Date of Award
12-1-2023
Language
English
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
College/School/Department
Department of Nanoscale Science and Engineering
Dissertation/Thesis Chair
Michael Fasullo
Committee Members
Thomas Begley, Benoit Boivin, Nathaniel Cady, Yubing Xie
Subject Categories
Molecular Biology
Abstract
Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred read meat and cigarette smoke. We hypothesize that HAAs may contribute to red meat being a risk factor for colorectal cancer (CRC). At present, few eukaryotic genes conferring resistance to HAAs have been identified. In this research, the genome of Saccharomyces cerevisiae (budding yeast) was profiled using next-generation sequencing to identify genes conferring resistance to one such HAA, 2-amino-3-methylimidazo[4,5-f] quinoline (IQ).CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. Deletion libraries expressing human CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 μM IQ for 5 or 10 generations. DNA barcodes were sequenced, and statistical significance was determined for exactly matched barcodes. 424 ORFs, including 337 genes of known function, were identified as conferring resistance in duplicate screens of the “humanized” collection for IQ resistance; resistance was further validated for a select group of 51 genes by growth curves, competitive growth, or trypan blue assays. Screens of the library not expressing human genes identified 143 ORFs conferring resistance to IQ per se. Ribosomal protein and protein modification genes were identified as IQ resistance genes in both the original and “humanized” libraries, while nitrogen metabolism, DNA repair, and growth control genes were also prominent in the “humanized” library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. Among genes conferring resistance to activated IQ were eleven genes with human orthologs implicated in colorectal cancer in humans. Among DNA Repair and checkpoint genes, we identified those that function in postreplication repair (RAD18, UBC13, REV7), base excision repair (NTG1), and checkpoint signaling (CHK1, PSY2). Strains deficient in more than one repair pathway (rad4 ntg1 and rad4 rad51 mutants) exhibited far greater sensitivity to IQ than strains deficient in only one pathway. IQ-induced mutagenesis was also measured. Mutagenesis was successfully demonstrated in bacteria using extracts from yeast expressing CYP1A2 and NAT2. IQ-induced recombination was also demonstrated in CYP-expressing yeast strains deficient in base-excision repair, but induction of recombination was only weakly increased above levels seen in base-excision competent strains. Overall, these studies indicate that multiple pathways participate in conferring resistance to IQ and supports the notion that IQ increases risk for CRC.
Recommended Citation
Dolan, Michael J., "Identification Of Genes Conferring Resistance To 2-Amino-3-Methylimidazo [4,5-F] Quinoline Genotoxicity In Budding Yeast Using High-Throughput And Targeted Approaches" (2023). Legacy Theses & Dissertations (2009 - 2024). 3118.
https://scholarsarchive.library.albany.edu/legacy-etd/3118
