Role of RAG1 Ubiquitin Ligase Activity in Recombination

RAG1 泛素连接酶活性在重组中的作用

• 批准号: 7071064
• 负责人: JESSICA Morgan JONES
• 金额: $ 26.52万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7071064
• 关键词: DNA binding protein; enzyme activity; genetic recombination; immunoprecipitation; laboratory mouse; ligase; mass spectrometry; nuclear magnetic resonance spectroscopy; posttranslational modifications; protein protein interaction; surface plasmon resonance; ubiquitin; yeast two hybrid system

DESCRIPTION (provided by applicant): `The RAG1 protein is part of a multi-functional complex that carries out V(D)J recombination, a programmed DNA rearrangement that is required for development of the immune system in humans. Either alone or in the context of its recombinase partner RAG2, RAG1 encompasses multiple biochemical activities, including DNA binding, DNA cleavage, and ubiquitin ligation. The working hypothesis of this laboratory is that RAGl's integral ubiquitin ligase (E3) activity works with the ubiquitin conjugation machinery to regulate V(D)J recombination and lymphocyte development. This is based on the observations that 1) the zinc binding RING finger domain of RAG1 acts as a ubiquitin ligase (E3) in vitro and promotes covalent attachment of the small ubiquitin protein to a specific RAG1 lysine residue, 2) RAG1 is ubiquitylated in intact cells, 3) mutation or deletion of the RING finger in model systems leads to altered protein expression and decreased recombination, and 4) mutation of the RING finger or deletion of the region that is ubiquitylated can lead to severe immune deficiency in human patients. The specific hypothesis of this proposal is that RAG1's RING domain interacts with ubiquitin conjugating (E2) enzymes to promote V(D)J recombination. This hypothesis will be tested through a detailed functional and physical analysis of the interaction between the RAG1 RING finger and various E2 enzymes, followed by functional analysis of V(D)J recombination. 1. Test the prediction that the RAG1-E2 binding interface uses structural elements analogous to other RING-E2 pairs. Two independent methods will be used to identify the binding interface. 2. Test the hypothesis that alterations to the zinc binding RING finger domain change the affinity and specificity of RAG1-E2 binding and E3 activity. Multiple independent methods will be used to analyze mutants at the binding interface. 3. Test the hypothesis that RAG1 ubiquitin ligase activity/ubiquitylation promotes V(D)J recombination in cells and that ubiquitylation modulates RAGl's other biochemical activities in vitro. Mutations that diminish E3 activity will be examined in V(D)J recombination and cleavage asays.

描述（由申请人提供）：`rag1蛋白是一种多功能复合物的一部分，该复合物进行了V（d）J重组，这是一种编程的DNA重排，是人类免疫系统开发所需的。 RAG1单独或在其重组酶伴侣RAG2的背景下，包括多种生化活性，包括DNA结合，DNA裂解和泛素连接。该实验室的工作假设是RAGL的积分泛素连接酶（E3）活性与泛素结合机械起作用，以调节V（d）J重组和淋巴细胞的发展。 This is based on the observations that 1) the zinc binding RING finger domain of RAG1 acts as a ubiquitin ligase (E3) in vitro and promotes covalent attachment of the small ubiquitin protein to a specific RAG1 lysine residue, 2) RAG1 is ubiquitylated in intact cells, 3) mutation or deletion of the RING finger in model systems leads to altered protein expression and重组降低，4）环形泛素化区域的无名指或缺失突变会导致人类患者的严重免疫缺陷。该提议的具体假设是Rag1的环域与泛素结合（E2）酶相互作用，以促进V（d）J重组。该假设将通过对RAG1环手指和各种E2酶之间相互作用的详细功能和物理分析进行检验，然后进行V（d）J重组的功能分析。 1。测试RAG1-E2结合界面使用类似于其他环-E2对的结构元件的预测。将使用两种独立的方法来识别绑定界面。 2。检验以下假设：对锌结合环手指结构域的改变改变了RAG1-E2结合和E3活性的亲和力和特异性。多种独立的方法将用于分析结合界面处的突变体。 3。检验以下假设：RAG1泛素连接酶活性/泛素化促进细胞中的V（d）J重组，并且泛素化调节RAGL在体外的其他生化活性。在V（d）J重组和裂解Asay中将检查减少E3活性的突变。