Biology and Biomedical Sciences: Immunology
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
The generation of a functional antigen receptor gene in developing lymphocytes requires that the second exon be assembled through a process known as V(D)J recombination, a process that necessarily involves the generation and repair of DNA double-strand breaks made by the Rag endonuclease. Double strand breaks incurred during G1 of the cell cycle activate ATM, a PI3-kinase-like kinase that, in response to genotoxic DNA damage, is known to phosphorylate hundreds of proteins with unique and diverse functions. Notably, deficiencies in ATM lead to ataxia-telangiectasia, a syndrome characterized by lymphopenia, genomic instability and a predisposition to tumors involving antigen receptor loci suggesting that ATM likely plays a similar critical role downstream of Rag-mediated DNA DSBs during V(D)J recombination. In this body of work, I describe three distinct pathways activated by ATM in response to Rag DNA DSBs and how defects in these pathways would impair lymphocyte development and contribute to the phenotype of ATM-deficient mice and humans. First, ATM-deficient lymphocytes have a defect in the repair of Rag DNA DSBs characterized by an accumulation of unrepaired coding ends and an increase in aberrant hybrid joint formation during rearrangement by inversion. Furthermore, these coding ends drift apart and are frequently aberrantly resolved as translocations. We determine that ATM contributes to the repair of Rag DNA DSBs by maintaining the unrepaired ends in a stable post-cleavage complex possibly through the phosphorylation of components of the MRN complex. Secondly, in G1-phase lymphocytes, the ATM-dependent phosphorylation of histone H2AX inhibits the robust CtIP-dependent opening and resection of hairpin-sealed coding ends thereby preventing their aberrant resolution by alternative repair pathways. Finally, in response to Rag DSBs, through the activation of NFkB and other transcription factors, ATM activates a broad genetic program that transcends the canonical DNA damage response and includes genes whose known functions are integral to lymphocyte development. While this work has significantly increased our understanding of the cellular response to Rag DNA DSBs, given the diversity of the other known substrates of ATM, it seems likely that we have only begun to unravel the complexities of the ATM-deficient phenotype.
Helmink, Beth, "ATM Deficiency -- A Multifaceted Defect in Lymphocyte Development" (2012). All Theses and Dissertations (ETDs). 587.