Single-stranded DNA structure and positional context of the target cytidine determine the enzymatic efficiency of AID.
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.
Larijani
Mani
M
Department of Immunology, University of Toronto, Medical Sciences Bldg. 5265, Toronto, Canada M5S 1A8.
Martin
Alberto
A
D020137:Base Pair Mismatch; D001483:Base Sequence; D003562:Cytidine / Q000378:metabolism*; D003564:Cytidine Deaminase / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics; D003641:Deamination; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
D004277:DNA, Single-Stranded; D003562:Cytidine; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase