Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The African swine fever virus DNA polymerase X (pol X), a member of the X family of DNA polymerases, is thought to be involved in base excision repair. Kinetics data indicate that pol X catalyzes DNA polymerization with low fidelity, suggesting a role in viral mutagenesis. Though pol X lacks the fingers domain that binds the DNA in other members of the X family, it binds DNA tightly. To help interpret details of this interaction, molecular dynamics simulations of free pol X at different salt concentrations and of pol X bound to gapped DNA, in the presence and in the absence of the incoming nucleotide, are performed. Anchors for the simulations are two NMR structures of pol X without DNA and a model of one NMR structure plus DNA and incoming nucleotide. Our results show that, in its free form, pol X can exist in two stable conformations that interconvert to one another depending on the salt concentration. When gapped double stranded DNA is introduced near the active site, pol X prefers an open conformation, regardless of the salt concentration. Finally, under physiological conditions, in the presence of both gapped DNA and correct incoming nucleotide, and two divalent ions, the thumb subdomain of pol X undergoes a large conformational change, closing upon the DNA. These results predict for pol X a substrate-induced conformational change triggered by the presence of DNA and the correct incoming nucleotide in the active site, as in DNA polymerase beta. The simulations also suggest specific experiments (e.g., for mutants Phe-102Ala, Val-120Gly, and Lys-85Val that may reveal crucial DNA binding and active-site organization roles) to further elucidate the fidelity mechanism of pol X.
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PMID:In silico studies of the African swine fever virus DNA polymerase X support an induced-fit mechanism. 1621 65

We recently demonstrated that African swine fever virus DNA polymerase X (Pol X) is extremely error-prone during single-nucleotide gap-filling and that the downstream ASFV DNA ligase seals 3' mismatched nicks with high efficiency. To further assess the credence of our hypothesis that these proteins may promote viral diversification by functioning within the context of an aberrant DNA repair pathway, herein we characterize the third protein expected to function in this system, a putative AP endonuclease (APE). Assays of the purified protein using oligonucleotide substrates unequivocally establish canonical APE activity, 3'-phosphatase and 3'-phosphodiesterase activities (in the context of a single-nucleotide gap), 3' --> 5' exonuclease activity (in the context of a nick), and nucleotide incision repair activity against 5,6-dihydrothymine. The 3' --> 5' exonuclease activity is shown to be highly dependent upon the identity of the nascent 3' base pair and to be inhibited when 2-deoxyribose-5-phosphate, rather than phosphate, constitutes the 5' moiety of the nick. ASFV APE retains activity when assayed in the presence of EDTA but is inactivated by incubation with 1,10-phenanthroline in the absence of a substrate, suggesting that it is an endonuclease IV homologue possessing intrinsic metal cofactors. The activities of ASFV APE, when considered alongside those of Pol X and ASFV DNA ligase, provide an enhanced understanding of (i) the types of damage that are likely to be sustained by the viral genome and (ii) the mechanisms by which the minimalist ASFV DNA repair pathway, consisting of just these three proteins, contributes to the fitness of the virus.
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PMID:Contributions of an endonuclease IV homologue to DNA repair in the African swine fever virus. 1650 34

We show here that the African swine fever virus (ASFV) protein pE296R, predicted to be a class II apurinic/apyrimidinic (AP) endonuclease, possesses endonucleolytic activity specific for AP sites. Biochemical characterization of the purified recombinant enzyme indicated that the K(m) and catalytic efficiency values for the endonucleolytic reaction are in the range of those reported for Escherichia coli endonuclease IV (endo IV) and human Ape1. In addition to endonuclease activity, the ASFV enzyme has a proofreading 3'-->5' exonuclease activity that is considerably more efficient in the elimination of a mismatch than in that of a correctly paired base. The three-dimensional structure predicted for the pE296R protein underscores the structural similarities between endo IV and the viral protein, supporting a common mechanism for the cleavage reaction. During infection, the protein is expressed at early times and accumulates at later times. The early enzyme is localized in the nucleus and the cytoplasm, while the late protein is found only in the cytoplasm. ASFV carries two other proteins, DNA polymerase X and ligase, that, together with the viral AP endonuclease, could act as a viral base excision repair system to protect the virus genome in the highly oxidative environment of the swine macrophage, the virus host cell. Using an ASFV deletion mutant lacking the E296R gene, we have determined that the viral endonuclease is required for virus growth in macrophages but not in Vero cells. This finding supports the existence of a viral reparative system to maintain virus viability in the infected macrophage.
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PMID:African swine fever virus protein pE296R is a DNA repair apurinic/apyrimidinic endonuclease required for virus growth in swine macrophages. 1664 Dec 76

We previously demonstrated that the DNA repair system encoded by the African swine fever virus (ASFV) is both extremely error-prone during the single-nucleotide gap-filling step (catalyzed by ASFV DNA polymerase X) and extremely error-tolerant during the nick-sealing step (catalyzed by ASFV DNA ligase). On the basis of these findings we have suggested that at least some of the diversity known to exist among ASFV isolates may be a consequence of mutagenic DNA repair, wherein damaged nucleotides are replaced with undamaged but incorrect nucleotides by Pol X and the resultant mismatched nicks are sealed by ASFV DNA ligase. Recently, this hypothesis appeared to be discredited by Salas and co-workers [(2003) J. Mol. Biol. 326, 1403-1412], who reported the fidelity of Pol X to be, on average, 2 orders of magnitude higher than what we previously published. In an effort to address this discrepancy and provide a definitive conclusion about the fidelity of Pol X, herein we examine the fidelity of Pol X-catalyzed single-nucleotide gap-filling in both the steady state and the pre-steady state under a diverse array of assay conditions (varying pH and ionic strength) and within different DNA sequence contexts. These studies corroborate our previously published data (demonstrating the low fidelity of Pol X to be independent of assay condition/sequence context), do not reproduce the data of Salas et al., and therefore confirm Pol X to be one of the most error-prone polymerases known. These results are discussed in light of ASFV biology and the mutagenic DNA repair hypothesis described above.
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PMID:ASFV DNA polymerse X is extremely error-prone under diverse assay conditions and within multiple DNA sequence contexts. 1714 76

After extensive studies spanning over half a century, there is little consensus on the kinetic mechanism of DNA polymerases. Using stopped-flow fluorescence assays for mammalian DNA polymerase beta (Pol beta), we have previously identified a fast fluorescence transition corresponding to conformational closing, and a slow fluorescence transition matching the rate of single-nucleotide incorporation. Here, by varying pH and buffer viscosity, we have decoupled the rate of single-nucleotide incorporation from the rate of the slow fluorescence transition, thus confirming our previous hypothesis that this transition represents a conformational event after chemistry, likely subdomain reopening. Analysis of an R258A mutant indicates that rotation of the Arg258 side chain is not rate-limiting in the overall kinetic pathway of Pol beta, yet is kinetically significant in subdomain reopening. We have extended our kinetic analyses to a high-fidelity polymerase, Klenow fragment (KF), and a low-fidelity polymerase, African swine fever virus DNA polymerase X (Pol X), and showed that they follow the same kinetic mechanism as Pol beta, while differing in relative rates of single-nucleotide incorporation and the putative conformational reopening. Our data suggest that the kinetic mechanism of Pol beta is not an exception among polymerases, and furthermore, its delineated kinetic mechanism lends itself as a platform for comparison of the kinetic properties of different DNA polymerases and their mutants.
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PMID:A unified kinetic mechanism applicable to multiple DNA polymerases. 1741 90

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase beta (Pol beta) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol beta-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol beta-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5'-dRP lyase domain of the second Pol beta molecule with the active site of the 1 : 1 Pol beta-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5'-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5'-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol beta-DNA complex enhances the function of Pol beta.
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PMID:Solution structures of 2 : 1 and 1 : 1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle X-ray scattering. 1808 22

A sequential ordered substrate binding established previously for several DNA polymerases is generally extended to all DNA polymerases, and the characterization of novel polymerases is often based on the assumption that the enzymes should productively bind DNA substrate first, followed by template-directed dNTP binding. The comprehensive kinetic study of DNA polymerase X (Pol X) from African swine fever virus reported here is the first analysis of the substrate binding order performed for a low-fidelity DNA polymerase. A classical steady-state kinetic approach using substrate analogue inhibition assays demonstrates that Pol X does not follow the bi-bi ordered mechanism established for other DNA polymerases. Further, using isotope-trapping experiments and stopped-flow fluorescence assays, we show that Pol X can bind Mg (2+).dNTPs in a productive manner in the absence of DNA substrate. We also show that DNA binding to Pol X, although rapid, may not always be productive. Furthermore, we show that binding of Mg (2+).dNTP to Pol X facilitates subsequent formation of the catalytically competent Pol X.DNA.dNTP ternary complex, whereas DNA binding prior to dNTP binding brings the enzyme into a nonproductive conformation where subsequent nucleotide substrate binding is hindered. Together, our results suggest that Pol X prefers an ordered sequential mechanism with Mg (2+).dNTP as the first substrate.
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PMID:Altered order of substrate binding by DNA polymerase X from African Swine Fever virus. 1859 57

DNA polymerase X (pol X) from the African swine fever virus is a 174-amino-acid repair polymerase that likely participates in a viral base excision repair mechanism, characterized by low fidelity. Surprisingly, pol X's insertion rate of the G*G mispair is comparable to that of the four Watson-Crick base pairs. This behavior is in contrast with another X-family polymerase, DNA polymerase beta (pol beta), which inserts G*G mismatches poorly, and has higher DNA repair fidelity. Using molecular dynamics simulations, we previously provided support for an induced-fit mechanism for pol X in the presence of the correct incoming nucleotide. Here, we perform molecular dynamics simulations of pol X/DNA complexes with different incoming incorrect nucleotides in various orientations [C*C, A*G, and G*G (anti) and A*G and G*G (syn)] and compare the results to available kinetic data and prior modeling. Intriguingly, the simulations reveal that the G*G mispair with the incoming nucleotide in the syn configuration undergoes large-scale conformational changes similar to that observed in the presence of correct base pair (G*C). The base pairing in the G*G mispair is achieved via Hoogsteen hydrogen bonding with an overall geometry that is well poised for catalysis. Simulations for other mismatched base pairs show that an intermediate closed state is achieved for the A*G and G*G mispair with the incoming dGTP in anti conformation, while the protein remains near the open conformation for the C*C and the A*G syn mismatches. In addition, catalytic site geometry and base pairing at the nascent template-incoming nucleotide interaction reveal distortions and misalignments that range from moderate for A*G anti to worst for the C*C complex. These results agree well with kinetic data for pol X and provide a structural/dynamic basis to explain, at atomic level, the fidelity of this polymerase compared with other members of the X family. In particular, the more open and pliant active site of pol X, compared to pol beta, allows pol X to accommodate bulkier mismatches such as guanine opposite guanine, while the more structured and organized pol beta active site imposes higher discrimination, which results in higher fidelity. The possibility of syn conformers resonates with other low-fidelity enzymes such as Dpo4 (from the Y family), which readily accommodate oxidative lesions.
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PMID:Mismatched base-pair simulations for ASFV Pol X/DNA complexes help interpret frequent G*G misincorporation. 1895 64

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.
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PMID:Modulation of the structure, catalytic activity, and fidelity of African swine fever virus DNA polymerase X by a reversible disulfide switch. 1941 58

Heterocapsa circularisquama DNA virus (HcDNAV; previously designated as HcV) is a giant virus (girus) with a approximately 356-kbp double-stranded DNA (dsDNA) genome. HcDNAV lytically infects the bivalve-killing marine dinoflagellate H. circularisquama, and currently represents the sole DNA virus isolated from dinoflagellates, one of the most abundant protists in marine ecosystems. Its morphological features, genome type, and host range previously suggested that HcDNAV might be a member of the family Phycodnaviridae of Nucleo-Cytoplasmic Large DNA Viruses (NCLDVs), though no supporting sequence data was available. NCLDVs currently include two families found in aquatic environments (Phycodnaviridae, Mimiviridae), one mostly infecting terrestrial animals (Poxviridae), another isolated from fish, amphibians and insects (Iridoviridae), and the last one (Asfarviridae) exclusively represented by the animal pathogen African swine fever virus (ASFV), the agent of a fatal hemorrhagic disease in domestic swine. In this study, we determined the complete sequence of the type B DNA polymerase (PolB) gene of HcDNAV. The viral PolB was transcribed at least from 6 h post inoculation (hpi), suggesting its crucial function for viral replication. Most unexpectedly, the HcDNAV PolB sequence was found to be closely related to the PolB sequence of ASFV. In addition, the amino acid sequence of HcDNAV PolB showed a rare amino acid substitution within a motif containing highly conserved motif: YSDTDS was found in HcDNAV PolB instead of YGDTDS in most dsDNA viruses. Together with the previous observation of ASFV-like sequences in the Sorcerer II Global Ocean Sampling metagenomic datasets, our results further reinforce the ideas that the terrestrial ASFV has its evolutionary origin in marine environments.
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PMID:Remarkable sequence similarity between the dinoflagellate-infecting marine girus and the terrestrial pathogen African swine fever virus. 1986 Sep 21


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