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)

In contrast to cellular or SV40 DNA replication, adenovirus type 5 (Ad5) or type 2 (Ad2) DNA synthesis in isolated nuclei is strongly inhibited by low concentrations of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP). On the basis of differential sensitivity of cellular DNA polymerases, a role of DNA polymerase gamma in adenovirus DNA replication has been proposed. We have investigated the mechanism of inhibition of adenovirus DNA synthesis, using [alpha-32P]ddTTP and other dNTP analogues. Both ddATP and ddGTP were as inhibitory as ddTTP, while ddCTP had an even stronger effect on adenovirus DNA replication. DNA polymerase alpha was resistant to all four ddNTP's, while DNA polymerase gamma was very sensitive. The inhibition by ddTTP in isolated infected nuclei was slowly reversible. [alpha-32P]ddTTP was incorporated into Ad5 DNA as a chain-terminating nucleotide, and the analogue could be used as a substrate by DNA polymerase gamma. Under similar conditions, incorporation in cellular DNA or using DNA polymerase alpha was not observed. The nucleoside analogues ddA and ddC suppressed adenovirus. DNA replication in intact cells and reduced plaque formation. These results provide further evidence for a function of DNA polymerase gamma in adenovirus DNA synthesis.
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PMID:Role of DNA polymerase gamma in adenovirus DNA replication. Mechanism of inhibition by 2',3'-dideoxynucleoside 5'-triphosphates. 723 28

Adenoviral vectors have recently been shown to effectively deliver genes into a variety of tissues. Since these vectors have some advantages over the more extensively investigated retroviruses, we studied the effect of two replication-defective adenovectors bearing human wild type tumor suppressor gene p53 (Adp53) and Escherichia coli beta-galactosidase gene (AdLacZ) on 9L glioma cells. Successful in vitro gene transfer was shown by DNA polymerase chain reaction (PCR), and expression was confirmed by reverse transcriptase RNA PCR and Western blot analyses. Transduction of 9L cells with the Adp53 inhibited cell growth and induced phenotypic changes consistent with cell death at low titers, while AdLacZ caused cytopathic changes only at high titers. Stereotactic injection of AdLacZ (10(7) plaque forming units) into tumor bed stained 25 to 30% of tumor cells at the site of vector delivery. Injection of Adp53 (10(7) plaque forming units), but not AdLacZ (controls), into established 4-day old 9L glioma brain tumors decreased tumor volume by 40% after 14 days. As a step toward gene therapy of brain tumors using replication-defective adenoviruses, these data support the use of tumor suppressor gene transfer for in vivo treatment of whole animal brain tumor models.
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PMID:Adenovirus-mediated p53 gene delivery inhibits 9L glioma growth in rats. 764 77

Resistance of human cytomegalovirus to approved antiviral drugs is becoming a problem of increasing concern. In order to further study drug resistance in a related virus, strains of murine cytomegalovirus (MCMV) have been prepared in vitro by extensive adaptation of the virus to increasingly higher concentrations of either ganciclovir, foscarnet, or (S)-9-(3-hydroxy-2-[phosphonylmethoxy]propyl)cytosine (HPMPC). Plaque reduction 50% effective concentrations (EC50) for the above inhibitors increased 9-, 7-, and 23-fold, respectively (against the corresponding virus), compared to wild-type MCMV. Each virus was then evaluated against other known anti-MCMV agents to determine cross-resistance patterns. These compounds included 3-hydroxy-phosphonylmethoxypropyl derivatives of adenine (HPMPA) and guanine (HPMPG), 2-phosphonylmethoxyethyl derivatives of adenine (PMEA) and 2,6-diaminopurine (PMEDAP), cyclobutylguanine, acyclovir, and the methylene phosphonate derivatives of acyclovir (SR3722) and ganciclovir (SR3773). The ganciclovir-resistant MCMV was cross-resistant to foscarnet, HPMPA, HPMPC, HPMPG, SR3722, and SR3773. The foscarnet-resistant virus was also resistant to acyclovir, PMEA, PMEDAP, SR3722, and SR3773. The HPMPC-resistant MCMV was cross-resistant to HPMPA, HPMPG, and SR3773. Changes in susceptibility were from 3- to 22-fold relative to the wild-type virus. Virus yield reduction data correlated with the plaque assay results. Only cyclobutylguanine was approximately equally active against wild-type and the three drug-resistant MCMVs. The patterns of cross-resistance correlated with resistance seen in human cytomegalovirus strains expressing altered DNA polymerase function. The GCV-resistant and HPMPC-resistant viruses were markedly attenuated in their ability to kill severe combined immunodeficient mice.
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PMID:Antiviral activities of nucleosides and nucleotides against wild-type and drug-resistant strains of murine cytomegalovirus. 774 17

Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo[a]pyrene, is a mutagenic in both bacterial and mammalian systems. Toward understanding the mutagenic effects of different stereoisomers of BPDE at specific sites in DNA, six stereochemically defined BPDE adducts were constructed on adenine N6 at position 2 of the human N-ras 61 codon within an 11-base oligonucleotide fragment. Both the nonadducted and BPDE-adducted N-ras 61 11-mers were inserted into a unique EcoRI site in single-stranded M13mp7L2 DNA and utilized for in vivo studies. The ligation efficiencies of BPDE-adducted 11-mers into the single-stranded vector were determined by Southern hybridization and confirmed by electron microscopy. Repair-deficient AB2480 E. coli cells were transformed with adducted and non-adducted DNA samples. The resultant plaque-forming abilities were used to evaluate the replication competence of the various BPDE adducts with respect to the nonadducted 11-mer. Point mutations due to aberrant replication at the adducted site were identified by the technique of differential DNA hybridization. All of the six BPDE adducts examined were mutagenic in vivo, generating exclusively A-->G mutations at frequencies ranging from 0.26 to 1.20%. In vitro replication studies using these BPDE-adducted 11-mers involved primer extension assays with Klenow fragment. All of the BPDE-modified templates demonstrated distinct blockage at the adducted site and/or 1 base 3' to the adducted site, allowing essentially no translesion synthesis to form fully extended polymerization products in vitro.
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PMID:In vivo and in vitro replication consequences of stereoisomeric benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide adducts on adenine N6 at the second position of N-ras codon 61. 789 Jun 5

Single residues of O6-methylguanine (O6-meG) were introduced into the first or second position of codon 12 (GGC; positions 12G1 or 12G2, respectively) or the first position of codon 13 (GGT; position 13G1) of the human Ha-ras oncogene in phage M13-based vectors. After transformation of E.coli, higher mutant plaque frequencies (MPF) were observed at 12G1 and 13G1 than at 12G2 if O6-alkylguanine-DNA alkyltransferase (AGT) had been depleted, while similar MPF were observed at all three positions in the presence of active AGT. Taken together, these observations suggest reduced AGT repair at 12G2. Kinetic analysis of in vitro DNA replication in the same sequences using E. coli DNA polymerase I (Klenow fragment) indicated that variation in polymerase fidelity may contribute to the overall sequence specificity of mutagenesis. By constructing vectors which direct methyl-directed mismatch repair to the (+) or the (-) strand and comparing the MPF values in bacteria proficient or deficient in mismatch repair and/or AGT, it was concluded that, while mutS-mediated mismatch repair did not remove O6-meG from O6-meG:C pairs, this repair mechanism can affect O6-meG mutagenesis by repairing G:T pairs generated through AGT-induced demethylation of O6-meG:T replication intermediates.
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PMID:Comparative study of mutagenesis by O6-methylguanine in the human Ha-ras oncogene in E. coli and in vitro. 793 3

In order to deduce the biological fate of adducts formed by the reaction of styrene oxide, a suspected carcinogen, with DNA, four oligodeoxynucleotides were synthesized which contained either R- or S-styrene oxide lesions on the N6 position of neighboring adenines within the human N-ras codon 61. When these adducted oligodeoxynucleotides were ligated into the single-stranded vector M13mp7L2 and the modified DNA used to transform repair-deficient Escherichia coli, the resultant plaque-forming abilities were found to vary as much as 300-fold, depending on the stereochemical configuration of the styrene oxide lesion and the sequence context in the vicinity of the damage. The frequency of mutations caused by the various styrene oxide adducts were similarly dependent on both their chirality and local sequence context. Oligodeoxynucleotide templates bearing these same four adducts were also constructed in order to evaluate their replication in vitro by the Klenow fragment. Three of the four styryl-modified templates yielded significant levels of fully extended primer upon polymerization. In contrast, the template containing R-styrene oxide at the second position of N-ras 61 was a very poor substrate for replication, a result which correlates well with the observed lethality of this lesion in vivo.
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PMID:The replication fate of R- and S-styrene oxide adducts on adenine N6 is dependent on both the chirality of the lesion and the local sequence context. 822 68

We have investigated the pathogenicity of a US3 protein kinase-deficient mutant (L1 BR1) of herpes simplex virus type 2 (HSV-2) for 4-week-old ICR mice to define the role of the viral protein kinase in virus-host interaction. When mice were intraperitoneally infected with 10(5)PFU of L1 BR1, the virus disappeared from the peritoneal cavity by 2 days postinfection and failed to induce any significant histopathological changes in the liver and spleen although viral antigens were occasionally detected in the epithelial cells of small bile ducts and small vascular wall. The parental virus (HSV-2 186) and a revertant of the mutant (L1 B-11) both caused severe hepatitis, and viral antigens were clearly detected in the hepatocytes and Kupffer cells in the focal necrotic areas. Both of the virulent viruses, unlike L1 BR1, could produce infectious progeny and cytopathic effects in freshly harvested peritoneal macrophages. The growth of L1 BR1 in peritoneal macrophages was restricted at a stage of or prior to viral DNA synthesis but after the induction of viral DNA polymerase. In addition, the production and/or the spread of mutant in mouse embryo fibroblasts (MEF) was found to be much more effectively suppressed by cocultivation of peritoneal macrophages than that of 186. An almost complete inhibition of L1 BR1-plaque formation was observed at a macrophage-to-MEF ratio of 4:1. These results suggest that the attenuation of L1 BR1 following intraperitoneal infection is primarily due to its high sensitivity to intrinsic and extrinsic inhibition of peritoneal macrophages and that the US3 protein kinase may play a role in viral DNA replication in peritoneal macrophages.
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PMID:The pathogenicity of a US3 protein kinase-deficient mutant of herpes simplex virus type 2 in mice. 825 88

Susceptibility of Oka varicella vaccine virus to antiherpetic drugs was determined by the effective dose for 50% plaque reduction (ED50) using cell-free virus preparation. ED50 values were 3.02 microM for acyclovir, 3.72 microM for vidarabine, 0.0035 microM for sorivudine, and 4.67 microM for penciclovir. Oka varicella vaccine virus was as susceptible to these drugs as wild-type viruses. Sensitivity of thymidine kinase (TK)-deficient virus to penciclovir and of some DNA polymerase (DPase) mutants to sorivudine suggested that these drugs might be used for the treatment of vaccine recipients, even if Oka varicella vaccine became acyclovir-resistant by mutations in the TK or DPase genes, respectively. This result encourages the wider use of Oka varicella vaccine even for immunocompromised hosts because of its attenuation and susceptibility to chemotherapeutic drugs.
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PMID:Susceptibility of Oka varicella vaccine strain to antiviral drugs. 831 Jul 56

In this report, we describe the isolation, molecular genetic mapping, and phenotypic characterization of vaccinia virus mutants resistant to cytosine arabinoside (araC) and phosphonoacetic acid (PAA). At 37 degrees C, 8 microM araC was found to prevent macroscopic plaque formation by wild-type virus and to cause a 10(4)-fold reduction in viral yield. Mutants resistant to 8 microM araC were selected by serial passage of a chemically mutagenized viral stock in the presence of drug. Because recovery of mutants required that initial passages be performed under less stringent selective conditions, and because plaque-purified isolates were found to be cross-resistant to 200 micrograms of PAA per ml, it seemed likely that resistance to araC required more than one genetic lesion. This hypothesis was confirmed by genetic and physical mapping of the responsible mutations. PAAr was accorded by the acquisition of one of three G-A transitions in the DNA polymerase gene which individually alter cysteine 356 to tyrosine, glycine 372 to aspartic acid, or glycine 380 to serine. AraCr was found to require one of these substitutions plus an additional T-C transition within codon 171 of the DNA polymerase gene, a change which replaces the wild-type phenylalanine with serine. Congenic viral stocks carrying one of the three PAAr lesions, either alone or in conjunction with the upstream araCr lesion, in an otherwise wild-type background were generated. The PAAr mutations conferred nearly complete resistance to PAA, a slight degree of resistance to araC, hypersensitivity to aphidicolin, and decreased spontaneous mutation frequency. Addition of the mutation at codon 171 significantly augmented araC resistance and aphidicolin hypersensitivity but caused no further change in mutation frequency. Several lines of evidence suggest that the PAAr mutations primarily affect the deoxynucleoside triphosphate-binding site, whereas the codon 171 mutation, lying within a conserved motif associated with 3'-5' exonuclease function, is postulated to affect the proofreading exonuclease of the DNA polymerase.
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PMID:Genetic characterization of the vaccinia virus DNA polymerase: cytosine arabinoside resistance requires a variable lesion conferring phosphonoacetate resistance in conjunction with an invariant mutation localized to the 3'-5' exonuclease domain. 838 30

African swine fever virus (ASFV) growth and plaque formation were inhibited by phosphonoacetic acid (PAA) concentrations of 200 micrograms/ml or more. One spontaneous mutant and two mutants isolated from mutagenized virus were resistant to PAA inhibition and showed practically normal viral DNA synthesis in the presence of PAA. DNA polymerase activity present in the cytoplasmic fraction from cells infected with the mutants required 10-fold higher concentrations of PAA for inhibition compared to equivalent inhibition of the wild-type enzyme. Like wild-type virus, the PAA-resistant mutants were resistant to inhibition by aphidicolin. Marker rescue analysis with mutant DNA fragments covering different regions of the ASFV DNA polymerase gene mapped the mutations within a fragment which was cloned and sequenced. A single nucleotide and amino acid change was assigned to each mutant. Two of the PAA-resistant mutations lie within the highly conserved region II common to alpha-like DNA polymerases, which has been implicated in pyrophosphate binding and probably also in dNTP binding. The other mutation was localized to within a region of moderate homology among viral DNA polymerases close to one of the motifs allegedly considered as constituting the 3'-5' exonuclease active site.
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PMID:Functional and molecular characterization of African swine fever virus mutants resistant to phosphonoacetic acid. 852 40


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