The ATM kinase is central to the DNA damage response; it follows that this large kinase is central to regulating how developing lymphocytes respond to their self-imposed DNA damage during VDJ recombination. Although the evidence is overwhelming and unequivocal that ATM contributes to accurate non-homologous end joining of VDJ coding segments, and restrains their participation in genomic translocations, a clear mechanistic understanding of how ATM actually does this job is lacking. Recently, we found that ATM ablation in many cultured cell strains results in increased VDJ joining in episomal assays, a completely counter-intuitive result if ATM were to have a direct functional role in end joining. We considered that if ATM's role was instead, to regulate the RAG post cleavage complex(s), loss of ATM might result in increased release of VDJ recombination intermediates and more rapid joining, explaining the increased recombination observed. We fine-tuned the assay so that a structure/function, reductionist approach could be employed to delineate if and how ATM directly affects the RAG complex. We find that ATM inhibition of signal joining requires the non-core C-termini of both RAG1 and RAG2. This prompted an examination of these regions for potential ATM/DNA-PK target sites. Blocking these sites not only blocks ATM's effect on signal joining in cellular assays, it also ablates robust phosphorylation of RAG1/RAG2 on highly purified and fully functional RAG complexes by ATM and DNA-PK in vitro. These data support our model that ATM directly regulates VDJ recombination by phosphorylation of the RAG complex. The experiments proposed in this application will use biochemical approaches to define how ATM affects cleavage and signal end release, and will use a cellular model of chromosomal VDJ recombination to further define how ATM regulates VDJ recombination.
B and T lymphocytes utilize special receptors to recognize pathogens called antibodies and T cell receptors; genes encoding these receptors do not exist in our genome as functional genes, but are assembled from small gene segments during lymphocyte development by a process called VDJ recombination. However, since this process requires breaking and repairing DNA, mistakes can occur that sometimes result in deleterious genome alterations, which have been shown to promote the development of lymphoid cancers. This application aims to elucidate how VDJ recombination is regulated so that these potentially cancer causing mistakes do not occur.
