Dataset Information

Replicative Bypass Studies of ?-Anomeric Lesions of 2'-Deoxyribonucleosides in Vitro.

ABSTRACT: Genomic integrity is constantly challenged by a variety of endogenous and exogenous DNA damaging agents, which can lead to the formation of 104-105 DNA lesions per cell per day. Reactive oxygen species (ROS) represent a major type of DNA damaging agent. Specifically, a hydroxyl radical can attack the C1' position of 2-deoxyribose, and the ensuing carbon-centered radical, if improperly repaired, can cause the inversion of stereochemical configuration at the C1' to give ?-anomeric lesions. In this study, we assessed the replicative bypass of ?-dA, ?-dT, ?-dC, and ?-dG in template DNA by conducting primer extension assays with the use of purified translesion synthesis DNA polymerases. Our results revealed that human polymerase (Pol) ?, but not human Pol ?, Pol ?, or yeast Pol ?, was capable of bypassing all of the ?-dN lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol ? was the most efficient in inserting the correct nucleotides opposite the modified nucleosides, with the relative efficiencies of nucleotide incorporation following the order of ?-dA > ?-dG > ?-dT > ?-dC. Additionally, human Pol ? was found to misincorporate dTMP opposite ?-dT and dCMP opposite ?-dC at frequencies of 66% and 24%, respectively, whereas ?-dA and ?-dG were weakly miscoding. These findings provided important knowledge about the effects these ?-dN lesions have on the fidelity and efficiency of DNA replication mediated by human Pol ?.

SUBMITTER: Williams NL

PROVIDER: S-EPMC5614518 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Replicative Bypass Studies of α-Anomeric Lesions of 2'-Deoxyribonucleosides in Vitro.

Williams Nicole L NL Amato Nicholas J NJ Wang Yinsheng Y

Chemical research in toxicology 20170414 5

Genomic integrity is constantly challenged by a variety of endogenous and exogenous DNA damaging agents, which can lead to the formation of 10<sup>4</sup>-10<sup>5</sup> DNA lesions per cell per day. Reactive oxygen species (ROS) represent a major type of DNA damaging agent. Specifically, a hydroxyl radical can attack the C1' position of 2-deoxyribose, and the ensuing carbon-centered radical, if improperly repaired, can cause the inversion of stereochemical configuration at the C1' to give α-ano ...[more]

PMID: 28388097

Similar Datasets

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.

Dataset Information

Replicative Bypass Studies of ?-Anomeric Lesions of 2'-Deoxyribonucleosides in Vitro.

Publications

Replicative Bypass Studies of α-Anomeric Lesions of 2'-Deoxyribonucleosides in Vitro.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets