Regulation of V(D)J recombination by Rag2 C terminus

描述(申请人提供)：V(D)J重组是一种特定部位的体细胞重组反应，其最终产物是编码细胞表面受体和抗体分子的基因，这些分子对免疫系统功能至关重要。适当的重组对于发展健康的免疫系统是绝对必要的：当反应没有发生或控制不佳时，就有可能发展为免疫缺陷、白血病和淋巴瘤。我们的长期目标是了解V(D)J重组及其调控的机制，我们的方法是通过RAG蛋白的生化分析。尽管在过去的二十年里，重组领域已经取得了很大的进展，但仍然存在许多悬而未决的问题，其中包括可及性问题和体内重组的调控问题。在这项拨款申请中，我们描述了一系列实验，这些实验将解决这些问题，同时扩展我们的发现，显示Rag2与核心组蛋白和MCM复合体相互作用。通过提出的特定目标，我们将：1)研究MCM2-7复合体和MCM相关蛋白在V(D)J重组中的需求和参与；2)研究Rag2 C末端、酸性区域和PhD结构域在V(D)J重组的不同步骤中的作用及其参与该区域介导的各种蛋白质-蛋白质相互作用；3)利用染色质底物分析依赖于Rag2全长的RSS切割机制。这些目的的重点是了解决定Rag2调控V(D)J重组的分子机制，特别是Rag2 C末端在这一过程中的贡献。

DESCRIPTION (provided by applicant): V(D)J recombination is a site-specific somatic recombination reaction whose end products are genes encoding cell surface receptors and antibody molecules that are central to immune system function. Proper recombination is absolutely required for development of a healthy immune system: when the reaction does not occur or is poorly controlled, there is the potential for development of immunodeficiencies, leukemias, and lymphomas. Our long-term goal is to understand the mechanisms of V(D)J recombination and its regulation, and our approach has been through a biochemical analysis of the RAG proteins. Though much progress has been made in the field of recombination in the last two decades, there are still numerous questions outstanding, among them are the issue of accessibility and the regulation of recombination in vivo. In this grant application, we describe a series of experiments that will address these issues while extending our findings that showed interaction of Rag2 with core histones and the MCM complex. Through specific aims proposed, we will: 1) Investigate the requirement and participation of MCM2-7 complex and MCM associated proteins in V(D)J recombination; 2) Study the role of Rag2 C-terminus, acidic region and PHD domain, in the different steps of V(D)J recombination as well as their participation in the various protein-protein interactions mediated by this region; 3) Analyze the RSS cleavage mechanisms that depend on Rag2 full-length using a chromatin substrate. The focus of these aims is to understand the molecular mechanism that determine regulation of V(D)J recombination by Rag2 and specifically the contribution of Rag2 C-terminus to this process.