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)

Fourteen mutants known or likely to contain mutations in the herpes simplex virus DNA polymerase gene were examined for their sensitivity to aphidicolin in plaque reduction assays. Eleven of these exhibited some degree of hypersensitivity to the drug; altered aphidicolin-sensitivity correlated with altered sensitivity to the pyrophosphate analog, phosphonoacetic acid. The DNA polymerase specified by one of these mutants, PAAr5, required roughly seven-fold less aphidicolin to inhibit its activity by 50% than did polymerase specified by its parental strain. Mutations responsible for the aphidicolin-hypersensitivity phenotype of PAAr5 were mapped to an 0.8 kbp region in the herpes simplex virus DNA polymerase locus. These data taken together indicate that 1) mutations in the herpes simplex virus DNA polymerase gene can confer altered sensitivity to aphidicolin, 2) that the HSV polymerase is sensitive to aphidicolin in vivo, and 3) that amino acid alterations which affect aphidicolin binding may affect the pyrophosphate exchange-release site as well, suggesting that aphidicolin binds in close proximity to this site.
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PMID:Mutations in the herpes simplex virus DNA polymerase gene conferring hypersensitivity to aphidicolin. 630 78

A DNA-relaxing enzyme was found to copurify along with herpes simplex virus type I (HSV-1)-induced DNA polymerase throughout a multistep purification scheme. Both the enzymes had similar sedimentation velocity, required high ionic strength for optimal enzymatic activities and showed time dependence of reaction. The DNA-relaxing enzyme however, differed from the HSV-1 DNA polymerase in its requirement for higher Mg2+ concentration, rATP and much broader pH dependence. Furthermore, phosphonoacetic acid, a potent inhibitor of HSV-1 DNA polymerase did not influence the DNA-relaxing activity even at a much higher concentration. On the other hand, the DNA-relaxing enzyme associated with the DNA polymerase may be specified by HSV-1 since IgG fraction of rabbit antisera against the virus-infected cells but not against the mock-infected cells strongly inhibited both the enzymatic activities. Thus, HSV-1-induced DNA polymerase which is known to be associated with a 3' to 5' exonuclease may also be associated with yet another enzymatic activity involved in DNA metabolism.
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PMID:A DNA topoisomerase activity copurifies with the DNA polymerase induced by herpes simplex virus. 630 34

The stimulation of host cell DNA synthesis was studied in permissive human embryonic lung (HEL) cells and in nonpermissive rabbit kidney (RK) cells infected with human cytomegalovirus (HCMV). Host cell DNA synthesis was induced by HCMV infection in resting cells of both types. In permissive cultures the stimulation of cellular DNA synthesis was detectable mainly in those cells which had not become productively infected and in which virus antigens were not detectable. In abortively infected RK cells, on the other hand, stimulation of host cell DNA synthesis and the expression of virus antigens were detected in the same cells. Infection of actively growing permissive HEL cells resulted in a shutdown of cellular DNA synthesis beginning approximately 10 hr postinfection. Shutdown of cellular DNA synthesis also occurred when the infected cells were treated with phosphonoacetic acid and was thus classified as an "early" virus function. In actively growing, abortively infected RK cells, on the other hand, host cell DNA synthesis was not affected, indicating that the early virus function(s) responsible for inhibition of cellular DNA synthesis was not expressed in these cells. Virus-encoded DNA polymerase activity, another early virus gene function, was also not detected in these abortively infected cultures. In RK cells the cellular DNA synthesized as a result of infection was capable of undergoing at least one further round of replication, indicating that the HCMV gene expression which occurred in abortively infected RK cells was not lethal for these cells.
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PMID:Correlation between stimulation of host cell DNA synthesis by human cytomegalovirus and lack of expression of a subset of early virus genes. 631 75

When human embryonic fibroblasts (HEF) were infected with herpes simplex virus type 2 (HSV-2), replicative viral DNA synthesis and some repair synthesis of cellular DNA were induced at the early stage of infection, but almost all DNA synthesis at the late stage of infection was derived from repair synthesis of cellular and viral DNA (Y. Nishiyama and F. Rapp, Virology 110, 466-475, 1981). In this study, we have assessed the effects of DNA polymerase inhibitors on repair DNA synthesis HSV-2-infected HEF. Both viral and cellular DNA syntheses during the late stage of infection were extremely resistant to aphidicolin and phosphonoacetic acid but partially sensitive to high concentrations of 1-beta-D-arabinofuranosylcytosine, while replicative viral DNA synthesis during the early stage of infection was very sensitive to all of those inhibitors. The results suggest that neither HSV-induced DNA polymerase nor cellular DNA polymerase alpha was involved in the repair synthesis of viral and cellular DNA but that cellular DNA polymerase beta was.
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PMID:Identification of DNA polymerase(s) involved in the repair of viral and cellular DNA in herpes simplex virus type 2-infected cells. 631 89

Mutants of Varicella-Zoster Virus (VZV) which are resistant to phosphonoacetic acid (PAA), bromodeoxyuridine (BuDR), and acyclovir (ACV) were obtained by serial passages of VZV with increasing concentrations of these drugs. A PAA-resistant mutant and a BuDR-resistant mutant were found also to be resistant to ACV. Five of 8 ACV-resistant mutants acquired resistance to PAA, but none acquired resistance to BuDR. The BuDR-resistant mutant did not induce viral thymidine kinase (TK) activity, but all the ACV-resistant mutants selected in ACV showed viral TK activity which was suppressed with anti-VZV serum and had almost the same electrophoretic mobility as that of the parent strain on polyacrylamide gel electrophoresis in non-denaturing conditions. However, in competitive TK assay with ACV, 2 of 8 ACV-resistant mutants showed no change of phosphorylation of radioactive thymidine, while the other 6 showed decreased phosphorylation of radioactive thymidine. It was suggested that TK induced by the former 2 ACV-resistant mutants had lost affinity to ACV, and so the mutants could grow in the presence of ACV. Thus of the 8 ACV-resistant mutants selected in ACV, 2 were sensitive to PAA with altered TK activity, 5 were resistant to PAA with unaltered TK activity, and 1 was sensitive to PAA with unaltered TK activity, and may have altered DNA polymerase activity to ACV, retaining sensitivity to PAA. These results suggest that resistance of VZV to ACV results from alterations in the virus-specified TK or DNA polymerase, as demonstrated in HSV resistant to ACV.
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PMID:Isolation of drug resistant mutants of varicella-zoster virus: cross resistance of acyclovir resistant mutants with phosphonoacetic acid and bromodeoxyuridine. 631 58

Mutations in five phenotypically distinct mutants derived from herpes simplex virus type 1 strain KOS which lie in or near the herpes simplex virus DNA polymerase (pol) locus have been fine mapped with the aid of cloned fragments of mutant and wild-type viral DNAs to distinct restriction fragments of 1.1 kilobase pairs (kbp) or less. DNA sequences containing a mutation or mutations conferring resistance to the antiviral drugs phosphonoacetic acid, acyclovir, and arabinosyladenine of pol mutant PAAr5 have been cloned as a 27-kbp Bg+II fragment in Escherichia coli. These drug resistance markers have been mapped more finely in marker transfer experiments to a 1.1-kbp fragment (coordinates 0.427 to 0.434). In intratypic marker rescue experiments, temperature-sensitive (ts), phosphonoacetic acid resistance, and acyclovir resistance markers of pol mutant tsD9 were mapped to a 0.8-kbp fragment at the left end of the EcoRI M fragment (coordinates 0.422 to 0.427). The ts mutation of pol mutant tsC4 maps within a 0.3-kbp sequence (coordinates 0.420 to 0.422), whereas that of tsC7 lies within the 1.1-kbp fragment immediately to the left (coordinates 0.413 to 0.420). tsC4 displays the novel phenotype of hypersensitivity to phosphonoacetic acid; however, the phosphonoacetic acid hypersensitivity phenotype is almost certainly not due to the mutation(s) conferring temperature sensitivity. The ts mutation of mutant tsN20--which does not affect DNA polymerase activity--maps to a 0.5-kbp fragment at the right-hand end of the EcoRI M fragment (coordinates 0.445 to 0.448). The mapping of the mutations in these five mutants further defines the limits of the pol locus and separates mutations differentially affecting catalytic functions of the polymerase.
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PMID:Fine mapping and molecular cloning of mutations in the herpes simplex virus DNA polymerase locus. 631 91

Aphidicolin, a tetracyclic diterpenoid which inhibits the DNA polymerase-alpha activities of many eukaryotic cells, inhibited herpes simplex virus growth and DNA synthesis in infected cultures and the activity of the virus DNA polymerase in vitro. A wide range of stable aphidicolin sensitivities was represented amongst a collection of virus strains with no prior exposure to this drug, but viruses with polymerase mutations selected for resistance to phosphonoacetic acid (PAA) or to acycloguanosine typically showed increased sensitivity to aphidicolin. Of 16 unrelated PAA-resistant variants, 7 were hypersensitive to aphidicolin. A number of mutants with temperature-sensitive (ts) lesions in the polymerase gene also showed increased aphidicolin sensitivity (e.g. HSV-1[mP17]tsH) or aphidicolin hypersensitivity (e.g. HSV-1[KOS]tsD9, tsC4). Resistance or hypersensitivity of virus growth and DNA synthesis in vivo were correlated with resistance or hypersensitivity of virus DNA polymerase reactions in vitro. Resistance phenotypes were closely linked to the polymerase gene during recombination with outside markers. Moreover, the selection of aphidicolin-resistant mutants from hypersensitive variants with independent PAA resistance or ts mutations in the polymerase gene could result in co-selection for PAA-sensitive and ts+ phenotypes. Confirmation that multiple independent mutations could determine aphidicolin hypersensitivity was obtained by studies of recombination between independent hypersensitive variants. Aphidicolin-resistant recombinant progeny were formed with recombination frequencies (0.4 to 2.6%) compatible with intragenic events. With parental hypersensitive variants which were products of limited PAA selection, or with the ts polymerase mutations, aphidicolin-resistant recombinants were PAA-sensitive and/or ts+. The segregation of other markers (ts, plaque morphology) amongst recombinant progeny permitted the orientation of multiple determinants of PAA resistance and aphidicolin hypersensitivity with respect to other markers in the polymerase gene and in other genes. The nature of residues determined at any one of a constellation of separate sites within the polymerase locus can determine resistance or sensitivity to antiviral drugs and aphidicolin hypersensitivity associated with changes at the polymerase locus facilitates high resolution genetic analysis of this locus.
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PMID:Single mutations at many sites within the DNA polymerase locus of herpes simplex viruses can confer hypersensitivity to aphidicolin and resistance to phosphonoacetic acid. 631 64

We have measured the spontaneous production of mutants in derivatives of herpes simplex virus type 1 resistant to phosphonoacetic acid. Six such derivatives produced 9- to 123-fold fewer iododeoxycytidine (ICdR-)-resistant progeny (i.e., thymidine kinase deficient) than their wild-type parents. To locate the mutation which controls mutant production in one of the strains (PAAr-5), we constructed phosphonoacetic acid-resistant, recombinant viruses by marker transfer, using wild-type viral DNA and DNA restriction fragments conferring the resistance phenotype. The resultant recombinants also produced very low levels of ICdR-resistant progeny during growth, indicating a close linkage (within 1.1 kilobase pairs) between the drug resistance locus and the sequences controlling production of mutant progeny. Evidence is presented that the low mutant yield in PAAr-5 is not due to abnormal expression of mutants, hypersensitivity to ICdR, altered thymidine kinase activity, or slow replication rates. Since the locus conferring resistance to phosphonoacetic acid in PAAr-5 has been shown previously to be the DNA polymerase gene, we hypothesize that the reduced yield of mutants results from enhanced replication fidelity by the altered DNA polymerase. The existence of antimutator derivatives of herpes simplex indicates that the observed high mutation rate for wild-type strains is an intrinsic property of the virus and may provide a selective advantage during growth in animal hosts.
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PMID:Generation of genetic diversity in herpes simplex virus: an antimutator phenotype maps to the DNA polymerase locus. 632 May 35

An RNAase-sensitive DNA polymerase from rat cells transformed by avian sarcoma virus has been characterized. The enzyme requires RNA for its activity, as shown by its sensitivity to RNAase with endogenous as well as exogenous DNA templates. This sensitivity is maintained after its purification by sucrose gradients and ion exchange columns. A molecular weight of about 100 000 has been estimated. This DNA polymerase requires high salt concentration for its activity, is resistant to high concentrations of phosphonoacetic acid (400 micrograms/ml), is partially inhibited by 5 mM N-ethylmaleimide, and is completely inhibited by 0.3 mM parahydroxymercuribenzoate.
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PMID:RNAase-sensitive DNA-dependent DNA polymerase from rat cells transformed by avian sarcoma virus. 632 Aug 97

A herpes simplex virus type 2 (HSV-2) mutant TS6 (strain HG52) induces a heat-labile viral DNA polymerase at the nonpermissive temperature and is markedly resistant to 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-guanine [2'-nor-2'-deoxyguanosine; 2'NDG]. This antiviral drug requires HSV thymidine kinase for phosphorylation to an active inhibitor (2'NDG-triphosphate), and thymidine kinase-deficient mutants of HSV exhibit varying degrees of resistance to 2'NDG, with the HSV type 1 (HSV-1) B2006 mutant (Kit) being markedly resistant. The ts6 mutation and the 2'ndgR-1 mutation within the viral DNA polymerase locus have been physically mapped by marker rescue and generation of HSV-1/HSV-2 intertypic recombinants. The physical map limits for the ts6 mutation and 2'ndgR-1 mutation are closely linked within a 2.2-kilobase-pair region of DNA sequences and are physically separate from the paaR-1 and acvR-1 mutations. Resistance to 2'NDG by HSV-2 ts6 can be overcome in the presence of combinations of 2'NDG and phosphonoacetic acid, indicating drug synergism within the viral DNA polymerase locus. These physical mapping studies expand the limits of DNA sequences defining an active center in the viral polymerase to 3.5 kilobase pairs, indicating that regions spanning the entire polymerase polypeptide may contribute to a specialized surface able to interact with nucleotides of different structure.
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PMID:Resistance of herpes simplex virus to 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine: physical mapping of drug synergism within the viral DNA polymerase locus. 632 3


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