Accurate and Efficient Bypass of 8,5′-Cyclopurine-2′-Deoxynucleosides by Human and Yeast DNA Polymerase η

Reactive oxygen species (ROS), which can be produced during normal aerobic metabolism, can induce the formation of tandem DNA lesions, including 8,5′-cyclo-2′-deoxyadenosine (cyclo-dA) and 8,5′-cyclo-2′-deoxyguanosine (cyclo-dG). Previous studies have shown that cyclo-dA and cyclo-dG accumulate in cells and can block mammalian RNA polymerase II and replicative DNA polymerases. Here, we used primer extension and steady-state kinetic assays to examine the efficiency and fidelity for polymerase η to insert nucleotides opposite, and extend primer past, these cyclopurine lesions. We found that <i>Saccharomyces cerevisiae</i> and human polymerase η inserted 2′-deoxynucleotides opposite cyclo-dA, cyclo-dG and their adjacent 5′ nucleosides at fidelities and efficiencies that were similar to those of their respective undamaged nucleosides. Moreover, the yeast enzyme exhibited similar processivity in DNA synthesis on templates housing a cyclo-dA or cyclo-dG to those carrying an unmodified dA or dG; the human polymerase, however, dissociated from the primer–template complex after inserting one or two additional nucleotides after the lesion. Pol η’s accurate and efficient bypass of cyclo-dA and cyclo-dG indicates that this polymerase is likely responsible for error-free bypass of these lesions, whereas mutagenic bypass of these lesions may involve other translesion synthesis DNA polymerases. Together, our results suggested that pol η may have an additional function in cells, i.e., to alleviate the cellular burden of endogenously induced DNA lesions, including cyclo-dA and cyclo-dG

Quantification of Azaserine-Induced Carboxymethylated and Methylated DNA Lesions in Cells by Nanoflow Liquid Chromatography-Nanoelectrospray Ionization Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method

Humans are exposed to <i>N</i>-nitroso compounds through environmental exposure and endogenous metabolism. Some <i>N</i>-nitroso compounds can be metabolically activated to yield diazoacetate, which is known to induce DNA carboxymethylation. DNA lesion measurement remains one of the core tasks in toxicology and in evaluating human health risks associated with carcinogen exposure. In this study, we developed a highly sensitive nanoflow liquid chromatography-nanoelectrospray ionization-multistage tandem mass spectrometry (nLC-nESI-MS³) method for the simultaneous quantification of <i>O</i>⁶-carboxymethyl-2′-deoxyguanosine (<i>O</i>⁶-CMdG), <i>O</i>⁶-methyl-2′-deoxyguanosine (<i>O</i>⁶-MedG), and <i>N</i>⁶-carboxymethyl-2′-deoxyadenosine (<i>N</i>⁶-CMdA). We were able to measure the levels of these three lesions with the use of low-microgram quantities of DNA from cultured human skin fibroblasts and human colorectal carcinoma cells treated with azaserine, a DNA carboxymethylating agent. Our results revealed that the levels of <i>O</i>⁶-CMdG and <i>O</i>⁶-MedG increased when the dose of azaserine was increased from 0 to 450 μM. We, however, did not observe an apparent dose-dependent induction of <i>N</i>⁶-CMdA, suggesting the presence of repair mechanism(s) for the rapid clearance of this lesion in cells. This is the first report about the application of nLC-nESI-MS³ technique for the simultaneous quantification of <i>O</i>⁶-CMdG, <i>O</i>⁶-MedG, and <i>N</i>⁶-CMdA. The method reported here will be useful for future investigations about the repair of the carboxymethylated DNA lesions and about the implications of these lesions in carcinogenesis

<i>In-Vitro</i> Replication Studies on <i>O</i>²‑Methylthymidine and <i>O</i>⁴‑Methylthymidine

<i>O</i>²- and <i>O</i>⁴-methylthymidine (<i>O</i>²-MdT and <i>O</i>⁴-MdT) can be induced in tissues of laboratory animals exposed with <i>N</i>-methyl-<i>N</i>-nitrosourea, a known carcinogen. These two <i>O</i>-methylated DNA adducts have been shown to be poorly repaired and may contribute to the mutations arising from exposure to DNA methylating agents. Here, <i>in vitro</i> replication studies with duplex DNA substrates containing site-specifically incorporated <i>O</i>²-MdT and <i>O</i>⁴-MdT showed that both lesions blocked DNA synthesis mediated by three different DNA polymerases, including the exonuclease-free Klenow fragment of <i>Escherichia coli</i> DNA polymerase I (Kf^–), human DNA polymerase κ (pol κ), and <i>Saccharomyces cerevisiae</i> 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 <i>O</i>²-MdT, while <i>O</i>⁴-MdT primarily directed dGMP misincorporation. While steady-state kinetic experiments showed that pol κ-mediated nucleotide insertion opposite <i>O</i>²-MdT and <i>O</i>⁴-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 <i>in vitro</i>. In addition, the results from our study revealed that, if left unrepaired, <i>O</i>-methylated thymidine lesions may constitute important sources of nucleobase substitutions emanating from exposure to alkylating agents