Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, O4-alkylthymidine (O4-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various O4-alkyldT lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, (R)-sBu, or (S)-sBu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all O4-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite O4-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite O4-iPrdT, O4-iBudT, and O4-(R)-sBudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in O4-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

O2- and O4-methylthymidine (O2-MdT and O4-MdT) can be induced in tissues of laboratory animals exposed with N-methyl-N-nitrosourea, a known carcinogen. These two O-methylated DNA adducts have been shown to be poorly repaired and may contribute to the mutations arising from exposure to DNA methylating agents. Here, in vitro replication studies with duplex DNA substrates containing site-specifically incorporated O2-MdT and O4-MdT showed that both lesions blocked DNA synthesis mediated by three different DNA polymerases, including the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (Kf–), human DNA polymerase κ (pol κ), and Saccharomyces cerevisiae DNA polymerase η (pol η). Results from steady-state kinetic measurements and LC-MS/MS analysis of primer extension products revealed that Kf– and pol η preferentially incorporated the correct nucleotide (dAMP) opposite O2-MdT, while O4-MdT primarily directed dGMP misincorporation. While steady-state kinetic experiments showed that pol κ-mediated nucleotide insertion opposite O2-MdT and O4-MdT is highly promiscuous, LC-MS/MS analysis of primer extension products demonstrated that pol κ favorably incorporated the incorrect dGMP opposite both lesions. Our results underscored the limitation of the steady-state kinetic assay in determining how DNA lesions compromise DNA replication in vitro. In addition, the results from our study revealed that, if left unrepaired, O-methylated thymidine lesions may constitute important sources of nucleobase substitutions emanating from exposure to alkylating agents.