title: Negative supercoiling creates single-stranded patches of DNA that are substrates for AID-mediated mutagenesis.
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.
authors.affiliation: Department of Immunology, University of Toronto, Toronto, Canada.
authors.forename: Jahan-Yar
authors.initials: JY
authors.lastname: Parsa
authors.forename: Shaliny
authors.initials: S
authors.lastname: Ramachandran
authors.forename: Ahmad
authors.initials: A
authors.lastname: Zaheen
authors.forename: Rajeev M
authors.initials: RM
authors.lastname: Nepal
authors.forename: Anat
authors.initials: A
authors.lastname: Kapelnikov
authors.forename: Antoaneta
authors.initials: A
authors.lastname: Belcheva
authors.forename: Maribel
authors.initials: M
authors.lastname: Berru
authors.forename: Diana
authors.initials: D
authors.lastname: Ronai
authors.forename: Alberto
authors.initials: A
authors.lastname: Martin
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics; D003562:Cytidine / Q000235:genetics / Q000378:metabolism; D003564:Cytidine Deaminase / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics* / Q000378:metabolism; D003641:Deamination; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D017578:Immunoglobulin Class Switching / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism; D027041:Somatic Hypermutation, Immunoglobulin / Q000235:genetics; D013379:Substrate Specificity; D013447:Sulfites / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D004277:DNA, Single-Stranded; D007135:Immunoglobulin Variable Region; D013447:Sulfites; D003562:Cytidine; D004247:DNA; D016914:Ribonuclease H; C078856:ribonuclease HI; D003564:Cytidine Deaminase; C042345:hydrogen sulfite