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

摘要

titleAbstract/titlepActivation‐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./p