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)

Phosphonoacetate (PAA), at concentrations of 200 micrograms/ml or more, prevented growth of vaccinia virus in HeLa and BSC-1 cells. Spontaneous vaccinia virus mutants, selected at high PAA levels, were resistant to the antiviral effects of the drug. The action of PAA was directed toward an early viral function, since the drug was inhibitory only during the first 4 h of the approximately 15-h growth cycle. Conversely, significant reversal of the antiviral effects was obtained only when the drug was removed at or before the fourth hour of infection. Incorporation of [3H]thymidine into cytoplasmic viral DNA was severely inhibited in cells infected with wild-type virus but not in cells infected with mutant virus. Virus-induced DNA polymerase isolated from the cytoplasm of cells infected with wild-type or mutant virus had indistinguishable chromatographic properties on DEAE-cellulose and phosphocellulose columns. However, the wild-type enzyme was inhibited by relatively low concentrations of PAA, whereas 10-fold higher concentrations were needed for equivalent inhibition of the mutant enzyme. Kinetic analysis indicated that PAA inhibition was noncompetitive with deoxyribonucleoside triphosphates; Ki values for wild-type and mutant DNA polymerases were approximately 25 and 300 microM, respectively. Inhibition of wild-type DNA polymerase was immediate and complete even when PAA was added after initiation of DNA synthesis in vitro, suggesting that chain elongation was affected. These results established that the DNA polymerase is a target of the antiviral action of PAA and provided genetic evidence that this enzyme is virus encoded.
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PMID:Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus. 710 36

The effect of the triphosphate of 9-(2-hydroxyethoxymethyl)guanine (acyclovir, acycloguanosine) on cellular alpha deoxyribonucleic acid (DNA) polymerases (DNA nucleotidyltransferases), DNA polymerases of several members of the herpes group, vaccinia virus DNA polymerase, and Friend leukemia virus ribonucleic acid-dependent DNA polymerase was examined. Several viruses, which were found to be susceptible to acyclovir, were found to induce DNA polymerases which were sensitive to acyclovir triphosphate (acyclo-GTP). Human cytomegalovirus and the H29R strain of herpes simplex virus type 1, however, were found to be relatively insusceptible to acyclovir, even though their induced DNA polymerases were inhibited by low concentrations of acyclo-GTP. The amount of acyclovir anabolized to acyclo-GTP was significantly lower for human cytomegalovirus and H29R than for the more susceptible viruses. Vaccinia virus and Friend leukemia virus induced DNA polymerases which were insensitive to inhibition by low concentrations of acyclo-GTP, anabolized little acyclovir to acyclo-GTP, and were found to be insensitive to inhibition by acyclovir. Uninfected WI-38 cells were not susceptible to inhibition by acyclovir, anabolized little acyclovir to acyclo-GTP, and had an alpha DNA polymerase which was insensitive to inhibition by low concentrations of acyclo-GTP.
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PMID:Inhibition of cellular alpha and virally induced deoxyribonucleic acid polymerases by the triphosphate of acyclovir. 719 34

We have overexpressed the vaccinia virus DNA polymerase using the hybrid vaccinia virus/T7 expression system. Accumulation of the DNA polymerase to levels as high as 10% of the total protein was observed following coinfection of BSC40 cells with the appropriate vaccinia recombinants. Although the DNA polymerase produced at 37 degrees C was largely insoluble, 25% of the recombinant protein could be recovered as soluble protein when infected cultures were maintained at 32 degrees C. Starting with cytoplasmic lysates of coinfected cells, a rapid and reproducible purification protocol that yielded apparently homogeneous preparations of the DNA polymerase after four chromatographic steps was established. Typically, 0.3 mg of purified DNA polymerase was obtained from 27 mg of total protein within 10 h after harvesting infected cells. As was previously described for the DNA polymerase purified from vaccinia-infected cells (Challberg and Englund, J. Biol. Chem., 254, 7812-7819, 1979), the purified recombinant enzyme displayed both polymerase and 3'-5' exonuclease activities but lacked detectable 5'-3' exonuclease activity. Kinetic analysis of nucleotide incorporation catalyzed by the vaccinia enzyme revealed apparent Km values of 0.9, 2.9, 4.0, and 2.7 microM for dGTP, dATP, TTP, and dCTP, respectively.
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PMID:Overexpression and purification of the vaccinia virus DNA polymerase. 795 Mar 89

Involvement of adenovirus DNA polymerase (AdPol) in viral DNA replication was initially demonstrated using two temperature-sensitive (ts) adenoviruses, Ad5ts36 and Ad5ts149. Ad5ts36 is particularly interesting because a single mutation mapping to Leu391-->Phe in the AdPol gene confers ts phenotype not only for DNA replication in human cells but also for transformation in rat cells. In order to elucidate the mechanism(s) involved in the ts phenotype, we introduced the ts36 mutation in AdPol of adenovirus type 2 and overexpressed the protein using recombinant vaccinia virus expression system in order to study its biochemical properties. The results show that the ts36 AdPol is defective in the in vitro DNA replication initiation and DNA polymerase elongation assays if the protein is expressed at the nonpermissive temperature (37 degrees) independent of whether the assays are performed at 32 or 37 degrees. In contrast, the ts36 AdPol expressed at 32 degrees had activities similar to that of the wild-type protein when assayed at either temperature. Furthermore, unlike the wild-type protein, ts36 AdPol expressed at 37 degrees failed to recognize the viral DNA replication origin, but bound to a single-stranded DNA cellulose column with greater affinity, suggesting that the defect in the ts36 AdPol for DNA replication can be attributed to its altered DNA-binding properties.
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PMID:Biochemical characterization of a temperature-sensitive adenovirus DNA polymerase. 797 33

The polymerization and proofreading activities of the vaccinia virus DNA polymerase reside within a 116-kDa catalytic polypeptide. We report here an investigation of the intrinsic processivity of this enzyme on both natural and homopolymeric DNA templates. Inclusion of the Escherichia coli helix destabilizing protein allowed the viral enzyme, which lacks strand displacement activity, to utilize a singly primed M13 DNA template. In the presence of either 10 mM MgCl2 or 1 mM MgCl2 + 40 mM NaCl, synthesis was achieved in a highly distributive manner. RFII formation required a significant excess of enzyme, and < or = 10 nucleotides (nt) were added per primer-template binding event. The apparent rate of primer elongation varied with the enzyme/template ratio and reached a maximum of 8 nt/s. A similar lack of processivity was observed on a poly(dA390)-oligo(dT12-18) template. In contrast, highly processive synthesis was achieved on both templates in the presence of 1 mM MgCl2 and the absence of NaCl. A primer extension rate of 30 nt/s was observed, and > or = 2000 nt were added per binding event. These studies suggest that the catalytic polypeptide of the vaccinia virus DNA polymerase will require accessory protein(s) to form a stable enzyme-template interaction and direct processive DNA synthesis under isotonic conditions in vivo.
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PMID:Vaccinia virus DNA polymerase. In vitro analysis of parameters affecting processivity. 798 61

Several of the vaccinia virus core proteins are synthesized as large precursor proteins which are subsequently processed to smaller products during the course of viral maturation. Amino acid alignment of these proteins reveals a conserved Ala-Gly-X motif (AG*X) at their confirmed cleavage sites. To better understand the regulation of cleavage site selection, the sequence of the entire vaccinia virus genome was searched for the occurrence of this AG*X motif in predicted open reading frames. Of the 82 sites found, 19 resembled cleavage sites which have previously been shown to be actively processed, namely AG*A of P25K and P4b, and AG*S and AG*T of P4a. To test the universality of the AG*X motif utilization, immunological methods in concert with N-terminal microsequencing procedures have been used to determine which of the subset of predicted proteins containing AG*A sites are utilized in vivo. Of the seven AG*A-containing substrates, four were cleaved and three were not. Considering all the known AG*X processing events, it appears that only those proteins expressed at late times during infection and associated with the assembling virion are candidate substrates for proteolytic cleavage. Such proteins include P4a, P4b, P25K, and the newly identified P21K and P17K (derived from genes A17L and A12L, respectively). Although proteins such as DNA polymerase, P37K, and a host range protein contain a consensus cleavage site, they are excluded from processing. This proteolytic exclusion presumably occurs because these proteins do not meet both of the above criteria, which suggests that temporal expression or compartmentalization (substrate presentation) in the assembling virion may play a regulatory role in proteolysis.
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PMID:Differential utilization of a conserved motif for the proteolytic maturation of vaccinia virus proteins. 812 19

The vaccinia virus genome encodes a DNA polymerase that is similar to other DNA polymerases. A mutation in the polymerase gene at a site that is adjacent to conserved residues allows viral replication in the presence of aphidicolin. Since wild-type virus is converted to aphidicolin-resistance by site-directed mutagenesis, it was feasible that active virus with substituted conserved residues could be detected by linking alterations to the aphidicolin-resistance mutation. Altered DNA, from a PCR, was introduced into virus by a marker transfer procedure. DNA from plaques of drug-resistant virus was amplified, and the product was sequenced to check for the conserved residue alteration. An alteration that introduced a Bg1I site was designed to facilitate the selection of drug-resistant virus containing substituted residues. One positive result was the replacement of two amino acids, tyrosine and alanine, by tryptophan and threonine. The failure to substitute aspartic acid for tyrosine indicates that drastic changes of the conserved sequence are not tolerated. Although the limitations associated with negative results apply, the method provides an in vivo assay for selecting a polymerase with conserved residue changes.
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PMID:A biological method for examining the effect of codon changes in a conserved region of DNA polymerase. 813 25

We have carried out a detailed analysis of viral mRNAs and proteins produced in cultured cells infected with a temperature-sensitive vaccinia virus mutant (ts36) containing a modified nucleoside triphosphate phosphohydrolase I (NPH-I), a nucleic acid-dependent ATPase. Using a recombinant virus (ts36LUC) which expresses the luciferase marker, we showed in seven different cell lines that early expression of the receptor gene is strongly inhibited (73.8 to 98.7%) at the nonpermissive temperature. The steady-state levels of different early viral polypeptides were also severely reduced. Analysis of steady-state mRNA levels for two early genes (DNA polymerase and D5) showed that inhibition of early polypeptide synthesis correlated with a reduction in the levels of mRNA accumulated at the nonpermissive temperature. Analysis of steady-state levels of late viral polypeptides and of mRNAs indicated that NPH-I regulation of intermediate and late gene expression is direct and not simply a consequence of its role in inhibiting early gene expression. Characterization of a rescued virus (R36) demonstrated that the temperature-sensitive phenotype of ts36 is due solely to the point mutation in the NPH-I gene. The mutant phenotype is not due to reduced levels of NPH-I present in ts36 virions or to the differential stability of this enzyme in cells infected at the nonpermissive temperature but to inhibition of normal enzymatic activity for this protein. Measurement of viral transcriptional activity in permeabilized purified virions demonstrated that NPH-I is required for normal rates of transcription in vaccinia virus. Our findings show ts36 to be a strongly defective early mutant of vaccinia virus and prove that NPH-I plays a key role in the control of early and late virus gene expression, possibly by way of an auxiliary function which regulates mRNA transcription during the virus growth cycle.
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PMID:Vaccinia virus nucleoside triphosphate phosphohydrolase I controls early and late gene expression by regulating the rate of transcription. 823 Apr 76

Adenovirus preterminal protein (pTP) exists as a heterodimer with the viral DNA polymerase (AdPol) and becomes covalently linked to a dCMP residue during initiation of DNA replication. The in vivo phosphorylation of pTP could be demonstrated when pTP is overproduced using recombinant vaccinia viruses, or by a large scale metabolic labeling of adenovirus 2 (Ad2)-infected HeLa cells. Phosphoserine was the only phosphoamino acid obtained by acid hydrolysis of 32P-labeled pTP immunoprecipitated from metabolically labeled HeLa cells infected with either Ad2 or recombinant vaccinia virus. Tryptic peptide maps of pTP expressed using recombinant vaccinia virus system in HeLa cells revealed that phosphorylation of pTP occurred on multiple sites. Dephosphorylation of pTP with calf intestinal alkaline phosphatase resulted in a significant decrease in its activity in the in vitro DNA replication initiation assays. Further characterization of the phosphatase-treated pTP indicated that although dephosphorylation did not affect its interaction with AdPol, the specific recognition of the DNA replication origin by pTP was significantly reduced as determined by gel electrophoresis-based DNA mobility shift assays.
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PMID:Phosphorylation-dependent interaction of adenovirus preterminal protein with the viral origin of DNA replication. 829 75

Adenovirus DNA polymerase (AdPol) exists as a complex with the preterminal protein (pTP) and is essential for both initiation and elongation stages of viral DNA replication. Recent evidence from our laboratory indicates that AdPol is a phosphoprotein and that the major in vivo phosphorylation site, serine 67, occurs within the consensus substrate recognition sequence for cdc2 kinases. In this study, we found that a protein kinase which also exhibits histone H1 phosphorylation activity is stably associated with AdPol. AdPol forms a multimeric complex with this histone H1 kinase and pTP in HeLa cells infected with adenovirus or coinfected with recombinant vaccinia viruses encoding AdPol and pTP. The associated protein kinase and the p34cdc2 kinase phosphorylate AdPol at the same sites which are utilized in vivo, suggesting that the p34cdc2 kinase or a related kinase may be involved in the in vivo phosphorylation of AdPol. Serine 67 is also one of the major in vitro phosphorylation sites, and the substitution of alanine for serine at this position abolishes DNA replication initiation activity of AdPol.
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PMID:Adenovirus DNA polymerase is phosphorylated by a stably associated histone H1 kinase. 834 26


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