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.
V(D)J recombination is a DNA rearrangement process that is responsible for generating a diverse population of B and T cells that together provide a highly specific and adaptive immunity to pathogenic microorganisms. V(D)J recombination is both essential and potentially dangerous to the host: defects in V(D)J recombination account for about 30% of human cases of severe combined immunodeficiency, yet certain forms of leukemia and lymphoma frequently bear molecular signatures implicating an aberrant V(D)J recombination event in their genesis. The research proposed here will investigate the biochemical mechanisms of V(D)J recombination, helping to improve our understanding of which steps of V(D)J recombination represent critical crossroads between appropriate, inappropriate or nonfunctional rearrangement outcomes that contribute to the development of a normal immune system, lymphoid malignancy, or immunodeficiency, respectively.
| Palmer, Victoria L; Aziz-Seible, Razia; Kassmeier, Michele D et al. (2015) VprBP Is Required for Efficient Editing and Selection of Ig?+ B Cells, but Is Dispensable for Ig?+ and Marginal Zone B Cell Maturation and Selection. J Immunol 195:1524-37 |
| Kassmeier, Michele D; Mondal, Koushik; Palmer, Victoria L et al. (2012) VprBP binds full-length RAG1 and is required for B-cell development and V(D)J recombination fidelity. EMBO J 31:945-58 |
