B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates from a somatic DNA rearrangement process, called V(D)J recombination, that assembles the antigen receptor genes during lymphocyte development. This process proceeds in two phases: a cleavage phase during which the RAG1 and RAG2 proteins bind and cleave a pair of receptor gene coding segments at specific recombination signal sequences, and a joining phase during which the four DNA ends resulting from RAG-mediated cleavage are reorganized, processed and rejoined via the non-homologous endjoining (NHEJ) repair pathway. While the basic biochemical steps of V(D)J recombination are known, a major gap in knowledge is in our understanding of how the RAG proteins guide the repair of RAG-mediated DNA breaks. We have identified two NHEJ repair factors, Ku70 and Ku80, and components of an E3 ubiquitin ligase complex as interaction partners with full-length RAG1. These findings leads us to hypothesize that the cleavage and joining phases of V(D)J recombination are connected and regulated by a ubiquitin modification pathway. Using biochemical and cellular approaches, as well as animal models, we will test this hypothesis by (i) establishing whether one of the E3 ubiquitin ligase components, called VprBP, is required for B cell development and V(D)J recombination;(ii) defining ubiquitination targets of the RAG-Ku-E3 ligase complex, and determining the functional role of the target proteins and the consequences of target ubiquitination on V(D)J recombination, and (iii) identifying the structural determinants required for maintaining association between the RAG, Ku and E3 ligase components and establishing the requirement for these interactions in V(D)J recombination. Greater knowledge of how the RAG proteins guide the repair of RAG-mediated DNA breaks will improve our understanding of the mechanisms contributing to impaired and aberrant V(D)J recombination that underlie certain forms of immunodeficiency and lymphoid malignancy, respectively.
|Effective start/end date||8/16/11 → 7/31/13|
- National Institutes of Health: $361,250.00
Protein Sorting Signals