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In vivo detection and replication studies of ?-anomeric lesions of 2'-deoxyribonucleosides.

ABSTRACT: DNA damage, arising from endogenous metabolism or exposure to environmental agents, may perturb the transmission of genetic information by blocking DNA replication and/or inducing mutations, which contribute to the development of cancer and likely other human diseases. Hydroxyl radical attack on the C1', C3' and C4' of 2-deoxyribose can give rise to epimeric 2-deoxyribose lesions, for which the in vivo occurrence and biological consequences remain largely unexplored. Through independent chemical syntheses of all three epimeric lesions of 2'-deoxyguanosine (dG) and liquid chromatography-tandem mass spectrometry analysis, we demonstrated unambiguously the presence of substantial levels of the ?-anomer of dG (?-dG) in calf thymus DNA and in DNA isolated from mouse pancreatic tissues. We further assessed quantitatively the impact of all four ?-dN lesions on DNA replication in Escherichia coli by employing a shuttle-vector method. We found that, without SOS induction, all ?-dN lesions except ?-dA strongly blocked DNA replication and, while replication across ?-dA was error-free, replicative bypass of ?-dC and ?-dG yielded mainly C?A and G?A mutations. In addition, SOS induction could lead to markedly elevated bypass efficiencies for the four ?-dN lesions, abolished the G?A mutation for ?-dG, pronouncedly reduced the C?A mutation for ?-dC and triggered T?A mutation for ?-dT. The preferential misincorporation of dTMP opposite the ?-dNs could be attributed to the unique base-pairing properties of the nucleobases elicited by the inversion of the configuration of the N-glycosidic linkage. Our results also revealed that Pol V played a major role in bypassing ?-dC, ?-dG and ?-dT in vivo. The abundance of ?-dG in mammalian tissue and the impact of the ?-dNs on DNA replication demonstrate for the first time the biological significance of this family of DNA lesions.

SUBMITTER: Amato NJ

PROVIDER: S-EPMC4787794 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 p53 mutations similar to those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase ?-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 this study, we examined how carboxymethylated DNA lesions, which included N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA), N(4)-carboxymethyl-2'-deoxycytidine (N(4)-CMdC), N3-carboxymethylthymidine (N3-CMdT), and O(4)-carboxymethylthymidine (O(4)-CMdT), perturbed the efficiency and fidelity of DNA replication mediated by Saccharomyces cerevisiae polymerase ? (pol ?). Our results from steady-state kinetic assay showed that pol ? could readily bypass and extend past N(6)-CMdA and incorporated the correct nucleotides opposite the lesion and its neighboring 5'-nucleoside with high efficiency. By contrast, the polymerase could bypass N(4)-CMdC inefficiently, with substantial misincorporation of dCMP followed by dAMP, though pol ? could extend past the lesion with high fidelity and efficiency when dGMP was incorporated opposite the lesion. On the other hand, yeast pol ? experienced great difficulty in bypassing O(4)-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. These results suggest that these lesions may contribute significantly to diazoacetate-induced mutations and those in the p53 gene observed in human gastrointestinal tumors.

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In vivo detection and replication studies of ?-anomeric lesions of 2'-deoxyribonucleosides.

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