Date of Award

Spring 4-1-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Immunobiology

First Advisor

Schatz, David

Abstract

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 RAG1215 mice exhibit reduced short-range Igh D-to-J recombination. In addition, we find the V gene segment repertoire is altered in RAG1215 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.

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