Exposure to the mycotoxin aflatoxin B1 (AFB₁) strongly correlates with hepatocellular carcinoma (HCC). P450 enzymes convert AFB₁ into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB₁-N⁷-Gua) DNA adducts, which convert to stable mutagenic AFB₁ formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB₁ is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB₁ resistance. Here, we profiled the yeast genome for AFB₁ resistance. We introduced the human CYP1A2 into ∼90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 μM AFB₁ for 20 hs. By using next generation sequencing (NGS) to count molecular barcodes, we initially identified 86 genes from the CYP1A2-expressing libraries, of which 79 were confirmed to confer AFB₁ resistance. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in postreplication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes also included those functioning in checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in postreplication repair, we observed that CSM2, a member of the CSM2(SHU) complex, functioned in AFB₁-associated sister chromatid recombination while suppressing AFB₁-associated mutations. These studies thus broaden the number of AFB₁ resistance genes and have elucidated a mechanism of error-free bypass of AFB₁-associated DNA adducts.

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Journal Article | Research Support, N.I.H., Extramural

Authors

St John N, Freedland J, Baldino H, Doyle F, Cera C, Begley T, Fasullo M

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