Single-Stranded DNA Structure and Positional Context of the Target Cytidine Determine the Enzymatic Efficiency of AID

Abstract

Activation-induced cytidine deaminase (AID) initiates antibody diversification processes by deaminating immunoglobulin sequences. Since transcription of target genes is required for deamination in vivo and AID exclusively mutates single-stranded DNA (ssDNA) in vitro, AID has been postulated to mutate transcription bubbles. However, since ssDNA generated by transcription can assume multiple structures, it is unknown which of these are targeted in vivo. Here we examine the enzymatic and binding properties of AID for different DNA structures. We report that AID has minimal activity on stem-loop structures and preferentially deaminates five-nucleotide bubbles. We compared AID activity on cytidines placed at various distances from the single-stranded/double-stranded DNA junction of bubble substrates and found that the optimal target consists of a single-stranded NWRCN motif. We also show that high-affinity binding is required for but does not necessarily lead to efficient deamination. Using nucleotide analogues, we show that AID's WRC preference (W = A or T; R = A or G) involves the recognition of a purine in the R position and that the carbonyl or amino side chains of guanosine negatively influence specificity at the W position. Our results indicate that AID is likely to target short-tract regions of ssDNA produced by transcription elongation and that it requires a fully single-stranded WRC motif.

We are grateful to members of the Martin lab and Marc Shulman, Gillian Wu, and David Pulleyblank for helpful discussions and to Oxana Kolenchenko, Maribel Berru, and Yin Ying Zhang for technical help.

This research was funded by a grant from the Canadian Institute of Health Research to A.M., who is supported by a Canada Research Chair award. M.L. was supported by the David Rae Memorial Award from the Leukemia and Lymphoma Society of Canada and is presently supported by a Terry Fox Foundation award from the National Cancer Institute of Canada.