Somatic Mutation of IG Variable Regions

DESCRIPTION (provided by applicant): A great deal has been learned about the biochemistry of activation induced cytidine deaminase (AID) and the process of somatic hypermutation of antibody variable regions, but the rules that govern the preferential targeting of AID to hot spots within the antibody V regions and the mechanisms that are responsible that targeting are still obscure. Here we propose to use the Ramos Burkitt's lymphoma cell line to examine the role of local DNA sequences and structures in recruiting AID to the variable region. We will also examine whether particular chromatin modifications play a role in targeting AID to different parts of the V region exon and its immediate flanking sequences and compare them to the switch regions. We will use recombinant mediated cassette exchange to manipulated the structure of the variable region in the endogenous heavy chain locus and chromatin immunoprecipitation to identify the chromatin modifications and the sites of interaction of AID with the heavy chain variable region. We will use purified AID and computational tools that we have developed to compare the preferred sites of action of AID in vitro and to those that are targeted in Ramos cells and to a large data base of human V region mutations that have arisen in vivo.