The aflatoxin B(1)-induced formamidopyrimidine adduct is repaired by transcription-coupled nucleotide excision repair in human cells.

The mycotoxin, aflatoxin B(1) (AFB(1)), is a potent mutagen that contaminates agricultural food supplies. After ingestion, AFB(1) is oxidized into a reactive electrophile that alkylates DNA, forming bulky lesions such as the genotoxic formamidopyrimidine lesion, AFB(1)-Fapy dG. This lesion is mainly repaired by nucleotide excision repair (NER) in bacteria; however, in humans the picture is less clear. We report a plasmid-based host cell reactivation assay containing a site-specific AFB(1)-Fapy dG lesion and present evidence that this lesion is mainly repaired by transcription-coupled NER (TC-NER) in human cells. Using a combination of isogenic knockout cell lines and immortalized fibroblasts from xeroderma pigmentosum and Cockayne syndrome patients, we show that the TC-NER factors CSA, CSB, and UVSSA are required for efficient AFB(1)-Fapy dG repair, while the global-genome NER protein, XPC, is dispensable. Furthermore, knockout of CSB or UVSSA impairs AFB(1)-Fapy dG repair to a similar degree as knockout of the core NER nuclease, XPF. Our data indicate that TC-NER is the major repair pathway for AFB(1)-Fapy dG adducts in human cells.