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

Exposure to the mycotoxin aflatoxin B1 (AFB(1)) strongly correlates with hepatocellular carcinoma (HCC). P450 enzymes convert AFB(1) into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB(1)-N-7-Gua) DNA adducts, which convert to stable mutagenic AFB(1) formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB(1) is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB(1) resistance. Here, we profiled the yeast genome for AFB(1) resistance. We introduced the human CYP1A2 into similar to 90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 mu M AFB(1) 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 AFB(1) 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 AFB(1)-associated sister chromatid recombination while suppressing AFB(1)-associated mutations. These studies thus broaden the number of AFB(1) resistance genes and have elucidated a mechanism of error-free bypass of AFB(1)-associated DNA adducts.