Genome Profiling for Aflatoxin B1 Resistance in Saccharomyces cerevisiae Reveals a Role for the CSM2/SHU Complex in Tolerance of Aflatoxin B1-associated DNA Damage

Abstract

Exposure to the mycotoxin aflatoxin B1 (AFB1) strongly correlates with hepatocellular carcinoma. 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 M AFB1 for 20 hs. By using next generation sequencing to count molecular barcodes, we identified 85 AFB1 resistant genes from the CYP1A2-expressing libraries. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in post-replication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes included those functioning in DNA damage tolerance, checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in error-free DNA damage tolerance, 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 resistant genes and have elucidated a mechanism of error-free bypass of AFB1-associated DNA adducts.