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 O2-methylthymidine (O2-Me-dT) and O2-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O2-POB-dT). To investigate the biological effects of these O2-alkylthymidines in humans, we have replicated plasmids containing a site-specifically incorporated O2-Me-dT or O2-POB-dT in human embryonic kidney 293T (HEK293T) cells. The bulkier O2-POB-dT exhibited high genotoxicity and only 26% translesion synthesis (TLS) occurred, while O2-Me-dT was less genotoxic and allowed 55% TLS. However, O2-Me-dT was 20% more mutagenic (mutation frequency (MF) 64%) compared to O2-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 O2-Me-dT and O2-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 O2-Me-dT and O2-POB-dT as the TLS efficiency decreased with knockdown of each pol. In contrast, MF of O2-Me-dT was decreased in pol ζ and Rev1 knockdown cells by 24% and 25%, respectively, while for O2-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.

DNA Polymerases η and ζ Combine to Bypass O2-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens

“…44 These results, along with known reactivities of the enzymes, led to the hypothesis illustrated in Scheme 2 in which pol η is involved in the insertion of dNTPs opposite the adducts, pol ζ is involved in the extension past the adduct, while Rev1 is a structural protein. To test the hypotheses that catalytic activities of pols η and ζ are critical to bypass and mutagenesis of O 2 -POB-dT, we evaluated the in vitro kinetics of insertion of dNTPs opposite and bypass of O 2 -POB-dT by pols η , ι , κ , and ζ .…”

“…42 More recently, the bypass of O 2 -Me-dT and O 2 -POB-dT were evaluated in mammalian cells. 44 Both dATP and dTTP were incorporated opposite the adducts, and pol η , ξ , and REV1 are the primary polymerases involved. To further investigate the mechanisms of mutagenicity of O 2 -POB-dT in humans, we evaluated the in vitro reactivity of O 2 -POB-dT in a defined oligodeoxynucleotide substrate with purified human DNA polymerases η , κ , and ι .…”

Replicative Bypass of O2-Alkylthymidine Lesions in Vitro

“…Pol IV. 28, 29, 36, 39–41 This may be attributed to the fact that, unlike the N 2 -modified dG derivatives which still pair favorably with dCTP, the O 2 -alkyldT lesions do not have strong tendency to form Watson-Crick hydrogen bonding with any of the four canonical nucleotides. 28, 29, 36, 39–41 …”

“…28, 29 Human Pol η is known to accurately bypass thymine-thymine cyclobutane pyrimidine dimers (CPD) with a similar accuracy and efficiency as bypassing the corresponding unmodified nucleosides. 16–21 Pol η is unique among the Y-family polymerases due to its spacious active site which has the ability to accommodate the two cross-linked thymine bases in the CPD lesion.…”

“…27 Recently, our laboratory showed that polymerase V (an ortholog of human polymerase η) plays a major role in the bypass of various O 2 -alkylthymidine lesions in Escherichia coli , and the polymerase was indispensable for the lesion-induced T→A and T→G mutations in E. coli cells. 28 In addition, Basu and coworkers 29 showed that Pol η promotes the replication across the O 2 -MedT and O 2 -POBdT lesions in human cells. Thus, in the present study, we chose to focus on human Pol η by characterizing biochemically how the efficiency and fidelity of nucleotide insertion opposite the O 2 -alkyldT lesions are modulated by the length and structure of the alkyl group.…”