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)

Seven temperature-sensitive mutants of vaccinia virus have been isolated after preselection for virus resistant to phosphonoacetic acid (PAA). In all seven mutants, the PAA-resistant (PAAr) and ts lesions represent separate mutations. In one mutant, NG26, the PAAr (NG26-PAAr) and ts (NG26-ts) mutations are very closely linked. Both NG26-ts and NG26-PAAr map in the HindIII E DNA fragment. NG26 has a DNA-negative phenotype at 40 degrees. NG26-ts is in the same complementation group as ts42, another DNA-negative mutant which maps in the HindIII E DNA fragment (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128, 000-000, 1983). The order of the mutations is (NG26-ts)-(NG26-PAAr)-ts42. The virus-coded DNA polymerase has been partially purified from wt- and NG26-infected cells. The DNA polymerase encoded by NG26 is temperature sensitive and PAA resistant in vitro as compared to the wt enzyme.
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PMID:Selection for temperature-sensitive mutations in specific vaccinia virus genes: isolation and characterization of a virus mutant which encodes a phosphonoacetic acid-resistant, temperature-sensitive DNA polymerase. 661 92

The very limited coding capacity of the HBV-DNA led us to study the nature of the HBV (Dane particle) -- associated DNA polymerase. The HBV-associated DNA polymerase met in many respects the characteristics of the repair enzyme of the host: the DNA polymerase beta. It operates under high salt conditions, and exhibits similar salt effects with NaCl, KCL, and PO4(3-). It is insensitive to sulfhydryl group blockers, such as p-hydroxymercuribenzoate and N-ethylmaleimide, is resistant to phosphonoacetic acid, and not inhibited by 5 mol/l urea. It requires a divalent cation (Mg2+) for activity, the Mg2+ concentration revealing optimal activity is somewhat higher than that described for most DNA polymerases beta. The HBV-associated DNA polymerase differs also from most DNA polymerase beta in its sensitivity ot ddTTP and its optimal pH. The fact that DNA polymerase beta of different origin vary considerably in their response to chemical agents and that the DNA polymerase beta from human liver has not been studied allows no definite conclusion as to the nature of the Dane particle-associated DNA polymerase.
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PMID:Hepatitis-B virus-associated deoxyribonucleic acid polymerase: a partial characterization by the use of chemical agents. 725 43

The genomic localization of two immunodominant genes encoding two proteins of the Epstein-Barr virus capsid antigen (VCA) complex, VCA-p18 and VCA-p40, has been identified. For that purpose, lambda gt11-based cDNA libraries were constructed from HH514.c16 cells induced for virus production. The libraries were screened with a monoclonal antibody, EBV.OT41A, directed against VCA-p40 or with affinity-purified human antibodies against VCA-p18. Sequencing of the inserts of positive plaques showed that VCA-p18 and VCA-p40 are encoded within open reading frames (ORFs) BFRF3 and BdRF1, respectively. Peptide scanning analysis of the predicted protein of ORF BdRF1 resulted in defining the epitope of monoclonal antibody EBV.OT41A at the C-terminal region. The dominant VCA-p18 reactivity of human sera can be completely inhibited by preadsorption with Escherichia coli-expressed BFRF3-beta-galactosidase. Serum of a rabbit immunized with BFRF3-beta galactosidase reacts with a VCA-specific protein of 18 kDa. In addition, BFRF3-beta-galactosidase affinity-purified antibodies react with VCA-p18 of virus-producing cells (HH514.c16). Complete inhibition of viral DNA polymerase activity by phosphonoacetic acid is associated with the absence of RNAs and protein products of both ORFs, indicating that VCA-p18 and VCA-p40 are true late antigens.
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PMID:Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins. 768 3

Herpes simplex virus encodes proteins, such as DNA polymerase, that are essential for its replication and proteins, such as thymidine kinase, that are not essential for replication in cell culture, but are important for pathogenesis in animal models. However, certain mutations affecting these proteins exert little or no effect on replication or pathogenesis. We tested the effects of combining two such mutations--one that alters DNA polymerase and one that decreases but does not abolish thymidine kinase activity--on replication in cultured cells and on acute and latent infections in mice. The double mutant replicated similarly to the single mutants and wild-type virus both in cell culture and acutely in the mouse eye. However, it was severely impaired for acute replication in trigeminal ganglia and for reactivatable latent infections. This impairment depended upon the polymerase mutation. Similarly, although Ro 31-5140, a thymidine kinase inhibitor, did not potentiate the antiviral effects of phosphonoacetic acid, a polymerase inhibitor, in cell culture, the two drugs in combination substantially inhibited viral reactivation from latency at concentrations that had little or no effect when used singly. These synergistic effects may have implications for viral functions during pathogenesis and for antiviral chemotherapy.
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PMID:Synergistic effects on ganglionic herpes simplex virus infections by mutations or drugs that inhibit the viral polymerase and thymidine kinase. 783 80

Repair of airway epithelium after viral infection involves migration of epithelial cells to cover injured, denuded areas. We determined whether viral infection reduces the capability of bronchial epithelial cells to migrate and to attach to extracellular matrix proteins. Inoculation of bovine bronchial epithelial cells in vitro with bovine herpesvirus-1 reduced their ability to migrate in two different assays of cell migration. When attachment assays were performed, fewer cells attached to both control wells and matrix protein-precoated wells, suggesting that general mechanisms of adherence to substrates were altered by viral infection. Focal contact points of epithelial cells with the underlying matrix were evaluated with epifluorescence microscopy and monoclonal antibodies to vinculin and alpha v, an integrin chain. Disruption of focal contact points was seen early after infection and was prevented by an inhibitor of viral DNA polymerase, phosphonoacetic acid. Cycloheximide did not cause similar disruptions of focal contacts at early time points. Viral infection thus has marked effects on the interactions of bronchial epithelial cells with extracellular matrix and the organization of matrix to cytoskeleton links. The effects appear to be dependent in part on viral replication in the cells and are not simply due to reductions in host cell protein synthesis.
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PMID:Bovine herpesvirus-1 infection reduces bronchial epithelial cell migration to extracellular matrix proteins. 786 42

The DNA polymerase gene of Epstein-Barr virus (EBV) was cloned into baculovirus transfer vector (pBlueBac). The recombinant baculovirus (AcEBP-15) was obtained by cotransfection of Spodoptera frugiperda (Sf9) cells with infectious DNA from Autographa californica multiple nuclear polyhedrin virus (AcMNPV) and pBlueBac plasmid carrying EBV polymerase gene. Infection of Sf9 cells with the recombinant virus produced substantial quantities of the EBV DNA polymerase protein of the expected size (110 kD). The identity of the EBV polymerase 110-kD polypeptide was determined by (a) immunoprecipitation and Western blot analyses with rabbit polyclonal antiserum specific for a synthetic peptide derived from the coding sequence of the polymerase gene; (b) identification of a polypeptide of identical size (110 kD) from EBV-infected cells; (c) measurement of DNA polymerase activity similar to that of the enzyme induced in EBV-infected cells; and (d) neutralization of the enzymatic activity by the rabbit antiserum and inhibition by phosphonoacetic acid. Our results indicate that the baculovirus expression system provides large quantities of functional polymerase suitable for biochemical and structural analyses, thereby furthering our understanding of the mechanism of viral DNA replication and its inhibition by antiviral drugs.
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PMID:Functional characterization of partially purified Epstein-Barr virus DNA polymerase expressed in the baculovirus system. 797 69

Polymerases in general share only a few regions of amino acid similarity. One of the most conserved regions, called motif A, has the sequence DXXSLYPSII or a similar sequence in many eukaryotic and viral DNA polymerases and in bacteriophage T4 DNA polymerase. We designed genetic techniques to isolate mutant T4 DNA polymerases with amino acid substitutions in this highly conserved motif. The mutant DNA polymerases differed from wild type T4 DNA polymerase in several ways. For one mutant DNA polymerase, the pyrophosphate analog, phosphonoacetic acid, was a potent inhibitor of DNA replication, and this mutant DNA polymerase replicated DNA with reduced fidelity. Another mutant DNA polymerase replicated DNA with increased accuracy, but this mutant DNA polymerase was less processive in primer extension reactions, and DNA replication required high concentrations of deoxynucleoside triphosphates. We provide evidence that indicates that all of these changes to DNA polymerase function are due to differences in how the mutant DNA polymerases partition between states active for DNA replication or exonucleolytic proofreading. These studies also provide further support for the hypothesis that the accuracy of DNA replication observed for DNA polymerases and 3'-->5' exonuclease activities (Muzyczka, N., Poland, R. L., and Bessman, M. J. (1972) J. Biol. Chem. 247, 7116-7122).
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PMID:Motif A of bacteriophage T4 DNA polymerase: role in primer extension and DNA replication fidelity. Isolation of new antimutator and mutator DNA polymerases. 811

The DNA polymerase gene of African swine fever virus (ASFV) was mapped by marker rescue experiments using a phosphonoacetic acid-resistant mutant and hybridization with an oligonucleotide probe designed from the most conserved motif of family B DNA polymerases. Viral DNA fragments mapping in this region were cloned and sequenced. An open reading frame coding for a 1244 amino acid long peptide with a molecular mass of 142.5 kDa was determined from the sequence. A unique feature of ASFV DNA polymerase is the presence of 13 tandem repeats of the sequence Ala-Gly-Asp-Pro near the carboxyl end of the molecule. Comparison with 30 sequences of alpha-like DNA polymerases of cellular and viral origin showed that ASFV DNA polymerase has all the conserved motifs of family B DNA polymerases. A 3.9 kb transcript was detected by Northern hybridization and the transcription initiation and termination sites were mapped by S1 analysis and primer extension. Late transcription was initiated at a site different from the early transcription initiation site. A 145 kDa protein, consistent with the size of the gene, was identified by an in situ enzyme assay after gel electrophoresis of infected cell extracts.
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PMID:Genetic identification and nucleotide sequence of the DNA polymerase gene of African swine fever virus. 812 6

phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid sequence similarity and sensitivity to inhibitors of eukaryotic DNA polymerase alpha. In this paper, site-directed mutants in the phi 29 DNA polymerase residues Asp249, Ser252, Leu253, and Pro255 of the conserved amino acid motif "Dx2SLYP" are described. Two mutants, D249E and S252R, were drastically affected in all the synthetic activities, whereas their 3' to 5' exonuclease activity and interaction with the TP primer was normal. Mutant D249E, slightly affected in template-primer binding, was completely inactive in all conditions tested, suggesting that Asp249 could be playing a direct role in catalysis. On the other hand, mutant S252R, strongly affected in template-primer binding, showed some DNA polymerization activity in the presence of Mn2+. Mutants S252G and P255S showed a reduced template-primer binding ability; these mutants, together with mutant L253V, showed metal ion-dependent phenotypes in their synthetic activities and altered sensitivities to the PPi analog phosphonoacetic acid. All these results support the hypothesis that the Dx2SLYP motif forms part of the polymerization active site of the phi 29 DNA polymerase, being the Asp249 residue critical both for protein-primed initiation and DNA polymerization.
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PMID:Phi 29 DNA polymerase active site. Residue ASP249 of conserved amino acid motif "Dx2SLYP" is critical for synthetic activities. 822 57

Most point substitutions in the highly-conserved 885-GDTDS motif of the HSV-1 DNA polymerase inactivate polymerase elongation activity. However, in an assay system based on expression by in vitro transcription-translation, the mutant GDTDA (S889A) possessed wild-type elongation activity which was highly resistant to phosphonoacetic acid and acyclovir triphosphate, but retained sensitivity to aphidicolin.
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PMID:Resistance to antiviral inhibitors caused by the mutation S889A in the highly-conserved 885-GDTDS motif of the herpes simplex virus type 1 DNA polymerase. 833 48


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