Background:The 8,5Ј-cyclopurine-2Ј-deoxynucleosides (cPus) are important types of oxidative DNA damage. Results: cPus exhibit both inhibitory and mutagenic effects on replication; polymerases (Pols) , , and are involved in translesion synthesis of these lesions. Conclusion: Pols , , and cooperatively promote translesion synthesis across cPu lesions. Significance: This work revealed the effects of cPus on DNA replication in mammalian cells.

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 Saccharomyces cerevisiae and human polymerase η inserted 2'-deoxynucleotides opposite cyclo-dA, cyclo-dG, and their adjacent 5' nucleosides at fidelities and efficiencies that were similar as their respective undamaged nucleosides. Moreover, the yeast enzyme exhibited similar processivity in DNA synthesis on templates housing a cyclo-dA or cyclo-dG as 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 indicate 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.

Humans are exposed to N-nitroso compounds (NOCs) both endogenously and exogenously from a number of environmental sources, and NOCs are both mutagenic and carcinogenic. After metabolic activation, some NOCs can induce carboxymethylation of nucleobases through a diazoacetate intermediate, which could give rise to similar p53 mutations as those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase η-deficient human cells was inhibited by treatment with azaserine, a DNA carboxymethylation agent, suggesting the importance of this polymerase in bypassing the azaserine-induced carboxymethylated DNA lesions. In the present study, we examined how carboxymethylated DNA lesions, which included N6-carboxymethyl-2′-deoxyadenosine (N6-CMdA), N4-carboxymethyl-2′-deoxycytidine (N 4-CMdC), N3-carboxymethylthymidine (N3-CMdT) and O4-carboxymethylthymidine (O4-CMdT), perturbed the efficiency and fidelity of DNA replication mediated by Saccharomyces cerevisiae polymerase η (pol η). Our results from steady-state kinetic assay showed that pol η could readily bypass and extend past N6-CMdA, and incorporated the correct nucleotides opposite the lesion and its neighboring 5′ nucleoside with high efficiency. By contrast, the polymerase could bypass N4-CMdC inefficiently, with substantial misincorporation of dCMP followed by dAMP, though pol η could extend past the lesion with high fidelity and efficiency when dGMP was incorporated opposite the lesion. On the other hand, yeast pol η experienced great difficulty in bypassing O4-CMdT and N3-CMdT and the polymerase inserted preferentially the incorrect dGMP opposite these two DNA lesions; the extension step, nevertheless, occurred with high fidelity and efficiency when the correct dAMP was opposite the lesion, as opposed to the preferentially incorporated incorrect dGMP. The above results suggest that these lesions may contribute significantly to diazoacetate-induced mutations and those in p53 gene observed in human gastrointestinal tumors.