Abstract
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.
Highlights
The generation of high affinity antibodies is an important feature of the adaptive immune response to 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
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
Summary
The generation of high affinity antibodies is an important feature of the adaptive immune response to pathogens. The iterative process of somatic hypermutation (SHM) introduces mutations into the DNA encoding the variable (V) region of the antibody molecule that confer higher affinity for pathogen-derived antigen. Immunoglobulin (Ig) genes undergo class switch recombination (CSR), which creates antibodies of different isotypes with distinct effector functions. The importance of these secondary diversification processes is demonstrated in hyper-IgM type II patients that lack these diversification processes and are immunocompromised [1]. Through in vitro SHM studies, the RNA exosome, a complex of factors that specialize in an array of diverse RNA processing events, has been shown to direct AID to both template and non-template strands of DNA [4]. The role of transcription in SHM has long been suspected [5,6,7,8,9]
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