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)

Biochemical characterization of the herpes simplex virus (HSV) DNA polymerase, a model DNA polymerase and an important target for antiviral drugs, has been limited by a lack of pure enzyme in sufficient quantity. To overcome this limitation, the HSV DNA polymerase gene was introduced into the baculovirus, Autographa californica nuclear polyhedrosis virus, under the control of the polyhedrin promoter to give rise to a recombinant baculovirus, BP58. BP58-infected Spodoptera frugiperda insect cells expressed a polypeptide that was indistinguishable from authentic polymerase by several immunological and biochemical properties, at levels approximately ten-fold higher per infected cell than found in HSV-infected Vero cells. The DNA polymerase was purified to apparent homogeneity from BP58-infected insect cells. Using activated DNA as primer-template, the purified enzyme exhibited specific activity similar to that of enzyme isolated from HSV-infected Vero cells, indicating that additional polymerase-associated proteins from HSV-infected cells are not critical for activity with this primer-template. 3'-5' exonuclease activity co-purified with the BP58-expressed HSV DNA polymerase, demonstrating that this activity is intrinsic to the polymerase polypeptide. The purified enzyme also exhibited RNAse H activity. The recombinant baculovirus should permit detailed biochemical and biophysical studies of this enzyme.
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PMID:Enzymatic activities of overexpressed herpes simplex virus DNA polymerase purified from recombinant baculovirus-infected insect cells. 215 92

We have derived Vero cell lines containing the herpes simplex virus DNA polymerase (pol) gene that complement temperature-sensitive pol mutants. These cell lines were used to recover viruses containing new mutations at the pol locus. Two spontaneously arising host-range mutants, 6C4 and 7E4, were isolated. These mutants did not grow efficiently on Vero cells or synthesize late polypeptides but formed plaques on a cell line containing the pol gene (DP6 cells). Whereas mutant 6C4 specified a wild-type-size Pol protein, we detected no full-length Pol protein in 7E4-infected cell extracts. Complementation studies demonstrated that 6C4 and 7E4 contain different mutations and indicated that 6C4 is in a complementation group different from that of pol temperature-sensitive mutant tsC7 or tsD9. A mutant in which 2.2 kilobases of pol sequences were replaced with the Escherichia coli lacZ gene under the control of the herpes simplex virus thymidine kinase promoter was constructed. This mutant formed blue plaques on DP6 cells in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. Using this virus in marker rescue experiments, we engineered three mutants containing deletions in the pol coding region which grew efficiently on DP6 cells but not on Vero cells and which differed in their synthesis of Pol polypeptides. The lacZ insertion virus was also used to introduce a deletion in the region upstream of the pol long open reading frame, which removes a short open reading frame that could encode a 10-amino-acid peptide. This mutant grew to similar titers on Vero and DP6 cells, indicating that these sequences are not essential for growth of the virus in tissue culture.
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PMID:Isolation and characterization of herpes simplex virus mutants containing engineered mutations at the DNA polymerase locus. 215 81

The potent inhibition of herpes simplex type 1 (HSV-1) DNA polymerase by acyclovir triphosphate has previously been shown to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3'-end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism of inhibition of HSV-1 DNA polymerase has been used here to design an affinity column for the enzyme. A DNA hook template-primer containing an acyclovir monophosphate residue on the 3'-primer terminus has been synthesized and attached to a resin support. In the absence of added nucleotides, the column behaves as a simple DNA-agarose column, and HSV-1 DNA polymerase can be chromatographed using a salt gradient. The presence of the next required nucleotide encoded by the template (dGTP) increases the affinity of HSV-1 DNA polymerase for the acyclovir monophosphate terminal primer-template attached to the resin, and the enzyme is retained even in the presence of 1 M salt. The enzyme can be eluted from the column with a salt gradient after removal of the nucleotide from the buffer. Traditionally, the affinity purification of an enzyme relies on elution by a salt gradient, pH gradient, or more selectively by addition of a competing ligand (substrate/inhibitor) to the elution buffer. In the present example, elution of HSV-1 polymerase is facilitated by removal of the substrate from the buffer. This represents an example of mechanism-based affinity chromatography.
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PMID:Herpes simplex virus type 1 DNA polymerase. Mechanism-based affinity chromatography. 215 86

Herpes simplex virus (HSV) induces within the host cell genome DNA amplification which can be suppressed by coinfection with adeno-associated virus (AAV). To characterize the AAV functions mediating this effect, cloned AAV type 2 wild-type or mutant genomes were transfected into simian virus 40 (SV40)-transformed hamster cells together with the six HSV replication genes (encoding UL5, UL8, major DNA-binding protein, DNA polymerase, UL42, and UL52) which together are necessary and sufficient for the induction of SV40 DNA amplification (R. Heilbronn and H. zur Hausen, J. Virol. 63:3683-3692, 1989). The AAV rep gene was identified as being responsible for the complete inhibition of HSV-induced SV40 DNA amplification. Likewise, rep inhibited origin-dependent HSV replication. rep neither killed the transfected host cells nor interfered with gene expression from the cotransfected amplification genes. This points to a specific interference with HSV-induced DNA amplification.
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PMID:The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification. 215 59

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) is a potent and selective inhibitor of the replication of herpes simplex virus type 1 (HSV-1). CEDU is preferentially phosphorylated by HSV-infected (Vero) cells, as compared with mock-infected cells or cells infected with a thymidine kinase-deficient strain of HSV-1. The end product of this phosphorylation process, CEDU 5'-triphosphate, is a competitive inhibitor of HSV-1 DNA polymerase activity and, to a lesser extent, of cellular DNA polymerase alpha activity. However, in the absence of the natural substrate dTTP, CEDU 5'-triphosphate also serves as an alternative substrate for viral and cellular DNA polymerase. When exposed to HSV-1-infected cells, [2-14C]CEDU was incorporated into both viral and cellular DNA. The extent to which [2-14C]CEDU was incorporated remained approximately constant over a concentration range of 0.5 to 50 microM. Within this concentration range, CEDU effected a concentration-dependent inhibition of viral DNA synthesis that closely paralleled the inhibition of viral progeny formation. It is postulated that CEDU owes (i) its selectivity as an antiviral agent to its preferential phosphorylation by the virus-infected cell and (ii) its antiviral potency to an inhibition of viral DNA synthesis at the level of the viral DNA polymerization reaction.
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PMID:Mechanism of action of 5-(2-chloroethyl)-2'-deoxyuridine, a selective inhibitor of herpes simplex virus replication. 216 59

A study was undertaken to determine whether genes associated with herpes simplex virus (HSV) neuroinvasiveness in mice influence the growth of HSV in man, the virus's natural host. HSV-2(186), a nonneuroinvasive HSV strain, was found to replicate poorly (less than 3-fold) in cultures of phytohemagglutinin (PHA) stimulated human peripheral blood mononuclear cells (PBMC). In contrast, seven other HSV strains all multiplied 40- to 100-fold. The paucity of HSV-2(186) growth in PBMC was not due to a failure of this strain to grow in primary human cells because high titers (greater than 10(8) PFU/ml) were obtained following infection of human foreskin fibroblasts. The genetic basis for the deficient growth was analyzed by marker rescue experiments. Recombinant HSV-2 strains were generated in marker rescue experiments utilizing HSV-2(186) DNA and plasmids containing a cloned DNA polymerase gene isolated from a neuroinvasive HSV strain possessing the capacity to replicate in human PBMC. Progeny which rescued DNA from the cloned HSV DNA polymerase gene replicated 40- to 100-fold in PHA-stimulated PBMC. Moreover, unlike the HSV-2(186) parent, HSV-2(186) isolates possessing rescued DNA grew well in the eye, trigeminal ganglion, and brain of mice and induced fatal encephalitis. The results indicate that nucleotide sequences responsible for increasing the capacity of HSV-2(186) to grow in PBMC of man are identical to those responsible for increasing the capacity of this strain to grow in mouse tissues and to spread from the eye to the brain.
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PMID:Nucleotide sequences responsible for the inability of a herpes simplex virus type 2 strain to grow in human lymphocytes are identical to those responsible for its inability to grow in mouse tissues following ocular infection. 216 Nov 43

Synthetic peptides corresponding to amino acid sequences present in the herpes simplex virus type-1 (HSV-1) DNA polymerase (pol) were used to raise polyclonal rabbit antisera. The three peptides described in detail in this report were among seven sequences chosen for initial studies designed to generate reagents capable of recognizing discrete regions of the HSV-1 pol protein from the amino to carboxy termini. Two of the peptides, designated P6 and P7, representing amino acid residues 1100-1108 and 1216-1224 of the deduced HSV-1 (strain KOS) DNA pol sequence (1235 residues) produced antisera that could not only recognize the native HSV-1 pol enzyme but also could specifically neutralize purified HSV-1 pol activity in a dose-dependent manner. An additional peptide, designated P3, representing residues 548-557, produced an antiserum that was unable to recognize the native protein but could react with HSV-1 pol in a denatured form by immunoblot assay.
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PMID:Neutralization of purified herpes simplex virus DNA polymerase by two antipeptide sera. 216 84

Phosphorothioate homo-oligodeoxynucleotides were found to be potent inhibitors of herpes simplex virus type 2 (HSV-2) but less potent for HSV-1 in cell culture studies. Oligomers with longer chain lengths were more active against HSV-2 than those with shorter ones. Of all the compounds examined, the 28-mer phosphorothioate homo-oligodeoxynucleotides were the strongest inhibitors of HSV-2. The degree of inhibition was related to the base moiety on the order of deoxycytidine = thymidine greater than deoxyadenosine. The inhibition of HSV-2 growth by S-dC28 was dose dependent with a 90% inhibitory dose of 1 microM. At 50 microM, S-dC28 inhibited HeLa S3 cell growth by less than 10%. The anti-HSV-2 activity was time and schedule dependent. The oligomer was most inhibitory to viral growth when present during the 1-h viral adsorption period, and this effect could be enhanced by continuous drug exposure after the adsorption period. S-dC28 was also an effective inhibitor of two HSV-2 drug-resistant mutants: a phosphonoformate-resistant mutant that induces an altered DNA polymerase and a 9-(1,3-dihydroxy-2-propoxymethyl)guanine-resistant mutant that does not induce the viral thymidine kinase. In drug combination studies, phosphonoformate was shown to potentiate the action of S-dC28 against HSV-2 growth. In conclusion, because of their potency and selectivity, phosphorothioate homo-oligodeoxynucleotides are a promising new class of anti-HSV agents.
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PMID:Inhibition of herpes simplex virus type 2 growth by phosphorothioate oligodeoxynucleotides. 216 43

(E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-triphosphate (BrVdUTP) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil 5'-triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase gamma from human KB and murine myeloma cells. In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase gamma than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic alpha and beta, or prokaryotic) origin. The mode of inhibition of DNA polymerase gamma by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate. Whereas BrVdUTP was an efficient substrate for DNA polymerase gamma and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis. Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase gamma, as determined with (rA)n.(dT) as the template.primer, were much smaller than the Km values for dTTP (0.16 microM and 0.71 microM for murine and human DNA polymerase gamma, respectively). Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase gamma was much stronger than that of dTTP.
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PMID:Potent inhibitory effects of the 5'-triphosphates of (E)-5-(2-bromovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on DNA polymerase gamma. 216 28

The exonucleolytic activities associated with herpes simplex virus type-1 (HSV-1) DNA polymerase and DNase were compared. The unique properties of these nucleases were assessed by applying biochemical and immunological methods as well as by genetics. In contrast to the viral DNA polymerase, HSV DNase is equipped with a 5'-3'-exonuclease activity. Under reaction conditions optimal for HSV DNA polymerase, i.e. at high ionic strength, HSV DNase exhibited only limited endonucleolytic activity and degraded double-stranded DNA in a very processive manner and exclusively in the 5'-3' direction, producing predominantly mononucleotides. Both viral enzymes displayed significant RNase activity which could be correlated with the endogenous endonucleolytic and 5'-3'-exonucleolytic activities of the DNase and the polymerase-associated 3'-5' exonuclease. The tight linkage of polymerizing and exonucleolytic functions of the viral DNA polymerase was demonstrated by their identical response to (a) thermal inactivation, (b) drug inhibition and (c) neutralization by polyclonal antibodies reacting specifically with the N-terminal, central and C-terminal polypeptide domains of HSV-1 DNA polymerase. From the data presented it can be concluded that the cryptic 3'-5' exonuclease is the only exonucleolytic activity associated with the viral DNA polymerase.
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PMID:Comparison of exonucleolytic activities of herpes simplex virus type-1 DNA polymerase and DNase. 216 60


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