title: Roles of translesion synthesis DNA polymerases in the potent mutagenicity of tobacco-specific nitrosamine-derived O2-alkylthymidines in human cells.
abstract: The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent human carcinogen. Metabolic activation of NNK generates a number of DNA adducts including O(2)-methylthymidine (O(2)-Me-dT) and O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT). To investigate the biological effects of these O(2)-alkylthymidines in humans, we have replicated plasmids containing a site-specifically incorporated O(2)-Me-dT or O(2)-POB-dT in human embryonic kidney 293T (HEK293T) cells. The bulkier O(2)-POB-dT exhibited high genotoxicity and only 26% translesion synthesis (TLS) occurred, while O(2)-Me-dT was less genotoxic and allowed 55% TLS. However, O(2)-Me-dT was 20% more mutagenic (mutation frequency (MF) 64%) compared to O(2)-POB-dT (MF 53%) in HEK293T cells. The major type of mutations in each case was targeted T → A transversions (56% and 47%, respectively, for O(2)-Me-dT and O(2)-POB-dT). Both lesions induced a much lower frequency of T → G, the dominant mutation in bacteria. siRNA knockdown of the TLS polymerases (pols) indicated that pol η, pol ζ, and Rev1 are involved in the lesion bypass of O(2)-Me-dT and O(2)-POB-dT as the TLS efficiency decreased with knockdown of each pol. In contrast, MF of O(2)-Me-dT was decreased in pol ζ and Rev1 knockdown cells by 24% and 25%, respectively, while for O(2)-POB-dT, it was decreased by 44% in pol ζ knockdown cells, indicating that these TLS pols are critical for mutagenesis. Additional decrease in both TLS efficiency and MF was observed in cells deficient in pol ζ plus other Y-family pols. This study provided important mechanistic details on how these lesions are bypassed in human cells in both error-free and error-prone manner.
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, CT 06269, United States.
authors.forename: Savithri
authors.initials: S
authors.lastname: Weerasooriya
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, CT 06269, United States.
authors.forename: Vijay P
authors.initials: VP
authors.lastname: Jasti
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, CT 06269, United States.
authors.forename: Arindam
authors.initials: A
authors.lastname: Bose
authors.affiliation: Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, PA 17033, United States.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Spratt
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, CT 06269, United States. Electronic address: ashis.basu@uconn.edu.
authors.forename: Ashis K
authors.initials: AK
authors.lastname: Basu
mesh_terms: D002273:Carcinogens / Q000633:toxicity*; D018736:DNA Adducts / Q000138:chemical synthesis; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000502:physiology*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis / Q000187:drug effects; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D009602:Nitrosamines / Q000633:toxicity*; D034741:RNA, Small Interfering / Q000235:genetics; D014026:Nicotiana / Q000737:chemistry*
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D009153:Mutagens; D009602:Nitrosamines; D034741:RNA, Small Interfering; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D004259:DNA-Directed DNA Polymerase
keywords: HEK293T; Mutagenesis; NNK; O(2)-Methylthymidine; O(2)-POB-thymidine; TLS polymerase; Tobacco-specific nitrosamine; siRNA knockdown