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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 antibodies1. Purified AID catalyses the deamination of C to U on single-stranded (ss)DNA2,3,4. 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 mutation5,6, and the requirement for active transcription to obtain mutagenesis7,8.

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Figure 1: Processive AID-catalysed C deamination on ssDNA recapitulates SHM hotspots and clonal mutational heterogeneity.
Figure 2: AID-catalysed C → U deamination spectrum.
Figure 3: AID preferentially deaminates C in the non-transcribed strand during in vitro transcription of dsDNA.

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Acknowledgements

This work was supported by National Institutes of Health Grants. P.P. and R.B. were supported on an NIH-NIA training grant. The authors acknowledge the intellectual contributions and encouragement of M. D. Scharff regarding all aspects of this work.

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Correspondence to Myron F. Goodman.

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Pham, P., Bransteitter, R., Petruska, J. et al. Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424, 103–107 (2003). https://doi.org/10.1038/nature01760

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