Activation-induced cytidine deaminase can target multiple topologies of double-stranded DNA in a transcription-independent manner

FASEB J. 2020 Jul;34(7):9245-9268. doi: 10.1096/fj.201903036RR. Epub 2020 May 21.


Activation-induced cytidine deaminase (AID) mutates immunoglobulin genes and acts genome-wide. AID targets robustly transcribed genes, and purified AID acts on single-stranded (ss) but not double-stranded (ds) DNA oligonucleotides. Thus, it is believed that transcription is the generator of ssDNA for AID. Previous cell-free studies examining the relationship between transcription and AID targeting have employed a bacterial colony count assay wherein AID reverts an antibiotic resistance stop codon in plasmid substrates, leading to colony formation. Here, we established a novel assay where kb-long dsDNA of varying topologies is incubated with AID, with or without transcription, followed by direct sequencing. This assay allows for an unselected and in-depth comparison of mutation frequency and pattern of AID targeting in the absence of transcription or across a range of transcription dynamics. We found that without transcription, AID targets breathing ssDNA in supercoiled and, to a lesser extent, in relaxed dsDNA. The most optimal transcription only modestly enhanced AID action on supercoiled dsDNA in a manner dependent on RNA polymerase speed. These data suggest that the correlation between transcription and AID targeting may reflect transcription leading to AID-accessible breathing ssDNA patches naturally occurring in de-chromatinized dsDNA, as much as being due to transcription directly generating ssDNA.

Keywords: RNA polymerase; breathing DNA structures; cytidine deamination; genome mutagenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cytidine Deaminase / genetics
  • Cytidine Deaminase / metabolism*
  • DNA / chemistry*
  • DNA / genetics
  • DNA / metabolism
  • DNA, Single-Stranded / chemistry*
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • Humans
  • Plasmids / chemistry
  • Plasmids / genetics*
  • Plasmids / metabolism
  • Substrate Specificity
  • Transcription, Genetic*


  • DNA, Single-Stranded
  • DNA
  • Cytidine Deaminase