High affinity antibody responses require secondary immunoglobulin gene (Ig) diversification by somatic hypermutation (SHM) and class switch recombination (CSR) in germinal centers. Both of these reactions are mediated by activation induced cytidine deaminase (AID), an enzyme that deaminates cytosine residues in single-stranded DNA (ssDNA) exposed by stalled RNA polymerase II during transcription (1-3). Although AID has a strong preference for targeting Ig genes, it also mutates a large number of non-Ig loci. AID induced U:G mismatches are processed by one of several repair pathways to yield non-templated mutations or DSBs, which are obligate intermediates in CSR that are normally recognized by DNA damage response proteins and repaired by non-homologous end joining (NHEJ). However, these DSBs can also serve as substrates for chromosome translocations (4-6). 53BP1 is a DNA damage response protein that is recruited to DNA double strand breaks (DSBs) and is essential for their efficient repair. We, and others have shown that 53BP1 contributes to DNA repair during CSR by preventing end resection and by bringing together DSBs separated by as much as 200kb (7-13). 53BP1 is a large protein that is implicated in the etiology of malignancy and that contains a number of domains that are essential to its function in CSR (7, 8). Most importantly, the N terminal domain of 53BP1 is a target for phosphorylation by ataxia telangiectasia mutated (ATM) and is essential for both end protection and synapsis (7, 8), but its function is not known. We have discovered that phosphorylated 53BP1 acts as an adaptor for Rif1 and that this interaction is required for DNA end protection and CSR. The long-term goals of the proposed research are to define the genes that are damaged by AID in germinal center B cells, understand the mechanism of AID targeting, and obtain a molecular understanding of how Rif1 contributes to CSR. To accomplish these goals we propose three specific aims. First, we will define the genes that are damaged by AID and that serve as substrates for translocation in GC B cells in chronic infection using malaria as a model. Second, we will define the mechanism of AID targeting and the role of Spt5. Third, we will define the mechanism by which Rif1 contributes to CSR. Taken together, these experiments should provide an understanding of antibody gene diversification reactions and how they contribute to the genesis of B cell malignancy.
High affinity, AID dependent, antibody responses are essential to nearly all vaccines. The proposed research aims to develop an understanding of how antibodies are modified and repaired after AID mediated DNA damage with the long-term goal of being able to impact vaccine development and prevent the off target effects of AID that are associated with malignancy.
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