Date of Award
Doctor of Philosophy (PhD)
Immunoglobulin and T cell receptor gene assembly depend on V(D)J recombination initiated by the RAG1-RAG2 recombinase. The RAG1 N-terminal region (NTR; aa 1-383) has been implicated in regulatory functions whose influence on V(D)J recombination and lymphocyte development in vivo are poorly understood. We generated mouse strains in which RAG1 lacks its first 215 residues (215), E3 ubiquitin ligase activity (P326G), or the major site of autoubiquitination (K233R). RAG1 protein levels are increased in all three mutants and RAG1-P326G B cells exhibit increased cell death during development and an increased Ig:Ig ratio. Previous studies have demonstrated that recombination of the immunoglobulin heavy chain gene (Igh) occurs by two distinct reaction modes: long-range, cohesin-dependent scanning and short-range collision. We find that RAG1215 mice exhibit reduced short-range Igh D-to-J recombination. In addition, we find the V gene segment repertoire is altered in RAG1215 mice at the Igh and Ig loci, while loss of integrity of the E3 ubiquitin ligase domain leads to a decrease in out-of-frame recombination events at the Igh locus. We also demonstrate preliminary findings indicating that off-target recombination events may also be dictated by the same mechanisms of short- and long-range recombination. Our findings indicate that the RAG1 NTR regulates V(D)J recombination and lymphocyte development by several mechanisms, including a multifaceted control of the balance between short- and long-range recombination.
Beilinson, Helen Alexander, "The Role of the RAG1 Non-Core Domain in V(D)J Recombination and Lymphocyte Development" (2021). Yale Graduate School of Arts and Sciences Dissertations. 11.