Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli . Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID. Author Summary Creating an effective antibody-mediated immune response relies on processes that create antibodies of high affinity and of different functions in order to clear pathogens. Activation-induced cytidine deaminase (AID) is an essential B cell–specific factor that is known to initiate these processes by deaminating dC on single-stranded DNA of actively transcribed genes. AID has also been implicated in deaminating dC at non-antibody genes, resulting in the disregulation of genes that may lead to B cell–related cancers. Until now, it has remained unknown what the source, structure, and distribution of the single-stranded DNA is that AID acts upon. By using a novel assay that allows direct detection of single-stranded DNA within intact cell nuclei, we observed patches of single-stranded DNA that are strongly correlated to the preferred activity of AID. Furthermore, we find that the activity of AID and single-stranded DNA patch formation can be enhanced by negative supercoiling of the DNA, which is a typical consequence of transcription. These findings allow us to better understand how AID is recruited to and mutates antibody genes as well as other genes implicated in cancers of B cell origin.
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