尚未完全阐明。阐明V（D）J重组的两个阶段之间的相互作用的部分困难可能是由于在这些反应的生物化学测定中RAG 1和RAG 2的截短的、催化活性的“核心”形式的历史使用，其可能不能完全稳定与调节因子和NHEJ因子的关联。我们已经开发了策略，以纯化全长RAG蛋白，增加其适用于生化分析，并使用这些RAG制剂获得的证据表明，全长RAG 1协会与Ku 70/Ku 80复杂的NHEJ，可能通过新的相互作用的合作伙伴。为了进一步了解控制和连接V（D）J重组的切割和连接阶段的分子机制，我们提出：（i）确定RAG蛋白的“非核心”部分如何有助于切割后复合物的稳定性，并调节在含有一对RSS的DNA底物上组装的离散RAG复合物中的组成、酶活性、蛋白-DNA接触;（iii）表征发现与全长RAG 1相互作用的新鉴定的因子在V（D）J重组中的生物活性和生理重要性。更多地了解RAG蛋白如何启动切割和引导修复RAG介导的DNA断裂，将提高我们对导致受损和异常V（D）J重组的机制的理解，这些重组被怀疑分别是某些形式的免疫缺陷和淋巴恶性肿瘤的基础。

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.