The role of DNA replication in B cell genome instability

Antibodies form the basis of all long-term serum immunity. Successful immune responses to pathogens, such as viruses and bacteria, nearly always involve the generation of high-affinity antibodies which bind and neutralize the pathogens. The process by which a vast repertoire of antibodies is generated is called antibody diversification and is the focus of the proposed research. Antibody diversification is a unique genomic phenomenon which occurs exclusively at the immunoglobulin (Ig) loci in B lymphocytes. The master regulator of this phenomenon is the enzyme, Activation Induced Deaminase (AID), which introduces mutations at discrete sites within the Ig loci. However, this crucial immunological process is accompanied by collateral damage to the genome because AID is a promiscuous enzyme that can mutate non-Ig genes. This can result in genome instability manifested by the occurrence of deleterious chromosomal translocations that can result in B cell cancers, such as Burkitts lymphoma and diffuse large cell lymphoma. Therefore, it is of vast clinical and biological significance to understand how AID activity and associated genomic processes lead to such malignancies. In this regard, we have recently identified DNA replication as an important player in antibody diversification and preliminary findings clearly demonstrate a role for DNA replication in AID-mediated translocations. Based on this, we hypothesize that DNA replication may have an important role in B cell tumorigenesis. The proposed research aims to address this as follows: 1. Elucidating the DNA replication landscape in B cells undergoing antibody diversification: Here, we will obtain a genome-wide map of where DNA replication initiates in B cells expressing AID and undergoing antibody diversification, and determine how their frequency and distribution are affected upon depletion of replication factors. This will be compared to previously identified sites of AID activity in the genome to determine if replication initiation sites correlate with AID activity. 2. Determining whether DNA replication is required for AID-mediated translocations: In this aim, we will determine if depletion of replication factors alters the frequency and distribution of AID-mediated translocations in B cells. In combination with data from Aim 1 this will allow us to determine if translocations occur at sites of AID activity and/or DNA replication initiation. In sum, we expect these studies to reveal new insights into the role of DNA replication in the genesis of AID-mediated genome instability, which we anticipate will have important implications for B cell lymphomagenesis research as well as the field of genome integrity in general.

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