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)

Human herpesvirus 6 (HHV-6) is a newly identified lymphotropic herpesvirus. We have analyzed viral and host DNA replication in peripheral blood lymphocytes infected in the absence of drugs or infected in the presence of phosphonoacetic acid (PAA) or acyclovir (ACV). The results revealed the following: (i) Infection with HHV-6 resulted in the shutoff of host DNA replication. (ii) PAA at concentrations of 100 and 300 micrograms/ml significantly reduced virus replication. The drug inhibited viral DNA replication, whereas host cell DNA replication was not affected. This strongly suggests that HHV-6 encodes a PAA sensitive viral DNA polymerase. (iii) ACV at 20 microM did not interfere with virus production and virus spread. ACV at 100 microM only partly interfered with virus replication, whereas at 400 microM the block was more complete. Viral DNA replication was not affected by ACV at 20 microM. However, approximately 60 and 85% inhibition in viral DNA replication was observed in the presence of 100 and 400 microM of ACV. (iv) Assays for viral thymidine kinase (TK) revealed no significant increase in TK activity, whereas increased TK activity was noted following infection of the same peripheral blood lymphocytes with herpes simplex virus. Thus, either HHV-6 does not encode a tk enzyme which can phosphorylate ACV or the inefficient block may reflect lower sensitivity of the HHV-6 DNA polymerase to the drug.
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PMID:The replication of viral and cellular DNA in human herpesvirus 6-infected cells. 215 9

AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization.
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PMID:[Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs]. 216 89

Using indirect immunofluorescence, well-characterized monoclonal and polyclonal antibodies, and temperature-sensitive (ts) mutants of herpes simplex virus type 1, we demonstrated that the 65-kilodalton DNA-binding protein (65KDBP), the major DNA-binding protein (infected cell polypeptide 8 [ICP8]), and the viral DNA polymerase (Pol) colocalize to replication compartments in the nuclei of infected cells under conditions which permit viral DNA synthesis. When viral DNA synthesis was blocked by incubation of the wild-type virus with phosphonoacetic acid, the 65KDBP, Pol, and ICP8 failed to localize to replication compartments. Instead, ICP8 accumulated nearly exclusively to prereplication sites, while the 65KDBP was only diffusely localized within the nuclei. Although some of the Pol accumulated in prereplication sites occupied by ICP8 in the presence of phosphonoacetic acid, a significant amount of Pol also was distributed throughout the nuclei. Examination by double-labeling immunofluorescence of DNA- ts mutant virus-infected cells revealed that the 65KDBP also did not colocalize with ICP8 to prereplication sites at temperatures nonpermissive for virus replication. These results are in disagreement with the hypothesis that ICP8 is the major organizational protein responsible for attracting other replication protein to prereplication sites in preparation for viral DNA synthesis (A. de Bruyn Kops and D. M. Knipe, Cell 55:857-868, 1988), and they suggest that other viral proteins, perhaps in addition to ICP8, or replication fork progression per se are required to organize the 65KDBP.
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PMID:Localization of the herpes simplex virus type 1 65-kilodalton DNA-binding protein and DNA polymerase in the presence and absence of viral DNA synthesis. 217 66

A cell-free system that catalyzes DNA replication was prepared from cytoplasmic extracts of Vero cells infected with African swine fever virus (ASFV). The cells were permeabilized with lysolecithin and disrupted by mild mechanical action and the nuclei were removed by low-speed centrifugation. Extracts prepared from infected cells at the time of maximal DNA replication incorporated [alpha-32P]dTTP into acid-insoluble material that was sensitive to DNase and resistant to RNase. The reaction was inhibited by phosphonoacetic acid, an inhibitor of ASFV-specific DNA polymerase. Extracts from mock-infected cells had a negligible activity. Micrococcal nuclease-treated extracts were able to replicate added virion DNA or viral replicative DNA. An increase in the mass of DNA detected by ethidium bromide staining and by dot blot hybridization with ASFV DNA showed that the incorporation was due to true replication. Plasmid DNA was also replicated, which indicates that ASFV-specific DNA polymerase does not require a virus-specific origin of replication. The pattern of fragments generated by EcoRI digestion of the in vitro product was characteristic of viral replicative DNA. Hybridization with a recombinant plasmid containing a terminal fragment of ASFV DNA confirmed the presence of dimer terminal ASFV fragments presumably generated from concatemeric replicative intermediates.
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PMID:In vitro DNA replication by cytoplasmic extracts from cells infected with African swine fever virus. 221 42

5-(Phosphonomethyl)-1H-tetrazole and a number of related tetrazoles have been prepared and their effects on the replication of Herpes Simplex Viruses-1 and -2 have been investigated as well as their abilities to inhibit the DNA polymerases induced by these viruses and the RNA transcriptase activity of influenza virus A. Contrary to an earlier report, 5-(phosphonomethyl)-1H-tetrazole was not an efficient inhibitor of the replication of HSV-1 and HSV-2 in tissue culture. Analogues of 5-(phosphonomethyl)-1H-tetrazole were also devoid of significant antiviral activity. Only 5-(phosphonomethyl)-1H-tetrazole and 5-(thiophosphonomethyl)-1H-tetrazole inhibited the influenza virus transcriptase, and both were more effective as inhibitors than phosphonoacetic acid under the same conditions. The DNA polymerases induced by HSV-1 and HSV-2 were inhibited slightly by 5-(phosphonomethyl)-1H-tetrazole and to a lesser extent by its N-ethyl analogue and 3-(phosphonomethyl)-1H-1,2,4-triazole. None of these compounds were as effective as phosphonoacetic acid. 5-(Thiophosphonomethyl)-1H-tetrazole was a better inhibitor of the DNA polymerase induced by HSV-1 than 5-(phosphonomethyl)-1H-tetrazole.
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PMID:The antiviral activity of tetrazole phosphonic acids and their analogues. 241 98

The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1, encoded by gene UL42, is required for herpes simplex virus origin-dependent DNA replication (C.A. Wu, N.J. Nelson, D.J. McGeoch, and M.D. Challberg, J. Virol. 62:435-443, 1988). We found by indirect immunofluorescence with monoclonal antibody to 65KDBP that the protein was first detectable at 3 h postinfection. It localized first to the inner periphery of the nucleus, but accumulated in large globular compartments within the nucleus by 6 h postinfection in a pattern similar to that displayed by the major DNA-binding protein ICP8. Immune electron microscopy revealed that 65KDBP was associated with the marginated heterochromatin at the early times, but migrated further into the nucleus at late times when the only discernible areas devoid of 65KDBP were the nucleoli and heterochromatin. The 65KDBP gene is a member of the beta kinetic class as determined by the ability of the mRNA to be expressed at significant levels even in the absence of viral DNA synthesis. Furthermore, in the presence or absence of the DNA polymerase inhibitor phosphonoacetic acid, the patterns of accumulation of protein as well as mRNA were virtually indistinguishable from those displayed by the model beta genes encoding ICP8 and thymidine kinase. Nuclear run-on experiments demonstrated that maximum rates of 65KDBP gene transcription occurred prior to the maximum rate of progeny viral DNA synthesis and confirmed that the expression of the 65KDBP gene is regulated at the level of transcriptional initiation.
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PMID:Kinetics of expression of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1. 253 21

Freshly isolated or overnight cultured bovine peripheral blood mononuclear (PBM) cells lysed bovine herpes virus 1 (BHV-1)-infected allogeneic and xenogeneic target cells but not non-infected target cells. To determine if late viral genes contribute to target cell lysis, phosphonoacetic acid (PAA), an inhibitor of DNA polymerase activity, was used to block DNA replication that is required for expression of late viral proteins. Both adherent and non-adherent (NA) cell populations mediated lysis against PAA-treated BHV-1-infected target cells in both 4- and 20-hr assays, indicating recognition and killing occurred in the absence of expression of late BHV-1 glycoproteins. Thus recognition of BHV-1 by bovine natural cytolytic effector cells does not require recognition of late BHV-1 glycoproteins for killing virally infected target cells.
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PMID:Bovine naturally cytolytic cell activation against bovine herpes virus type 1-infected cells does not require late viral glycoproteins. 254 Oct 73

A series of herpes simplex virus isolates were recovered from a bone marrow transplant patient who received prolonged acyclovir therapy for indolent herpes simplex mouth and throat ulceration. Of 14 isolates received 10 were resistant to acyclovir and partially resistant to phosphonoacetic acid. Biochemical characterization revealed that resistance was due to an alteration in the virus DNA polymerase. DNA sequence analysis of the polymerase gene of a plaque-purified resistant virus isolate revealed a single nucleotide change when compared with the sequence of the gene of a plaque-purified sensitive isolate. This single base change resulted in a predicted amino acid substitution of Gly to Ser at residue number 841, a putative functional region of the polymerase.
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PMID:Characterization of a DNA polymerase mutant of herpes simplex virus from a severely immunocompromised patient receiving acyclovir. 254 43

DNA-mediated transfer of a drug resistance marker (phosphonoacetate resistance) for the HCMV (Towne) DNA polymerase (pol) gene has been used to genetically confirm the physical localization of the HCMV (Towne) DNA pol gene. The HCMV (Towne) genomic region homologous to the pol genes of other herpesviruses was first identified by moderate stringency Southern hybridization. Restriction fragments from this region were molecularly cloned from previously characterized phosphonoacetic acid resistant (PAAr) HCMV genomes (R. T. D'Aquilla and W. C. Summers, J. Virol., 61, 1291-1295, 1987). A high frequency of recombinant PAAr virus was found among the progeny of cotransfections of infectious, wild-type HCMV (Towne) DNA only with pol-homologous restriction fragments cloned from PAAr HCMV. The co-transfection technique described here may facilitate further gene mapping in HCMV. The results presented here provide functional proof that the HCMV pol gene is encoded by the sequences previously identified as homologous to other herpesvirus pool genes.
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PMID:Physical mapping of the human cytomegalovirus (HCMV) (Towne) DNA polymerase gene: DNA-mediated transfer of a genetic marker for an HCMV gene. 254 37

A series of clinical isolates of herpes simplex virus type 2 were taken from a patient with chronic lymphocytic leukemia. Acyclovir (ACV) susceptibility assays revealed that some isolates were resistant to ACV and cross-resistant to ganciclovir but not to phosphonoacetic acid. The nature of the resistance was examined further. A number of cloned variants were generated, and thymidine kinase and DNA polymerase assays were carried out. Variants that were resistant to ACV were found to be thymidine kinase deficient. Evidence for alteration in the DNA polymerase was not found when ACV triphosphate or phosphonoacetic acid was used as the inhibitor. In vivo studies with the plaque-purified viruses showed that ACV resistance was associated with a reduced neurovirulence. In a zosteriform model, virus resistant to ACV was unable to induce secondary spread in the same dermatome, to invade the peripheral nervous system or the central nervous system, or to establish latent infections.
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PMID:Biological and biochemical characterization of clinical isolates of herpes simplex virus type 2 resistant to acyclovir. 254 86


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