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Nucleic Acids Res ; 47(20): 10815-10829, 2019 11 18.
Article in English | MEDLINE | ID: mdl-31566237

ABSTRACT

Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted.


Subject(s)
Cytidine Deaminase/metabolism , DNA/genetics , Immunoglobulin Variable Region/genetics , RNA Polymerase II/metabolism , Transcription, Genetic , DNA-Directed RNA Polymerases/metabolism , Deamination , HeLa Cells , Humans , Models, Biological , Mutation/genetics , Nuclear Proteins/metabolism , Substrate Specificity , Transcriptional Elongation Factors/metabolism , Viral Proteins/metabolism
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