Replication across Regioisomeric Ethylated Thymidine Lesions by Purified DNA Polymerases

Causal links exist between smoking cigarettes and cancer development. Some genotoxic agents in cigarette smoke are capable of alkylating nucleobases in DNA, and higher levels of ethylated DNA lesions were observed in smokers than in nonsmokers. In this study, we examined comprehensively how the regioisomeric O-2-, N3-, and O-4-ethylthymidine (O-2-, N3-, and O-4-EtdT, respectively) perturb DNA replication mediated by purified human DNA polymerases (hPols) eta, kappa, and iota, yeast DNA polymerase zeta (yPol zeta), and the exonuclease-free Klenow fragment (Kf(-)) of Escherichia coli DNA polymerase I. Our results showed that hPol eta and Kf(-) could bypass all three lesions and generate full-length replication products, whereas hPol iota stalled after inserting a single nucleotide opposite the lesions. Bypass conducted by hPol kappa and yPol zeta differed markedly among the three lesions. Consistent with its known ability to efficiently bypass the minor groove N-2-substituted 2'-deoxyguanosine lesions, hPol kappa was able to bypass O-2-EtdT, though it experienced great difficulty in bypassing N3-EtdT and O-4-EtdT. yPol zeta was only modestly blocked by O-4-EtdT, but the polymerase was strongly hindered by O-2-EtdT and N3-EtdT. LC-MS/MS analysis of the replication products revealed that DNA synthesis opposite O-4-EtdT was highly error-prone, with dGMP being preferentially inserted, while the presence of O-2-EtdT and N3-EtdT in template DNA directed substantial frequencies of misincorporation of dGMP and, for hPol iota and Kf(-), dTMP. Thus, our results suggested that O-2-EtdT and N3-EtdT may also contribute to the AT -> TA and AT -> GC mutations observed in cells and tissues of animals exposed to ethylating agents.