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

描述（由申请人提供）：V(D)J重组是一种位点特异性体细胞重组反应，其最终产物是编码细胞表面受体和抗体分子的基因，这些基因对免疫系统功能至关重要。适当的重组对健康免疫系统的发育是绝对必要的：当反应没有发生或控制不良时，就有可能发生免疫缺陷、白血病和淋巴瘤。我们的长期目标是了解V(D)J重组及其调控机制，我们的方法是通过对RAG蛋白的生化分析。虽然近二十年来重组领域取得了很大的进展，但仍有许多问题亟待解决，其中包括重组的可及性和体内重组的调控问题。在这项拨款申请中，我们描述了一系列的实验，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复合物的相互作用。通过提出具体目标，我们将：1)研究MCM2-7复合物和MCM相关蛋白在V(D)J重组中的需求和参与；2)研究Rag2 c端、酸性区和PHD结构域在V(D)J重组不同步骤中的作用及其参与该区域介导的各种蛋白-蛋白相互作用；3)利用染色质底物分析依赖于Rag2全长的RSS裂解机制。这些目标的重点是了解Rag2调控V(D)J重组的分子机制，特别是Rag2 c端在这一过程中的作用。

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. PUBLIC HEALTH RELEVANCE: V(D)J recombination is an essential process that takes place in B and T lymphocytes. This process is mediated by recombination factors that play unique and fundamental roles; deregulation of any of the required factors results in severe immunodeficiency. This project is focused on understanding how Rag2, one of the recombination factors, regulated the reaction by mediating multiple protein interactions and thus modulating the generation of a diverse immune system.