The adaptive immune system requires a large and diverse antigen receptor repertoire in order to protect against infection. Humans maintain ∼108 T cell clones, each with unique receptor specificities, yet the genome contains only ∼20,000 genes. This conundrum is overcome during lymphocyte development through V(D)J recombination, whereby the recombination activating gene (RAG) complex rearranges arrays of V, D, and J gene segments at the T cell receptor (TCR) and immunoglobulin (Ig) loci, generating diverse receptors. Though maximal diversity could be accomplished by random permutations of these genes, they are non-randomly utilized. Segment usage is partly regulated by sequence recognition. The recombination efficiency of the RAG complex fluctuates with alterations in the recombination signal sequences (RSSs) that flag each segment yet also with variations in the coding flank, the sequence of each gene itself adjacent to the RSS. The precise coding flank preferences and physiological implications of such sequence sensitivity have not been thoroughly characterized. Consequently, I systematically investigated the coding flank preferences of the murine RAG complex. I show that the frequency of rearrangement by the RAG proteins varies over a 30-fold range depending on the coding flank and is partially controlled at the DNA cleavage step. Second, since RAG1 mutations near a catalytic residue alter coding flank sensitivity, I examined other critical regions, discovering two new mutants, RAG1 T933A and RAG1 R972Q. I show that R972Q is defective precisely between DNA nicking and double-strand break formation. Finally, I assessed the physiological significance of altered coding flank sensitivity by R972Q, found in Omenn Syndrome, an immunodeficiency combined with allergy and autoimmunity that is characterized by an oligoclonal, restricted T cell repertoire. I found that the coding flanks of murine V, D, and J segments are slightly biased toward preferred sequences and that the R972Q mutant skews TCRβ segment usage in vivo, though additional factors appear to regulate the accessibility of these segments. Based on these data, I propose a novel model for the pathogenesis of OS wherein the altered coding flank sensitivity of R972Q causes immunodeficiency and autoimmunity via restriction and skewing of the antigen receptor repertoire.
|Advisor:||Roth, David B.|
|Commitee:||Dustin, Michael L., Lafaille, Juan J., Littman, Dan R., Sadofsky, Moshe J.|
|School:||New York University|
|Department:||Basic Medical Science|
|School Location:||United States -- New York|
|Source:||DAI-B 70/07, Dissertation Abstracts International|
|Keywords:||Antigen receptor, V(D)J recombination|
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