title: Replication across regioisomeric ethylated thymidine lesions by purified DNA polymerases.
abstract: 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) η, κ, and ι, yeast DNA polymerase ζ (yPol ζ), and the exonuclease-free Klenow fragment (Kf(-)) of Escherichia coli DNA polymerase I. Our results showed that hPol η and Kf(-) could bypass all three lesions and generate full-length replication products, whereas hPol ι stalled after inserting a single nucleotide opposite the lesions. Bypass conducted by hPol κ and yPol ζ differed markedly among the three lesions. Consistent with its known ability to efficiently bypass the minor groove N(2)-substituted 2'-deoxyguanosine lesions, hPol κ was able to bypass O(2)-EtdT, though it experienced great difficulty in bypassing N3-EtdT and O(4)-EtdT. yPol ζ 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 ι 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.
authors.affiliation: Department of Chemistry and ‡Environmental Toxicology Graduate Program, University of California , Riverside, California 92521-0403, United States.
authors.forename: Nisana
authors.initials: N
authors.lastname: Andersen
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Wang
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects*; D004269:DNA, Bacterial / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry; D013936:Thymidine / Q000031:analogs & derivatives / Q000032:analysis / Q000633:toxicity*; D000094769:DNA Polymerase iota; D000099270:Y-Family DNA Polymerases
chemical_list: D004247:DNA; D004256:DNA Polymerase I; D017931:DNA Primers; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase; D013936:Thymidine; D000094769:DNA Polymerase iota; C425929:DNA polymerase zeta; C404302:POLK protein, human; D000099270:Y-Family DNA Polymerases; C112338:Rad30 protein