Role of RAG1 Ubiquitin Ligase Activity in Recombination

Georgetown University, Washington DC

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Abstract

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.