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)

Vaccinia virus infection results in the synthesis of a protein that promotes joint molecule formation and strand-transfer reactions in vitro. We show here that this activity is also expressed by vaccinia DNA polymerase (gpE9L). Recombinant vaccinia polymerase was produced using a hybrid vaccinia/T7 expression system and purified to homogeneity. This protein catalyzed joint molecule formation and strand transfer in vitro in reactions containing single-stranded circular and linear duplex DNAs. The reaction required homologous substrates and magnesium ions and was stimulated by DNA aggregating agents such as spermidine HCl and Escherichia coli single-strand DNA binding protein. There was no requirement for a nucleoside triphosphate cofactor. The reaction ceased when approximately 20% of the double-stranded substrate had been incorporated into joint molecules and required stoichiometric quantities of DNA polymerase (0.5-1 molecules of polymerase per double-stranded DNA end). Electron microscopy showed that the joint molecules formed during these reactions contained displaced strands and thus represented the products of a strand-exchange reaction. We also reexamined the link between replication and recombination using a luciferase-based transfection assay and cells infected with DNA polymerase Cts42 mutant viruses. These data substantiate the claim that there exists an inextricable link between replication and recombination in poxvirus-infected cells. Together, these biochemical and genetic data suggest a way of linking poxviral DNA replication with genetic recombination.
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PMID:Vaccinia virus DNA polymerase promotes DNA pairing and strand-transfer reactions. 1032 61

The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases. In order to investigate this potential kinase activity, BGLF4 was expressed in Escherichia coli and the purified protein was used to generate a specific antiserum. Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter. Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay. In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells. Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity. BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay. BGLF4 can phosphorylate histone and casein in vitro. Among the potential viral protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro. Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4.
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PMID:A protein kinase activity associated with Epstein-Barr virus BGLF4 phosphorylates the viral early antigen EA-D in vitro. 1070 24

An enhancer-like element VV16 from Vaccinia virus genome DNA was obtained by using the plasmid with CAT reporter gene. Sequence analysis showed the element of 112 bp is a part of the DNA-dependent RNA polymerase, polyA polymerase and DNA polymerase (RPO30 gene). It contains 4 AT-rich regions. Detection of beta-galactosidase activity showed that VV16 in the positive direction can increase the activity 9.0 times and VV16 in the negative direction can increase 4.1 times. The RNA dot blotting confirmed the enhancing activity of the element are on the transcription level. DNA deletion experiment indicated the sequences of 10 bp at the 5' end and 12 bp at the 3' end in the element are important to its function and the sequence from nt76 to nt82 is essential to its activity.
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PMID:[Functional and structural study of the prokaryotic enhancer-like element VV16 from vaccinia virus genome]. 1105 76

During poxvirus infection, both viral genomes and transfected DNAs are converted into high-molecular-weight concatemers by the replicative machinery. However, aside from the fact that concatemer formation coincides with viral replication, the mechanism and protein(s) catalyzing the reaction are unknown. Here we show that vaccinia virus DNA polymerase can catalyze single-stranded annealing reactions in vitro, converting linear duplex substrates into linear or circular concatemers, in a manner directed by sequences located at the DNA ends. The reaction required > or =12 bp of shared sequence and was stimulated by vaccinia single-stranded DNA-binding protein (gpI3L). Varying the structures at the cleaved ends of the molecules had no effect on efficiency. These duplex-joining reactions are dependent on nucleolytic processing of the molecules by the 3'-to-5' proofreading exonuclease, as judged by the fact that only a 5'-(32)P-end label is retained in the joint molecules and the reaction is inhibited by dNTPs. The resulting concatemers are joined only through noncovalent bonds, but can be processed into stable molecules in E. coli, if the homologies permit formation of circular molecules. This reaction provides a starting point for investigating the mechanism of viral concatemer formation and can be used to clone PCR-amplified DNA.
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PMID:In vitro concatemer formation catalyzed by vaccinia virus DNA polymerase. 1111 78

Replicating poxviruses catalyze high-frequency recombination reactions by a process that is not well understood. Using transfected DNA substrates we show that these viruses probably use a single-strand annealing recombination mechanism. Plasmids carrying overlapping portions of a luciferase gene expression cassette and luciferase assays were first shown to provide an accurate method of assaying recombinant frequencies. We then transfected pairs of DNAs into virus-infected cells and monitored the efficiencies of linear-by-linear, linear-by-circle, and circle-by-circle recombination. These experiments showed that vaccinia virus recombination systems preferentially catalyze linear-by-linear reactions much more efficiently than circle-by-circle reactions and catalyze circle-by-circle reactions more efficiently than linear-by-circle reactions. Reactions involving linear substrates required surprisingly little sequence identity, with only 16-bp overlaps still permitting approximately 4% recombinant production. Masking the homologies by adding unrelated DNA sequences to the ends of linear substrates inhibited recombination in a manner dependent upon the number of added sequences. Circular molecules were also recombined by replicating viruses but at frequencies 15- to 50-fold lower than are linear substrates. These results are consistent with mechanisms in which exonuclease or helicase processing of DNA ends permits the forming of recombinants through annealing of complementary single strands. Our data are not consistent with a model involving strand invasion reactions, because such reactions should favor mixtures of linear and circular substrates. We also noted that many of the reaction features seen in vivo were reproduced in a simple in vitro reaction requiring only purified vaccinia virus DNA polymerase, single-strand DNA binding protein, and pairs of linear substrates. The 3'-to-5' exonuclease activity of poxviral DNA polymerases potentially catalyzes recombination in vivo.
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PMID:Effects of DNA structure and homology length on vaccinia virus recombination. 1143 72

In vitro analysis of the catalytic DNA polymerase encoded by vaccinia virus has demonstrated that it is innately distributive, catalyzing the addition of <10 nucleotides per primer-template binding event in the presence of 8 mM MgCl(2) or 40 mM NaCl (W. F. McDonald and P. Traktman, J. Biol. Chem. 269:31190-31197, 1994). In contrast, cytoplasmic extracts isolated from vaccinia virus-infected cells contain a highly processive form of DNA polymerase, able to catalyze the replication of a 7-kb template per binding event under similar conditions. To study this holoenzyme, we were interested in purifying and characterizing the vaccinia virus processivity factor (VPF). Our previous studies indicated that VPF is expressed early after infection and has a native molecular mass of approximately 48 kDa (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). Using these criteria, we established a six-step chromatographic purification procedure, in which a prominent approximately 45-kDa band was found to copurify with processive polymerase activity. This species was identified as the product of the A20 gene. By use of recombinant viruses that direct the overexpression of A20 and/or the DNA polymerase, we verified the physical interaction between the two proteins in coimmunoprecipitation experiments. We also demonstrated that simultaneous overexpression of A20 and the DNA polymerase leads to a specific and robust increase in levels of processive polymerase activity. Taken together, we conclude that the A20 gene encodes a component of the processive DNA polymerase complex. Genetic data that further support this conclusion are presented in the accompanying report, which documents that temperature-sensitive mutants with lesions in the A20 gene have a DNA(-) phenotype that correlates with a deficit in processive polymerase activity (A. Punjabi et al, J. Virol. 75:12308-12318, 2001).
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PMID:The A20R protein is a stoichiometric component of the processive form of vaccinia virus DNA polymerase. 1171 20

Although the vaccinia virus DNA polymerase is inherently distributive, a highly processive form of the enzyme exists within the cytoplasm of infected cells (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168-175, 1997). In the accompanying report we outline the purification of the 49-kDa A20 protein as a stoichiometric component of the processive polymerase complex (N. Klemperer, W. McDonald, K. Boyle, B. Unger, and P. Traktman, J. Virol. 75:12298-12307, 2001). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these, tsA20-6 and tsA20-ER5, have tight temperature-sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with the tsA20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA(-) phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication.
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PMID:Clustered charge-to-alanine mutagenesis of the vaccinia virus A20 gene: temperature-sensitive mutants have a DNA-minus phenotype and are defective in the production of processive DNA polymerase activity. 1171 21

Cidofovir ([(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] [HPMPC])-resistant forms of camelpox, cowpox, monkeypox, and vaccinia viruses were developed by prolonged passage in Vero 76 cells in the presence of drug. Eight- to 27-fold-higher concentrations of cidofovir were required to inhibit the resistant viruses than were needed to inhibit the wild-type (WT) viruses. Resistant viruses were characterized by determining their cross-resistance to other antiviral compounds, examining their different replication abilities in two cell lines, studying the biochemical basis of their drug resistance, and assessing the degrees of their virulence in mice. These viruses were cross resistant to cyclic HPMPC and, with the exception of vaccinia virus, to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine. Three of the four resistant cowpox and monkeypox viruses exhibited reduced abilities to infect and replicate in 3T3 cells compared to their abilities in Vero 76 cells. Compared to the WT virus polymers the resistant cowpox virus DNA polymerase was 8.5-fold less sensitive to inhibition by cidofovir diphosphate, the active form of the drug. Intracellular phosphorylation of [3H]cidofovir was not stimulated or inhibited by infection with resistant cowpox virus. In intranasally infected BALB/c mice, WT cowpox virus was 80-fold more virulent than the resistant virus. Cidofovir treatment (100 mg/kg of body weight, given one time only as early as 5 min after virus challenge) of a resistant cowpox virus infection could not protect mice from mortality. However, the drug prevented mortality in 80 to 100% of the mice treated with a single 100-mg/kg dose at 1, 2, 3, or 4 days after WT virus challenge. By application of these results to human orthopoxvirus infections, it is anticipated that resistant viruses may be untreatable with cidofovir but their virulence may be attenuated. Studies will need to be conducted with cidofovir-resistant monkeypox virus in monkeys to further support these hypotheses.
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PMID:Characterization of wild-type and cidofovir-resistant strains of camelpox, cowpox, monkeypox, and vaccinia viruses. 1195 64

The vaccinia virus A20R protein is required for DNA replication, is associated with the processive form of the viral DNA polymerase, and directly interacts with the viral proteins encoded by the D4R, D5R, and H5R open reading frames as determined by a genome-wide yeast two-hybrid analysis. The purpose of the present study was to further analyze the latter protein-protein interactions. Association of an epitope-tagged A20R protein with an epitope-tagged D4R or H5R protein, expressed in vaccinia virus-infected cells, was demonstrated by binding the complex to one mAb followed by Western blotting with another. Interaction between the A20R and D5R proteins, which was weakest in the yeast two-hybrid analysis, could not be demonstrated by this method. A panel of N- and C-terminal truncated forms of the A20R protein was tested for interaction with the D4R, H5R, and D5R proteins using the yeast two-hybrid system. These studies revealed that nonoverlapping regions of A20R comprising amino acids 1 to 25, 26 to 76, and 201 to 251 were required for binding of D4R, H5R, and D5R, respectively. By contrast, no interaction of A20R with D4R could be detected after deletion of only 25 codons from either end of the latter open reading frame. A fusion protein containing either full-length A20R or only the N-terminal 25 amino acids of A20R was sufficient to capture the D4R protein, whereas the fusion protein containing A20R amino acids 26 to 426 was not, confirming the results of the yeast two-hybrid analysis. The distinct protein binding domains of the A20R protein may contribute to the assembly or stability of the multiprotein DNA replication complex.
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PMID:Mapping interaction sites of the A20R protein component of the vaccinia virus DNA replication complex. 1249 Mar 86

The era of antiviral chemotherapy started more than 50 years with the findings by Domagk and his colleagues that thiosemicarbazones showed activity against vaccinia virus. One of the derivatives, methisazone, was even investigated in the prophylaxis of smallpox. With the successful implementation of the smallpox vaccine, the use of methisazone was not further pursued. Should there be a threat of smallpox or other poxvirus infections, that could not be immediately controlled by vaccination, a therapeutic intervention could be envisaged based on several therapeutic strategies targeted at such cellular enzymes as IMP dehydrogenase, SAH hydrolase, OMP decarboxylase and CTP synthetase, as well as viral enzymes such as the DNA polymerase. Most advanced as a therapeutic or early prophylactic modality to tackle poxvirus infection is cidofovir, which was found active (i) in vitro against all poxviruses studied so far; (ii) in vivo, against vaccinia and cowpox virus infections in experimental animal models; as well as (iii) some human poxvirus infections, such as molluscum contagiosum. In case of an inadvertent poxvirus epidemic, antiviral therapy (i.e. with cidofovir) will offer the possibility to provide short-term prophylaxis, or therapy. Cidofovir should also allow to treat severe complications of vaccination as may happen in for example immunosuppressed patients.
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PMID:Therapy and short-term prophylaxis of poxvirus infections: historical background and perspectives. 1261


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