Activation-induced cytidine deaminase (AID) initiates secondary antibody diversification processes by deaminating cytidines on single-stranded DNA. AID preferentially mutates cytidines preceded by W(A/ T)R(A/G) dinucleotides, a sequence specificity that is evolutionarily conserved from bony fish to humans. To uncover the biochemical mechanism of AID, we compared the catalytic and binding kinetics of AID on WRC (a hot-spot motif, where W equals A or T and R equals A or G) and non-WRC motifs. We show that although purified AID preferentially deaminates WRC over non-WRC motifs to the same degree observed in vivo, it exhibits similar binding affinities to either motif, indicating that its sequence specificity is not due to preferential binding of WRC motifs. AID preferentially deaminates bubble substrates of five to seven nucleotides rather than larger bubbles and preferentially binds to bubble-type rather than to single-stranded DNA substrates, suggesting that the natural targets of AID are either transcription bubbles or stem-loop structures. Importantly, AID displays remarkably high affinity for single-stranded DNA as indicated by the low dissociation constants and long half-life of complex dissociation that are typical of transcription factors and singlestranded DNA binding protein. These findings suggest that AID may persist on immunoglobulin and other target sequences after deamination, possibly acting as a scaffolding protein to recruit other factors.Activation-induced cytidine deaminase (AID) is the B-cellspecific enzyme responsible for the conversion of cytidine to uridine, the initiating event in somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes (20,22,26,29). This initial lesion can either be replicated, producing a transition mutation, or be engaged by the base excision (i.e., UNG) (34) or mismatch repair pathways (i.e., MSH2) (21, 33, 52), resulting in transversion mutations or A-T mutations, respectively. Cytidine deamination by AID also leads to the generation of double-stranded lesions in the switch regions of antibody genes, leading to CSR (9,22,42,43).While necessary for SHM and CSR, AID-induced lesions have been shown to cause chromosomal translocations contributing to lymphoma (13,36,37). Because of this inherently dangerous property of AID, it is likely that the activity of AID is controlled at different levels to focus it on immunoglobulin genes, such as through protein kinase A phosphorylation (2, 27) or the interaction with cofactors, such as replication protein A (RPA) (7). At the target DNA level, high levels of transcription have been shown to be necessary but not sufficient for AID activity (1,22,23,28,38). Since AID is able to deaminate cytidines only on single-stranded DNA (ssDNA) (6,8,10,17,18,25,47), it is likely that the requirement for transcription reflects the generation of single-stranded regions by transcription bubbles (8,22) or the generation of G4 DNA structures (11,12).Even prior to the discovery of AID, it was noted that SHM occurs more freq...