Characterization of V(D)J cleavage and repair complexes

B和T淋巴细胞形成我们的适应性免疫系统的基础，并具有独特的能力， 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 IG和TCR基因是独特的，因为编码Ig和TCR的可变结构域的外显子必须是 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 重排过程，称为V（D）J重组，当两种蛋白质，称为RAG 1和RAG 2， 通过蛋白质-DNA相互作用用重组信号桥接两个抗原受体基因片段 每个基因片段的侧翼序列（RSS），然后切割DNA以将RSS与 基因编码段（裂解期）。随后，四个DNA末端被重组、加工， 并通过非同源末端连接（NHEJ）DNA修复途径重新连接，形成一个信号连接， RSS结束和一个从两个编码结束的编码联合（联合阶段）。虽然V（D）J的基本步骤 重组是相当好的理解，许多重要的细节仍然不清楚，特别是在哪里， 解理和接合相相交。例如，一些证据表明，RAG蛋白发挥作用， 在引导DNA末端转移到NHEJ机器中起着积极的作用，但对这一点的机械理解 过程缺乏。解释V（D）J两个阶段之间的相互作用的部分困难是 重组可能是由于RAG 1的截短的、催化活性的核心形式的历史使用， RAG 2在这些反应的生物化学测定中，可能无法完全稳定与 调节和NHEJ因素。我们已经开发了纯化全长RAG蛋白的策略， 他们的生物化学分析的适用性，并使用这些RAG制剂，以获得证据，全长 RAG 1与参与NHEJ的Ku 70/Ku 80复合物相关，可能是通过新的相互作用伙伴。 为了获得更多的了解控制和连接裂解和连接阶段的分子机制 V（D）J重组，我们建议：（i）检查潜在的一种新形式的结构特异性的机制， 而不是序列特异性，DNA裂解的RAG蛋白;（ii）确定如何非核心？ RAG蛋白的一部分有助于切割后复合物的稳定性，影响DNA修复，并调节 在DNA底物上组装的离散RAG复合物中的酶活性和蛋白质-DNA接触 （iii）表征发现与全长RAG 1相互作用的新鉴定的因子， 它们在V（D）J重组中的生物活性和生理重要性。更好地了解 RAG蛋白启动切割并指导修复，RAG介导的DNA断裂将改善我们的细胞增殖。 理解导致疑似V（D）J重组受损和异常的机制， 分别是某些免疫缺陷和淋巴恶性肿瘤的基础。

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 is lacking. 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) examine the mechanisms underlying a novel form of structurespecific, rather than sequence-specific, DNA cleavage by the RAG proteins; (ii) determine how the ¿non-core¿ portions of the RAG proteins contribute to post-cleavage complex stability, influence DNA repair, and modulate the enzymatic activity and 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.