Targeting of the activation-induced cytosine deaminase is strongly influenced by the sequence and structure of the targeted DNA.

Activation-induced deaminase (AID) initiates immunoglobulin somatic hypermutation (SHM). Since in vitro AID was shown to deaminate cytosines on single-stranded DNA or the nontranscribed strand, it remained a puzzle how in vivo AID targets both DNA strands equally. Here we investigate the roles of transcription and DNA sequence in cytosine deamination. Strikingly different results are found with different substrates. Depending on the target sequence, the transcribed DNA strand is targeted as well as or better than the nontranscribed strand. The preferential targeting is not related to the frequency of AID hot spots. Comparison of cytosine deamination by AID and bisulfite shows different targeting patterns suggesting that AID may locally unwind the DNA. We conclude that somatic hypermutation on both DNA strands is the natural outcome of AID action on a transcribed gene; furthermore, the DNA sequence or structure and topology play major roles in targeting AID in vitro and in vivo. On the other hand, the lack of mutations in the first approximately 100 nucleotides and beyond about 1 to 2 kb from the promoter of immunoglobulin genes during SHM must be due to special conditions of transcription and chromatin in vivo.