Dataset Information

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

ACCESS DATA

Publications

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

Similar Datasets

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.

Project description:Since the first human infection reported in 2013, H7N9 avian influenza virus (AIV) has been regarded as a serious threat to human health. In this study, we sought to identify the virulence determinant of the H7N9 virus in mammalian hosts. By comparing the virulence of the SH/4664 H7N9 virus, a non-virulent H9N2 virus, and various H7N9-H9N2 hybrid viruses in infected mice, we first pinpointed PB2 as the primary viral factor accounting for the difference between H7N9 and H9N2 in mammalian virulence. We further analyzed the in vivo effects of individually mutating H7N9 PB2 residues different from the closely related H9N2 virus and consequently found residue 473, alongside the well-known residue 627, to be critical for the virulence of the H7N9 virus in mice and the activity of its reconstituted viral polymerase in mammalian cells. The importance of PB2-473 was further strengthened by studying reverse H7N9 substitutions in the H9N2 background. Finally, we surprisingly found that species-specific usage of ANP32A, a family member of host factors connecting with the PB2-627 polymorphism, mediates the contribution of PB2 473 residue to the mammalian adaption of AIV polymerase, as the attenuating effect of PB2 M473T on the viral polymerase activity and viral growth of the H7N9 virus could be efficiently complemented by co-expression of chicken ANP32A but not mouse ANP32A and ANP32B. Together, our studies uncovered the PB2 473 residue as a novel viral host range determinant of AIVs via species-specific co-opting of the ANP32 host factor to support viral polymerase activity.IMPORTANCEThe H7N9 avian influenza virus has been considered to have the potential to cause the next pandemic since the first case of human infection reported in 2013. In this study, we identified PB2 residue 473 as a new determinant of mouse virulence and mammalian adaptation of the viral polymerase of the H7N9 virus and its non-pathogenic H9N2 counterparts. We further demonstrated that the variation in PB2-473 is functionally linked to differential co-opting of the host ANP32A protein in supporting viral polymerase activity, which is analogous to the well-known PB2-627 polymorphism, albeit the two PB2 positions are spatially distant. By providing new mechanistic insight into the PB2-mediated host range determination of influenza A viruses, our study implicated the potential existence of multiple PB2-ANP32 interfaces that could be targets for developing new antivirals against the H7N9 virus as well as other mammalian-adapted influenza viruses.

Project description:Background: Safety and significant improvement in overall glycated hemoglobin (A1C) and percentage of time spent in (TIR), below (TBR), and above (TAR) glucose range were demonstrated in the pivotal trial of adolescents and adults using the MiniMed™ advanced hybrid closed-loop (AHCL) system with the adjunctive, calibration-required Guardian™ Sensor 3. The present study evaluated early outcomes of continued access study (CAS) participants who transitioned from the pivotal trial investigational system to the approved MiniMed™ 780G system with the non-adjunctive, calibration-free Guardian™ 4 Sensor (MM780G+G4S). Study data were presented alongside those of real-world MM780G+G4S users from Europe, the Middle East, and Africa. Methods: The CAS participants (N = 109, aged 7-17 years and N = 67, aged >17 years) used the MM780G+G4S for 3 months and data of real-world MM780G+G4S system users (N = 10,204 aged ≤15 years and N = 26,099 aged >15 years) were uploaded from September 22, 2021 to December 02, 2022. At least 10 days of real-world continuous glucose monitoring (CGM) data were required for analyses. Glycemic metrics, delivered insulin and system use/interactions underwent descriptive analyses. Results: Time in AHCL and CGM use were >90% for all groups. AHCL exits averaged 0.1/day and there were few blood glucose measurements (BGMs) (0.8/day-1.0/day). Adults in both cohorts met most consensus recommendations for glycemic targets. Pediatric groups met recommendations for %TIR and %TBR, although not those for mean glucose variability and %TAR, possibly due to low use of recommended glucose target (100 mg/dL) and active insulin time (2 h) settings (28.4% in the CAS cohort and 9.4% in the real-world cohort). The CAS pediatric and adult A1C were 7.2% ± 0.7% and 6.8% ± 0.7%, respectively, and there were no serious adverse events. Conclusions: Early clinical use of the MM780G+G4S was safe and involved minimal BGMs and AHCL exits. Consistent with real-world pediatric and adult use, outcomes were associated with achievement of recommended glycemic targets. Clinical Trial Registration number: NCT03959423.

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

OmicsDI is part of the ELIXIR infrastructure

Tweets