Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation

Abstract

Activation-induced cytidine deaminase (AID) is a protein required for B cells to undergo class switch recombination and somatic hypermutation (SHM)--two processes essential for producing high-affinity antibodies. Purified AID catalyses the deamination of C to U on single-stranded (ss)DNA. Here, we show in vitro that AID-catalysed C deaminations occur preferentially on 5' WRC sequences in accord with SHM spectra observed in vivo. Although about 98% of DNA clones suffer no mutations, most of the remaining mutated clones have 10-70 C to T transitions per clone. Therefore, AID carries out multiple C deaminations on individual DNA strands, rather than jumping from one strand to another. The avid binding of AID to ssDNA could result from its large net positive charge (+11) at pH 7.0, owing to a basic amino-terminal domain enriched in arginine and lysine. Furthermore, AID exhibits a 15-fold preference for C deamination on the non-transcribed DNA strand exposed by RNA polymerase than the transcribed strand protected as a RNA-DNA hybrid. These deamination results on ssDNA bear relevance to three characteristic features of SHM: preferential mutation at C sites within WRC hotspot sequences, the broad clonal mutagenic heterogeneity of antibody variable regions targeted for mutation, and the requirement for active transcription to obtain mutagenesis.