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To counteract the deleterious effects of DNA damage, cells are equipped with specialized polymerases to bypass DNA lesions. Previous biochemical studies revealed that DinB family DNA polymerases, including Escherichia coli DNA polymerase IV and human DNA polymerase κ, efficiently incorporate the correct nucleotide opposite some N(2)-modified 2'-deoxyguanosine derivatives. Herein, we used shuttle vector technology and demonstrated that deficiency in Polk or Poli in mouse embryonic fibroblast (MEF) cells resulted in elevated frequencies of G→T and G→A mutations at N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) and N(2)-carboxymethyl-2'-deoxyguanosine (N(2)-CMdG) sites. Steady-state kinetic measurements revealed that human DNA polymerase ι preferentially inserts the correct nucleotide, dCMP, opposite N(2)-CEdG lesions. In contrast, no mutation was found after the N(2)-CEdG- and N(2)-CMdG-bearing plasmids were replicated in POLH-deficient human cells or Rev3-deficient MEF cells. Together, our results revealed that, in mammalian cells, both polymerases κ and ι are necessary for the error-free bypass of N(2)-CEdG and N(2)-CMdG. However, in the absence of polymerase κ or ι, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately.

Causallinks exist between smoking cigarettes and cancer development.Some genotoxic agents in cigarette smoke are capable of alkylatingnucleobases in DNA, and higher levels of ethylated DNA lesions wereobserved in smokers than in nonsmokers. In this study, we examinedcomprehensively how the regioisomeric O2-, N3-, and O4-ethylthymidine(O2-, N3-, and O4-EtdT, respectively) perturb DNA replicationmediated by purified human DNA polymerases (hPols) η, κ,and ι, yeast DNA polymerase ζ (yPol ζ), and theexonuclease-free Klenow fragment (Kf–) of Escherichia coliDNA polymerase I. Our results showed thathPol η and Kf–could bypass all three lesionsand generate full-length replication products, whereas hPol ιstalled after inserting a single nucleotide opposite the lesions.Bypass conducted by hPol κ and yPol ζ differed markedlyamong the three lesions. Consistent with its known ability to efficientlybypass the minor groove N2-substituted2′-deoxyguanosine lesions, hPol κ was able to bypass O2-EtdT, though it experienced great difficultyin bypassing N3-EtdT and O4-EtdT. yPol ζ was only modestly blocked by O4-EtdT, but the polymerase was strongly hindered by O2-EtdT and N3-EtdT. LC–MS/MSanalysis of the replication products revealed that DNA synthesis opposite O4-EtdT was highly error-prone, with dGMP beingpreferentially inserted, while the presence of O2-EtdT and N3-EtdT in template DNA directedsubstantial frequencies of misincorporation of dGMP and, for hPolι and Kf–, dTMP. Thus, our results suggestedthat O2-EtdT and N3-EtdTmay also contribute to the AT → TA and AT → GC mutationsobserved in cells and tissues of animals exposed to ethylating agents. [ABSTRACT FROM AUTHOR]

O2- and O4-methylthymidine (O2-MdTand O4-MdT) can be induced in tissuesof laboratoryanimals exposed with N-methyl-N-nitrosourea,a known carcinogen. These two O-methylated DNA adductshave been shown to be poorly repaired and may contribute to the mutationsarising from exposure to DNA methylating agents. Here, invitroreplication studies with duplex DNA substrates containingsite-specifically incorporated O2-MdTand O4-MdT showed that both lesions blockedDNA synthesis mediated by three different DNA polymerases, includingthe exonuclease-free Klenow fragment of Escherichia coliDNA polymerase I (Kf–), human DNA polymerase κ(pol κ), and Saccharomyces cerevisiaeDNA polymeraseη (pol η). Results from steady-state kinetic measurementsand LC-MS/MS analysis of primer extension products revealed that Kf–and pol η preferentially incorporated the correctnucleotide (dAMP) opposite O2-MdT, while O4-MdT primarily directed dGMP misincorporation.While steady-state kinetic experiments showed that pol κ-mediatednucleotide insertion opposite O2-MdT and O4-MdT is highly promiscuous, LC-MS/MS analysisof primer extension products demonstrated that pol κ favorablyincorporated the incorrect dGMP opposite both lesions. Our resultsunderscored the limitation of the steady-state kinetic assay in determininghow DNA lesions compromise DNA replication in vitro. In addition, the results from our study revealed that, if leftunrepaired, O-methylated thymidine lesions may constituteimportant sources of nucleobase substitutions emanating from exposureto alkylating agents. [ABSTRACT FROM AUTHOR]

To counteract the deleterious effects of DNA damage, cells are equipped with specialized polymerases to bypass DNA lesions. Previous biochemical studies revealed that DinB family DNA polymerases, including Escherichia coli DNA polyrnerase IV and human DNA polymerase κ, efficiently incorporate the correct nucleotide opposite some N2-modified 2′-deoxyguanosine derivatives. Herein, we used shuttle vector technology and demonstrated that deficiency in Polkor Poli in mouse embryonic fibroblast (MEF) cells resulted in elevated frequencies of G→T and G→A mutations at N2-(1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) and N2-carboxymethyl-2′-deoxyguanosine (N2-CMdG) sites. Steady-state kinetic measurements revealed that human DNA polymerase ɩ preferentially inserts the correct nucleotide, dCMP, opposite N2-CEdG lesions. In contrast, no mutation was found after the N2-CEdG- and N2-CMdG-bearing plasmids were replicated in POLH-deficient human cells or Rev3-deficient MEF cells. Together, our results revealed that, in mammalian cells, both polymerases κ and ɩ are necessary for the error-free bypass of N2-CEdG and N2-CMdG. However, in the absence of polymerase κ or ɩ, other translesion synthesis polymerase(s) could incorporate nucleotide(s) opposite these lesions but would do so inaccurately. [ABSTRACT FROM AUTHOR]