| The immune responses of jawed vertebrates demonstrate a remarkable diversity of antigenic specificity. The molecular basis of this diversity resides in the fact that the antigen-recognition portions of vertebrate immunoglobulin and T-cell receptor genes are assembled from numerous smaller discrete gene elements in semi-random fashion through the process of V(D)J recombination. Recombination is mediated by the products of the recombination activating genes, RAG1 and RAG2, which recognize, bind, and cleave recombination signal sequences (RSSs) in the DNA. Along the recombination pathway a number of distinct RAG-RSS complexes are generated, leading ultimately to formation of a cleavage-competent complex containing RAG proteins bound to a synapsed pair of RSSs. The complexity of the protein-DNA interactions established within these complexes is underscored by the fact that RSSs, despite their small size (28–39 bp) and remarkable sequence heterogeneity, confer a tremendous degree of sequence specificity on the recombination process. To better understand the functional intricacies of protein-DNA interactions within RAG-RSS complexes, my graduate work has focused on V(D)J recombination in the presence of atypical recombination substrates that are normally not recognized or cleaved by the RAG proteins. Two experimental approaches are presented in this dissertation: (1) a genetic screen for RAG1 gain-of-function mutants that recognize altered RSSs with crippling mutations, and (2) a mutational analysis of an inactive RSS, murine Jβ2.6 pseudo-RSS, to identify nucleotide changes required for conversion of this RSS into an active sequence. Also discussed is a third project, more global in scope, which examines the roles of transcription factors in regulating accessibility of the endogenous antigen receptor loci to the recombination machinery. The results of these efforts suggest that V(D)J recombination activity is a summation of numerous intricate functional contributions of individual RSS nucleotides, including positions within the RSS that were previously thought to be unimportant.{09}These interactions modulate the balance between sequence specificity of V(D)J recombination and heterogeneity of recombination signal sequences. |
