Investigations into the Targeting and Substrate Specificity of Activation-induced Deaminase

The processes of secondary antibody diversification are initiated by the mutagenic, B cell specific enzyme, Activation-Induced Deaminase (AID). AID deaminates deoxycytosine (dC) that is located in single-stranded DNA (ssDNA) in actively transcribed DNA to initiate the processes of somatic hypermutation (SHM), gene conversion (GCV) and class switch recombination (CSR) at the antibody gene loci. These processes lead to high affinity antibodies and antibodies of various effector functions that are required to efficiently neutralize invading pathogens. It is currently unclear how the antibody genes are specifically targeted by AID over other genes. I found that AID is able to mutate a non-immunoglobulin (Ig) transgene independent of its chromosomal integration site at rates that were above background mutation rates, but were ~10-fold lower than at the antibody variable (V) region. This result suggests that AID can mutate non-Ig genes at low rates, which may explain AID’s role in oncogenesis, but nevertheless shows that AID preferentially mutates the Ig locus over other loci. While it is understood that AID specifically deaminates dC bases in ssDNA, the size, distribution and origin of these ssDNA substrates is unknown. By utilizing a unique in situ sodium bisulfite assay to detect regions of ssDNA in intact nuclei, I characterized ssDNA regions and found that they are accurate predictors of AID activity during the processes of SHM and CSR in mammalian B cells and E.coli. Importantly, with the use of E.coli models, I show that these ssDNA substrates are the product of transcription-induced negative-supercoiled DNA that correlates strongly with the mutagenic activity of AID. While several underlying mechanisms exist to prevent the mistargeting of AID, my findings suggest that by simply gaining access to ssDNA that is produced by transcription-induced negative supercoiling, AID has the potential to mutate non-Ig genes, albeit at lower rates than the antibody V-region.Ph