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

Abstract

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.

Checkpoint Control of DNA Repair in Yeast

“…Interestingly, checkpoint signaling is required for stimulation of both AFB1-associated unequal sister chromatid recombination and mutation [152]. By profiling the yeast genome for AFB1 resistance using next generation sequencing, St. John et al [138] identified both HR genes, including Rad54, Rad55, and Csm2, and those encoding error-prone polymerases. Similar to alkylated induced damage, the Csm2(Shu) complex favors an error-free template switch mechanism [153]; thus, csm2 mutants are deficient in sister chromatid recombination but exhibit higher frequencies of AFB1-associated mutations.…”

“…Genes the confer AFB1 resistance included PSY3, CKB1 and CKB2, which function in DNA damage tolerance [138]. While the genes encoding the CKII substrates, Ptc2 and Ptc3, did not appear in the screen, the identification of both CKII and Pph3 suggest that tolerance to AFB1-associated DNA damage requires both phosphorylation and dephosphorylation of multiple proteins.…”