Browse
Submit Data
Databases
API
Help

Dataset Information

19 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Picornaviral polymerase structure, function, and fidelity modulation.

ABSTRACT: Like all positive strand RNA viruses, the picornaviruses replicate their genomes using a virally encoded RNA-dependent RNA polymerase enzyme known as 3Dpol. Over the past decade we have made tremendous advances in our understanding of 3Dpol structure and function, including the discovery of a novel mechanism for closing the active site that allows these viruses to easily fine tune replication fidelity and quasispecies distributions. This review summarizes current knowledge of picornaviral polymerase structure and how the enzyme interacts with RNA and other viral proteins to form stable and processive elongation complexes. The picornaviral RdRPs are among the smallest viral polymerases, but their fundamental molecular mechanism for catalysis appears to be generally applicable as a common feature of all positive strand RNA virus polymerases.

SUBMITTER: Peersen OB

PROVIDER: S-EPMC5476519 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Picornaviral polymerase structure, function, and fidelity modulation.

Peersen Olve B OB

Virus research 20170202

Like all positive strand RNA viruses, the picornaviruses replicate their genomes using a virally encoded RNA-dependent RNA polymerase enzyme known as 3D<sup>pol</sup>. Over the past decade we have made tremendous advances in our understanding of 3D<sup>pol</sup> structure and function, including the discovery of a novel mechanism for closing the active site that allows these viruses to easily fine tune replication fidelity and quasispecies distributions. This review summarizes current knowledge ...[more]

PMID: 28163093

Similar Datasets

RB69 DNA polymerase structure, kinetics, and fidelity.

Project description:This review will summarize our structural and kinetic studies of RB69 DNA polymerase (RB69pol) as well as selected variants of the wild-type enzyme that were undertaken to obtain a deeper understanding of the exquisitely high fidelity of B family replicative DNA polymerases. We discuss how the structures of the various RB69pol ternary complexes can be used to rationalize the results obtained from pre-steady-state kinetic assays. Our main findings can be summarized as follows. (i) Interbase hydrogen bond interactions can increase catalytic efficiency by 5000-fold; meanwhile, base selectivity is not solely determined by the number of hydrogen bonds between the incoming dNTP and the templating base. (ii) Minor-groove hydrogen bond interactions at positions n - 1 and n - 2 of the primer strand and position n - 1 of the template strand in RB69pol ternary complexes are essential for efficient primer extension and base selectivity. (iii) Partial charge interactions among the incoming dNTP, the penultimate base pair, and the hydration shell surrounding the incoming dNTP modulate nucleotide insertion efficiency and base selectivity. (iv) Steric clashes between mismatched incoming dNTPs and templating bases with amino acid side chains in the nascent base pair binding pocket (NBP) as well as weak interactions and large gaps between the incoming dNTPs and the templating base are some of the reasons that incorrect dNTPs are incorporated so inefficiently by wild-type RB69pol. In addition, we developed a tC°-tCnitro Förster resonance energy transfer assay to monitor partitioning of the primer terminus between the polymerase and exonuclease subdomains.

| S-EPMC4018061 | biostudies-literature

Novel roles of the picornaviral 3D polymerase in viral pathogenesis.

Project description:The RNA-dependent RNA-polymerase, 3D(pol), is an essential component in the picornavirus genome for the replication of single stranded RNA. However, transgenic expression of 3D(pol) in mice has antiviral effects. Here we discuss the structure and function of 3D(pol) during picornavirus replication, we review the evidence and consequence of a host immune response to epitopes in 3D(pol) after picornavirus infection, highlight data showing the antiviral effects of transgenic 3D(pol) from Theiler's murine encephalomyelitis virus (TMEV), and discuss potential mechanisms by which 3D(pol) is causing this antiviral effect in mice.

| S-EPMC2897181 | biostudies-literature

Modulation of the structure, catalytic activity, and fidelity of African swine fever virus DNA polymerase X by a reversible disulfide switch.

Project description:African swine fever virus polymerase X (pol X) is the smallest DNA polymerase known (174 amino acids), and its tertiary structure resembles the C-terminal half of prototypical X-family pol beta, which includes a catalytic dNTP-binding site (palm domain) and a finger domain. This structural similarity and the presence of viral genes coding for other base excision repair proteins suggest that pol X functions in a manner similar to pol beta, but inconsistencies concerning pol X catalysis have been reported. We examined the structural and functional properties of two forms of pol X using spectroscopic and kinetic analysis. Using (1)H-(15)N correlated NMR, we unambiguously demonstrated the slow interconversion of pol X between a reduced (pol X(red)) and an oxidized form (pol X(ox)), confirmed by mass spectrometry. Steady-state kinetic analysis revealed that pol X(ox), with a disulfide bond between Cys-81 and Cys-86, has approximately 10-fold lower fidelity than pol X(red) during dNTP insertion opposite a template G. The disulfide linkage is located between two beta-strands in the palm domain, near the putative dNTP-binding site. Structural alignment of pol X with a pol beta ternary structure suggests that the disulfide switch may modulate fidelity by altering the ability of the palm domain to align and stabilize the primer terminus and catalytic metal ion for deprotonation of the 3'-OH group and subsequent phosphoryl transfer. Thus, DNA polymerase fidelity is altered by the redox state of the enzyme and its related conformational changes.

| S-EPMC2709365 | biostudies-literature

Activity and fidelity of human DNA polymerase ? depend on primer structure.

Project description:DNA polymerase ? (Pol?) plays an important role in genome replication. In a complex with primase, Pol? synthesizes chimeric RNA-DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Pol? needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Pol?'s interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Pol? catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Pol? is in the open conformation. Pol? induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Pol? extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Pol? has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Pol? with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.

| S-EPMC5936803 | biostudies-literature

Temperature effect on polymerase fidelity.

Project description:The discovery of extremophiles helped enable the development of groundbreaking technology such as PCR. Temperature variation is often an essential step of these technology platforms, but the effect of temperature on the error rate of polymerases from different origins is underexplored. Here, we applied high-throughput sequencing to profile the error rates of DNA polymerases from psychrophilic, mesophilic, and thermophilic origins with single-molecule resolution. We found that the reaction temperature substantially increases substitution and deletion error rates of psychrophilic and mesophilic DNA polymerases. Our motif analysis shows that the substitution error profiles cluster according to phylogenetic similarity of polymerases, not the reaction temperature, thus suggesting that the reaction temperature increases the global error rate of polymerases independent of the sequence context. Intriguingly, we also found that the DNA polymerase I of psychrophilic bacteria exhibits higher polymerization activity than its mesophilic ortholog across all temperature ranges, including down to -19 °C, which is well below the freezing temperature of water. Our results provide a useful reference for how the reaction temperature, a crucial parameter of biochemistry, can affect DNA polymerase fidelity in organisms adapted to a wide range of thermal environments.

| S-EPMC8592868 | biostudies-literature

Structure-function studies of DNA polymerase λ.

Project description:DNA polymerase λ (pol λ) functions in DNA repair with its main roles considered to be filling short gaps during repair of double-strand breaks by nonhomologous end joining and during base excision repair. As indicated by structural and biochemical studies over the past 10 years, pol λ shares many common properties with other family X siblings (pol β, pol μ, and terminal deoxynucleotidyl transferase) but also has unique structural features that determine its specific functions. In this review, we consider how structural studies over the past decade furthered our understanding of the behavior and biological roles of pol λ.

| S-EPMC4018081 | biostudies-literature

Structure and function of SARS-CoV-2 polymerase.

Project description:Coronaviruses use an RNA-dependent RNA polymerase (RdRp) to replicate and express their genome. The RdRp associates with additional non-structural proteins (nsps) to form a replication-transcription complex (RTC) that carries out RNA synthesis, capping and proofreading. However, the structure of the RdRp long remained elusive, thus limiting our understanding of coronavirus genome expression and replication. Recently, the cryo-electron microscopy structure of SARS-CoV-1 RdRp was reported. Driven by the ongoing COVID-19 pandemic, structural data on the SARS-CoV-2 polymerase and associated factors has since emerged at an unprecedented pace, with more than twenty structures released to date. This review provides an overview of the currently available coronavirus RdRp structures and outlines how they have, together with functional studies, led to a molecular understanding of the viral polymerase, its interactions with accessory factors and the mechanisms by which promising antivirals may inhibit coronavirus replication.

| S-EPMC8023233 | biostudies-literature

An abridged transition state model to derive structure, dynamics, and energy components of DNA polymerase ? fidelity.

Project description:We show how a restricted reaction surface can be used to facilitate the calculation of biologically important contributions of active site geometries and dynamics to DNA polymerase fidelity. Our analysis, using human DNA polymerase beta (pol ?), is performed within the framework of an electrostatic linear free energy response (EFER) model. The structure, dynamics, and energetics of pol ?-DNA-dNTP interactions are computed between two points on the multidimensional reaction free energy surface. "Point 1" represents a ground state activation intermediate (GSA), which is obtained by deprotonating the terminal 3'OH group of the primer DNA strand. "Point 2" is the transition state (PTS) for the attack of the 3'O(-) (O(nuc)) on the P(?) atom of dNTP substrate, having the electron density of a dianionic phosphorane intermediate. Classical molecular dynamics simulations are used to compute the geometric and dynamic contributions to the formation of right and wrong O(nuc)-P chemical bonds. Matched dCTP·G and mismatched dATP·G base pairs are used to illustrate the analysis. Compared to the dCTP·G base pair, the dATP·G mismatch has fewer GSA configurations with short distances between O(nuc) and P(?) atoms and between the oxygen in the scissile P-O bond (O(lg)) and the nearest structural water. The thumb subdomain conformation of the GSA complex is more open for the mismatch, and the H-bonds in the mispair become more extended during the nucleophilic attack than in the correct pair. The electrostatic contributions of pol ? and DNA residues to catalysis of the right and wrong P-O(nuc) bond formation are 5.3 and 3.1 kcal/mol, respectively, resulting in an 80-fold contribution to fidelity. The EFER calculations illustrate the considerable importance of Arg183 and an O(lg)-proximal water molecule to pol ? fidelity.

| S-EPMC3477174 | biostudies-literature

Structure-function relationships of the viral RNA-dependent RNA polymerase: fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket.

Project description:Studies of the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV), 3Dpol, have shown that Asn-297 permits this enzyme to distinguish ribose from 2'-deoxyribose. All animal RNA viruses have Asn at the structurally homologous position of their polymerases, suggesting a conserved function for this residue. However, all prokaryotic RNA viruses have Glu at this position. In the presence of Mg2+, the apparent affinity of Glu-297 3Dpol for 2'-deoxyribonucleotides was decreased by 6-fold relative to wild type without a substantial difference in the fidelity of 2'-dNMP incorporation. The fidelity of ribonucleotide misincorporation for Glu-297 3Dpol was reduced by 14-fold relative to wild type. A 4- to 11-fold reduction in the rate of ribonucleotide incorporation was observed. Glu-297 PV was unable to grow in HeLa cells due to a replication defect equivalent to that observed for a mutant PV encoding an inactive polymerase. Evaluation of the protein-(VPg)-primed initiation reaction showed that only half of the Glu-297 3Dpol initiation complexes were capable of producing VPg-pUpU product and that the overall yield of uridylylated VPg products was reduced by 20-fold relative to wild-type enzyme, a circumstance attributable to a reduced affinity for UTP. These studies identify the first RdRp derivative with a mutator phenotype and provide a mechanistic basis for the elevated mutation frequency of RNA phage relative to animal RNA viruses observed in culture. Although protein-primed initiation and RNA-primed elongation complexes employ the same polymerase active site, the functional differences reported here imply significant structural differences between these complexes.

| S-EPMC2116994 | biostudies-literature

Regulation and Modulation of Human DNA Polymerase δ Activity and Function.

Project description:This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on the mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, polymerase delta interaction protein 46 (PDIP46) and polymerase delta interaction protein 38 (PDIP38), both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and Pol δ4 in leading and lagging strand DNA synthesis that integrate a role for PDIP46 are presented. PDIP38 has multiple cellular localizations including the mitochondria, the spliceosomes and the nucleus. It has been implicated in a number of cellular functions, including the regulation of specialized DNA polymerases, mitosis, the DNA damage response, mouse double minute 2 homolog (Mdm2) alternative splicing and the regulation of the NADPH oxidase 4 (Nox4).

| S-EPMC5541323 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data