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 mM 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.

Exposure to the mycotoxin aflatoxin B1 (AFB 1 ) strongly correlates with hepatocellular carcinoma. 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 ~90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 M AFB 1 for 20 hs. By using next generation sequencing to count molecular barcodes, we identified 85 AFB 1 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 AFB 1 -associated sister chromatid recombination while suppressing AFB 1 -associated mutations. These studies thus broaden the number of AFB 1 resistant genes and have elucidated a mechanism of error-free bypass of AFB 1associated DNA adducts.

Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cigarette smoke. However, few eukaryotic resistance genes have been identified. We usedSaccharomyces cerevisiae(budding yeast) to identify genes that confer resistance to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. We introduced an expression vector that contains human CYP1A2 and NAT2 genes into selected mutant strains and the diploid yeast deletion collection. The deletion libraries expressing CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 M IQ for five or ten generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. Four screens for IQ resistance in the humanized collection identified 1160 unique ORFs, of which 337 were validated or duplicated in at least two screens. Two screens of the original yeast library identified 101 genes that overlap with the 337 previously identified. Selected genes were validated by growth curves, competitive growth assays, or trypan blue assays. Prominent among both sets are ribosomal protein genes, while nitrogen metabolism, cell wall synthesis, and phosphatase genes were identified among the humanized library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. DNA repair genes includedNTG1,RAD18, RAD9, PSY2andUBC13. Polymorphisms in human NTHL1, theNTG1ortholog, andRAD18are risk factors for colon cancer. These studies thus provoke questions of whether genetic risk factors for colon cancer confer more HAA-associated toxicity.