The synthesis of high affinity antibodies requires activation-induced cytidine deaminase (AID) to initiate somatic hypermutation (SHM), targeting the variable region of immunoglobulin genes, and class-switch recombination (CSR). We have investigated AID-catlyzed deamination of C → U on single-stranded DNA and actively transcribed closed double-stranded DNA. Mutations are initially favored at canonical WRC (W = A or T, R = A or G) SHM hotspot motifs, but over time mutations at neighboring non-hotspot sites increase creating random clusters of mutated regions, in a seemingly processive manner. N-terminal AID mutants and appear less processive and have altered mutational specificity compared to wild type AID. In contrast, a C-terminal deletion mutant defective in CSR in vivo, but normal for SHM, closely resembles wild type AID. A mutational spectrum generated during transcription of closed circular double-stranded DNA indicates that AID retains its specificity for WRC hotspot motifs within the confines of a moving transcription bubble while introducing clusters of multiple deaminations predominantly on the non-transcribed strand. A model involving the discrete action of dimeric AID subunits is proposed to account for the accumulation of random clusters of mutations.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]