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A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition.

ABSTRACT: 4,6-Diamino-5-formamidopyrimidine (Fapy•dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapy•dG is in its nucleotide triphosphate form, Fapy•dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapy•dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymerases. Here, we evaluated insertion kinetics and high-resolution ternary complex crystal structures of a configurationally stable Fapy•dGTP analog, ?-C-Fapy•dGTP, with DNA polymerase ?. The crystallographic snapshots and kinetic data indicate that binding of ?-C-Fapy•dGTP impedes enzyme closure, thus hindering insertion. The structures reveal that an active site residue, Asp276, positions ?-C-Fapy•dGTP so that it distorts the geometry of critical catalytic atoms. Removal of this guardian side chain permits enzyme closure and increases the efficiency of ?-C-Fapy•dG insertion opposite dC. These results highlight the stringent requirements necessary to achieve a closed DNA polymerase active site poised for efficient nucleotide incorporation and illustrate how DNA polymerase ? has evolved to hinder Fapy•dGTP insertion.

SUBMITTER: Smith MR

PROVIDER: S-EPMC6451102 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition.

Smith Mallory R MR Shock David D DD Beard William A WA Greenberg Marc M MM Freudenthal Bret D BD Wilson Samuel H SH

Nucleic acids research 20190401 6

4,6-Diamino-5-formamidopyrimidine (Fapy•dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapy•dG is in its nucleotide triphosphate form, Fapy•dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapy•dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymer ...[more]

PMID: 30649431

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Project description:Postovulatory aging of the mammalian oocytes causes deterioration of oocytes through several factors including oxidative stress. Keeping that in mind, we aimed to investigate the potential of a well-known antioxidant, resveratrol (RV), to evaluate the adverse effects of postovulatory aging in porcine oocytes. After in vitro maturation (IVM), a group of (25–30) oocytes (in three replicates) were exposed to 0, 1, 2, and 4 μmol/L of RV, respectively. The results revealed that the first polar body (PB1) extrusion rate of the oocytes significantly increased when the RV concentration reached up to 2 μmol/L (p < 0.05). Considering optimum RV concentration of 2 μmol/L, the potential of RV was evaluated in oocytes aged for 24 and 48 h. We used fluorescence microscopy to detect the relative level of reactive oxygen species (ROS), while GHS contents were measured through the enzymatic method. Our results revealed that aged groups (24 h and 48 h) treated with RV (2 μmol/L) showed higher (p < 0.05) ROS fluorescence intensity than the control group, but lower (p < 0.05) than untreated aged groups. The GSH content in untreated aged groups (24 h and 48 h) was lower (p < 0.05) than RV-treated groups, but both groups showed higher levels than the control. Similarly, the relative expression of the genes involved in antioxidant activity (CAT, GPXGSH-Px, and SOD1) in RV-treated groups was lower (p < 0.05) as compared to the control group but higher than that of untreated aged groups. Moreover, the relative mRNA expression of caspase-3 and Bax in RV-treated groups was higher (p < 0.05) than the control group but lower than untreated groups. Furthermore, the expression of Bcl-2 in the RV-treated group was significantly lower than control but higher than untreated aged groups. Taken together, our findings revealed that the RV can increase the expression of antioxidant genes by decreasing the level of ROS, and its potent antiapoptotic effects resisted against the decline in mitochondrial membrane potential in aged oocytes.

Dataset Information

A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition.

Publications

A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition.

Similar Datasets

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