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)

We describe the mapping and sequencing of mutations within the DNA polymerase gene of herpes simplex virus type 1 which confer resistance to aphidicolin, a DNA polymerase inhibitor. The mutations occur near two regions which are highly conserved among DNA polymerases related to the herpes simplex enzyme. They also occur near other herpes simplex mutations which affect the interactions between the polymerase and deoxyribonucleoside triphosphate substrates. Consequently, we argue in favor of the idea that the aphidicolin binding site overlaps the substrate binding site and that the near-by conserved regions are functionally required for substrate binding. Our mutants also exhibit abnormal sensitivity to another DNA polymerase inhibitor, phosphonoacetic acid. This drug is thought to bind as an analogue of pyrophosphate. A second-site mutation which suppresses the hypersensitivity of one mutant to phosphonoacetic acid (but not its aphidicolin resistance) is described. This second mutation may represent a new class of mutations, which specifically affects pyrophosphate, but not substrate, binding.
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PMID:Aphidicolin resistance in herpes simplex virus type I reveals features of the DNA polymerase dNTP binding site. 255 88

The frequency of recombination between transfected plasmid DNAs was measured by using cultured cells infected with a variety of poxviruses. Plasmid derivatives of pBR322 containing XhoI linker insertion mutations in the tetracycline gene were used to assess recombination frequencies in rabbit cells infected with the leporipoxviruses Shope fibroma virus and myxoma virus and the orthopoxvirus vaccinia virus. Recombination frequencies were calculated by Southern blotting, which detects novel plasmid restriction fragments generated by genetic recombination, and by a plasmid rescue procedure in which the reconstruction of an intact tetracycline gene in the transfected rabbit cell was monitored by transformation back into Escherichia coli. The highest recombination frequencies were measured in cells infected with Shope fibroma virus and myxoma virus, and a minimum recombination frequency of at least one recombination event per 7 kilobases was calculated within 24 h posttransfection under these conditions. The deduced recombination frequency in vaccinia virus-infected cells was at least fivefold lower and was not detectable in mock-infected cells, suggesting that the induced recombination activity detected by these methods was under viral control. The results of kinetic studies, analysis with methylation-sensitive restriction enzymes, and the use of phosphonoacetic acid, a specific inhibitor of poxvirus DNA polymerase, indicated that recombination between transfecting DNAs occurred concomitantly with DNA replication but that the two processes could be partially uncoupled. We conclude that the dramatic expansion of recombination activities in the cytoplasm of poxvirus-infected cells is virus specific and offers a good model system with which to analyze the mechanism of recombination in a eucaryotic environment.
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PMID:High levels of genetic recombination among cotransfected plasmid DNAs in poxvirus-infected mammalian cells. 282 1

Primary sympathetic neuronal cultures were maintained for up to 5 weeks after inoculation with herpes simplex virus (HSV) without evidence of viral infection. Treatment with acyclovir for the first 7 days after viral inoculation prevented lytic infections in 100% of the cultures and resulted in viral latency in 100% of the cultures; reactivation occurred as the result of nerve growth factor (NGF) deprivation. Treatment of the cultures with several different inhibitors of viral DNA polymerase (acyclovir, aphidicolin, and phosphonoacetic acid) for 7 days after viral inoculation did not prevent the establishment of latency, which suggests that viral DNA replication was not required. During the latent phase of the infection, viral antigens were not detected with HSV-specific immunohistochemistry. However, 24 h after NGF deprivation, viral antigens were detected in essentially all of the neurons, indicating that the majority of neurons harbored latent HSV. The establishment of latency was not strain or type specific since latency was established with HSV type 2 and four strains of HSV type 1 and reactivation occurred in response to NGF deprivation.
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PMID:Characterization of nerve growth factor-dependent herpes simplex virus latency in neurons in vitro. 282 4

Phosphonoformic acid (PFA) and its congener phosphonoacetic acid (PAA) are inhibitors of viral replication whose mechanism of action appears to be the inhibition of viral DNA polymerase. These drugs inhibit mammalian DNA polymerase to a lesser extent. We sought to characterize the effects of phonoformic acid on mammalian cells by examining mutants of S49 cells (a mouse T-lymphoma line), which were selected by virtue of their resistance to phosphonoformic acid. The 11 mutant lines that were resistant to growth inhibition by 3 mM PFA had a range of growth rates, cell cycle distribution abnormalities, and resistance to the inhibitory effects of thymidine, acycloguanosine (acyclovir), aphidicolin, deoxyadenosine, and novobiocin. Most mutant lines had pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates similar to those of wild-type S49 cells. However, one line (PFA 3-9) had a greatly elevated dCTP pool. When this mutant line was further characterized, no apparent defect in DNA polymerase alpha activity was seen, but an increased ribonucleotide reductase activity, as assayed by CDP reduction in permeabilized cells, was observed. The CDP reductase activity in the PFA 3-9 cells decreased to wild-type control levels, and the CDP reductase activity of wild-type cells was also greatly reduced when PFA (2-3 mM) was added to permeabilized cells during the enzyme assay. These results demonstrate that PFA can directly inhibit ribonucleotide reductase activity in permeabilized cells. In addition, when PFA was added to exponentially growing cultures of either wild-type or PFA 3-9 mutant cells, the drug caused an arrest in S phase of the cell cycle and a decrease in all four deoxyribonucleotide pools, with the most dramatic decrease in the dCTP pools. The reduction in the dCTP pool level could be reversed by addition of exogenous deoxycytidine, but this reversed PFA toxicity only marginally. These observations suggest that PFA is an inhibitor of mammalian ribonucleotide reductase and that partial resistance to PFA can be effected by mutation to increased CDP reductase activity resulting in a large dCTP pool. This mutation results in less than twofold resistance to PFA, suggesting that other sites of inhibition coexist.
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PMID:Selection and characterization of mutant S49 T-lymphoma cell lines resistant to phosphonoformic acid: evidence for inhibition of ribonucleotide reductase. 293 95

Selective DNA extraction and hybridization procedures were used to estimate the relative number of covalently closed circular viral genomes in cultures of Epstein-Barr virus (EBV)-transformed cells. In virus-producing P3HR-1 cultures that were exposed for 11 days to phosphonoacetic acid or to acyclovir, the content of covalently closed circular EBV DNA was reduced ca. 70% relative to a control culture without drug. The EBV plasmid content of Raji, a virus nonproducer cell line, was not reduced by exposure to these compounds. When P3HR-1 cultures were exposed to 12-O-tetradecanoylphorbol-13-acetate, the number of circular genomes per cell increased. These findings indicate that two enzyme activities synthesize circular EBV DNA and that the virus-associated DNA polymerase synthesizes most of the circular EBV DNA in a virus producer culture. It is suggested that the circular genomes synthesized by the viral enzyme are intermediates in the syntheses of linear virus DNA.
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PMID:The circular intracellular form of Epstein-Barr virus DNA is amplified by the virus-associated DNA polymerase. 298 82

Five herpes simplex virus mutants containing temperature-sensitive mutations in the gene for the major DNA-binding protein were assayed for their sensitivities to the DNA polymerase inhibitors aphidicolin and phosphonoacetic acid (PAA). Four of the mutants (tsA1, tsA15, tsA24, and tsA42) exhibited altered sensitivity to one or both of the inhibitors relative to the wild-type parent. In tsA1, a mutation or mutations conferring aphidicolin and PAA hypersensitivity were mapped by corescue with the temperature-sensitivity marker of tsA1 to a region of the DNA-binding protein locus, between map coordinates 0.385 and 0.398. The mutation conferring PAA hypersensitivity in tsA24 similarly corescued with the tsA24 temperature-sensitivity marker, mapping to the DNA-binding protein locus between coordinates 0.398 and 0.413. Thus, mutations outside the DNA polymerase locus and within the DNA-binding protein locus can confer altered sensitivity to certain DNA polymerase inhibitors. Assays of the aphidicolin and PAA sensitivities of ts+ recombinants derived by marker rescue of the DNA-binding protein mutants revealed the presence of additional mutations, separable from the ts mutations, in each of three mutants examined. One such mutation, which contributed to the aphidicolin-hypersensitivity phenotype of tsA1, mapped between coordinates 0.422 and 0.448, and resides, most probably, within the DNA polymerase locus. These additional mutations possibly confer compensating modifications to the DNA polymerase such that functional interaction with altered DNA-binding protein is restored. These findings provide strong evidence that the major DNA-binding protein and the DNA polymerase of herpes simplex virus interact in infected cells.
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PMID:Mutations in the herpes simplex virus major DNA-binding protein gene leading to altered sensitivity to DNA polymerase inhibitors. 299 51

Virus-nonproducer Raji cells, when induced to early antigen synthesis by 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate, showed an increase in DNA polymerase activity. This enzyme has the characteristics of a typical Epstein-Barr virus DNA polymerase with regard to chromatographical pattern and biological properties: it is eluted from DEAE-cellulose at 0.08 M NaCl, has a high salt resistance, is sensitive to phosphonoacetic acid and phosphonoformate, and shows a substrate preference for poly(dC)-oligo(dG12-18). The resistance of Epstein-Barr virus polymerase activity to aphidicolin is a property distinct from that of HSV DNA polymerase. Viral DNA polymerase activity increases in the absence of Epstein-Barr virus DNA replication, indicating that this enzyme is an early viral protein.
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PMID:Epstein-Barr virus-specific DNA polymerase in virus-nonproducer Raji cells. 300 79

Cross-resistance data for a group of nine acyclovir-resistant variants of herpes simplex virus type 1 are reported. These mutants, which express either altered thymidine kinase (TK) or DNA polymerase, were all derived from the same wild-type (wt) strain after exposure to acyclovir in tissue culture. Furthermore, all variants have pathogenic properties similar to the wt parental strain as assessed using mouse model systems (G. Darby, H.J. Field, and S.A. Salisbury, Nature (London) 289:81-83, 1981; B.A. Larder and G. Darby, Virology 146:262-271, 1985). Two groups of antiherpes compounds were used: those requiring activation by TK and those whose action is independent of that enzyme. The TK substrate-specificity mutants were generally resistant to the TK-activated drugs but showed wt susceptibility to phosphonoacetic acid, 9-beta-D-arabinofuranosyladenine, and aphidicolin. The DNA polymerase mutants were relatively susceptible to most TK-activated drugs, although two were resistant to 5-(trifluoromethyl)-2'-deoxyuridine. The polymerase mutants showed a more complex pattern of susceptibility, however, to those compounds whose mode of action is independent of TK. In general, these variants showed similar responses to phosphonoacetic acid, phosphonoformate, and 9-beta-D-arabinofuranosyladenine, a particular variant being either resistant, susceptible, or hypertensive to all three. The response of each variant to aphidicolin, however, appeared to be the inverse of its response to the other three drugs. The cross-resistance patterns are discussed, and their implications for combined or successive therapies are considered.
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PMID:Susceptibility to other antiherpes drugs of pathogenic variants of herpes simplex virus selected for resistance to acyclovir. 301 9

The DNA polymerase activity, and susceptibilities to 9-beta-D-arabinofuranosyladenine(ara-A) and 1-beta-arabinofuranosylcytosine(ara-C) of a phosphonoacetic acid resistant mutant (PAA-R) of varicella-zoster virus (VZV) selected in the presence of PAA were examined. The DNA polymerase activity of PAA-R was inhibited less than that of the parent strain by PAA in vitro. PAA-R was resistant to acyclovir and also to both ara-A and ara-C. The susceptibilities to ara-A and ara-C of four acyclovir resistant mutants selected in the presence of acyclovir, and also resistant to PAA, were examined. Two variants were resistant, one was slightly resistant, and one was sensitive to both drugs. These cross-resistances and susceptibilities of VZV variants to PAA, ACV, ara-A and ara-C should be considered in chemotherapy of VZV infections.
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PMID:Susceptibilities of phosphonoacetic acid and acyclovir resistant varicella-zoster virus mutants to 9-beta-arabinofuranosyladenine and 1-beta-arabinofuranosylcytosine. 302 9

An aphidicolin-resistant (Aphr) mutant of herpes simplex virus (HSV) type 2 strain 186 previously has been shown to induce an altered viral DNA polymerase that is more resistant to aphidicolin and more sensitive to phosphonoacetic acid (PAA) than is wild-type DNA polymerase. In this study the mutation responsible for the aphidicolin-resistant phenotype was physically mapped by marker transfer experiments. The physical map limits for the Aphr mutation were contained in a 1.1-kilobase pair region within the HSV DNA polymerase locus. The 1.1-kilobase-pair fragment of the Aphr mutant also conferred hypersensitivity to PAA, and DNA sequence analysis revealed an AT to GC transition within this fragment of the Aphr mutant. Analysis of the three potential open reading frames within the 1,147-base-pair fragment and comparison with the amino acid sequence of DNA polymerase of HSV type 1 indicated that the Aphr mutant polymerase had an amino acid substitution from a tyrosine to a histidine in the well-conserved region of the DNA polymerase. These results indicate that this single amino acid change can confer altered sensitivity to aphidicolin and PAA and suggest that this region may form a domain that contains the binding sites for substrates, PPi, and aphidicolin.
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PMID:A single-base change within the DNA polymerase locus of herpes simplex virus type 2 can confer resistance to aphidicolin. 302 69


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