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)

DNA polymerases induced by herpes simplex virus (HSV)-1 (KOS) and by three phosphonoformic acid-resistant strains were purified and the interaction of these enzymes with aphidicolin was examined. Incorporation of dATP, dCTP, and dTTP into activated DNA by parental enzyme was inhibited competitively by aphidicolin whereas dGTP incorporation was inhibited noncompetitively. Phosphonoformic acid-resistant enzymes were altered in KM and KI values for substrate and inhibitor, and two were inhibited by aphidicolin via the same modes as parental enzyme. However, aphidicolin competitively inhibited incorporation of dGTP by the third phosphonoformic acid-resistant enzyme under identical assay conditions. Two phosphonoformic acid-resistant enzymes were more sensitive than parental enzyme to inhibition by aphidicolin, indicating a close association between binding determinants for aphidicolin and for phosphonoformic acid on the virus DNA polymerase molecule. Aphidicolin inhibited hydrolysis of polynucleotide by HSV-1 DNA polymerase-associated nuclease. Inhibition was uncompetitive with DNA and the KI value (0.09 microM) was within the range of those calculated during nucleotide incorporation (0.071-0.74 microM). Therefore, aphidicolin may produce antiviral effects both by inhibition of deoxynucleotide incorporation and by deleterious effects resulting from inhibition of polymerase-associated nuclease.
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PMID:Novel interaction of aphidicolin with herpes simplex virus DNA polymerase and polymerase-associated exonuclease. 609 71

The fluorescence-plus-Giemsa (FPG) technique for analysis of sister chromatid exchange (SCE) is widely used as an assay for mutagenic carcinogens. There is very little information, however, on whether incorporation of the bromodeoxyuridine (BrdU) necessary for visualization of SCEs affects the sensitivity of the SCE test system to different chemical agents. We have investigated the effect of BrdU incorporation on SCE induction by labeling cells with BrdU for either the first cell cycle or the first and second cell cycles. The cells were then treated with bleomycin, which produces DNA strand breakage; proflavine, which intercalates into DNA; mitomycin C, which produces monoadducts and DNA crosslinks; or aphidicolin, which inhibits DNA polymerase alpha. Chemicals were added before BrdU exposure or during the first, second, or both cell cycles. Only mitomycin C, which induces long-lived lesions, elevated the SCE frequency when cells were treated before BrdU labeling. When bleomycin, proflavine, or mitomycin C was present concurrently with BrdU, the frequency of SCEs was increased independently of the BrdU labeling protocol. Aphidicolin, on the other hand, induced more SCEs when present for the second cell cycle, when DNA replicates on a template DNA strand containing BrdU. We also examined the induction of SCEs in the first cell cycle (twins) and in the second cell cycle (singles) after continuous treatment of cells with BrdU and the test chemicals. Only aphidicolin increased SCE frequency in the second cell cycle. These results indicate that aphidicolin, but not bleomycin, proflavine, or mitomycin C, affects BrdU-substituted DNA and unsubstituted DNA differently. This type of interaction should be taken into consideration when the SCE test is used as an assay system.
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PMID:Effect of 5-bromodeoxyuridine substitution on sister chromatid exchange induction by chemicals. 620 21

Aphidicolin, a specific inhibitor of eucaryotic alpha DNA polymerase, inhibits the growth of halophilic arachaebacteria. In Halobacterium halobium, aphidicolin prevents cell division and DNA synthesis. These results suggest that arachaebacterial replicases are of the eucaryotic type.
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PMID:Aphidicolin inhibits growth and DNA synthesis in halophilic arachaebacteria. 620 69

Aphidicolin, a mycotoxin that inhibits eucaryotic DNA polymerase alpha, blocked the growth of Toxoplasma gondii in confluent cultured human fibroblasts. Aphidicolin immediately inhibited DNA synthesis by T. gondii while it had a delayed and less dramatic effect on RNA synthesis. A mutant of T. gondii resistant to aphidicolin was isolated with the aid of mutagenesis by ethylnitrosourea. Parasite growth measured three days after drug treatment and parasite DNA synthesis measured immediately after drug treatment were, respectively, five- and four-fold more resistant to aphidicolin in the mutant as compared with the wild type parasite. The mutant had a three-fold greater capacity than the wild type to incorporate uracil into its deoxycytidine triphosphate pool. This increased deoxycytidine triphosphate pool is the probable explanation for the mutant's resistance because this deoxynucleotide is known, in mammalian cells, to reverse the inhibition of DNA synthesis by aphidicolin in a competitive manner.
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PMID:Characterization of a mutant of Toxoplasma gondii resistant to aphidicolin. 620 26

DNA polymerase delta from rabbit bone marrow has an associated 3'-5'-exonuclease. Previous studies demonstrated a Stokes radius of 45.5 A by gel filtration and a sedimentation coefficient of 6.5 S by zone sedimentation. Thus, a molecular weight of 122000 and a frictional coefficient of 1.39 were calculated [Byrnes, J. J., & Black, V. L. (1978) Biochemistry 17, 4226-4231]. Several problems obstructed further purification and definition of DNA polymerase delta. The small amount of protein obtained limited further purification as the nonspecific loss of enzyme in subsequent procedures was excessive. Furthermore, the amount of protein recovered was insufficient for conventional analysis. These difficulties have been overcome, and DNA polymerase delta has been purified to apparent homogeneity. Under conditions of nondenaturing microgel electrophoresis, DNA polymerase b aggregates to molecular weight species of 300000 and higher. In situ assays for DNA polymerase and exonuclease in these gels generate concordant activity profiles. Upon sodium dodecyl sulfate gel electrophoresis, delta is a single polypeptide of 122000 apparent molecular weight. The DNA polymerase incorporates between 250000 and 300000 nmol of thymidine deoxyribonucleoside monophosphate (dTMP) into poly(dA)/oligo(dT) (mg of protein)-1 h-2 at 37 degrees C; the exonuclease simultaneously hydrolyzes 13% of the newly synthesized DNA. Aphidicolin, considered to be a specific inhibitor of DNA polymerase alpha, inhibits both the DNA polymerase and 3'-5'-exonuclease activities of delta. DNA polymerase alpha from rabbit bone marrow does not share a common subunit with delta. Therefore, aphidicolin binding is not specific for alpha, and conclusions based upon the supposition that it is must be reconsidered.
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PMID:DNA polymerase delta: one polypeptide, two activities. 628 2

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

Aphidicolin is a potent inhibitor of both host cell DNA polymerase alpha and herpes simplex virus (HSV)-induced DNA polymerase but has no effect on DNA polymerases beta and gamma of host cells. By using an aphidicolin-resistant mutant (Aphr) of HSV, a possible involvement of DNA polymerase alpha in host cell reactivation of UV-damaged HSV was studied. Plaque formation by UV-irradiated Aphr was markedly inhibited by 1 microgram of aphidicolin per ml, which did not affect the plating efficiency of nonirradiated Aphr. Aphidicolin added before 12 h postinfection inhibited plaque formation by irradiated Aphr, which became aphidicolin insensitive after 36 h postinfection. The results strongly suggest that host cell DNA polymerase alpha is involved in the repair of UV-irradiated HSV DNA.
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PMID:Involvement of DNA polymerase alpha in host cell reactivation of UV-irradiated herpes simplex virus. 631 62

Chromatographic analysis of poly(dT) replication activity in fresh yeast extracts showed that the activities required co-fractionate with the yeast DNA polymerase I. Since poly(dT) replication requires both a primase and a DNA polymerase, the results of the fractionation studies suggest that these two enzymes might exist as a complex in the yeast extract. Sucrose gradient analysis of concentrated purified yeast DNA polymerase I preparations demonstrates that the yeast DNA polymerase I does sediment as a complex with DNA primase activity. Two DNA polymerase I peptides estimated at 78,000 and 140,000 Da were found in the complex that were absent from the primase-free DNA polymerase fraction. Rabbit anti-yeast DNA polymerase I antibody inhibits DNA polymerase I but not DNA primase although rabbit antibodies are shown to remove DNA primase activity from solution by binding to the complex. Mouse monoclonal antibody to yeast DNA polymerase I binds to free yeast DNA polymerase I as well as the complex, but not to the free DNA primase activity. These results suggest that these two activities exist as a complex and reside on the different polypeptides. Replication of poly(dT) and single-stranded circular phage DNA by yeast DNA polymerase I and primase requires ATP and dNTPs. The size of the primer produced is 8 to 9 nucleotides in the presence of dNTPs and somewhat larger in the absence of dNTPs. Aphidicolin, an inhibitor of yeast DNA polymerase I, is not inhibitory to the yeast DNA primase activity. The primase activity is inhibited by adenosine 5'-(3-thio)tri-phosphate but not by alpha-amanitin. The association of yeast DNA polymerase I and yeast DNA primase can be demonstrated directly by isolation of the complex on a column containing yeast DNA polymerase I mouse monoclonal antibody covalently linked to Protein A-Sepharose. Both DNA polymerase I and DNA primase activities are retained by the column and can be eluted with 3.5 M MgCl2. Part of the primase activity can be dissociated from DNA polymerase on the column with 1 M MgCl2 and this free primase activity can be detected as poly(dT) replication activity in the presence of Escherichia coli polymerase I.
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PMID:DNA polymerase I and DNA primase complex in yeast. 637 90

Aphidicolin, a specific inhibitor of DNA polymerase alpha, was found to induce high frequencies of endoreduplication in Chinese hamster V79 cells in a dose-dependent manner. The aphidicolin-induced endoreduplication was observed when cells were incubated at 37 degrees but not at 41 degrees. Since it is known that DNA polymerase beta is more thermally labile than is DNA polymerase alpha, the data are consistent with the hypothesis that DNA polymerase beta might be responsible for endoreduplication as was reported in mouse trophoblast cells. From the induced diplochromosomes, it was observed that the two unifilarly 5-bromodeoxyuridine-substituted chromatids are generally paired and located inside, whereas the two bifilarly 5-bromodeoxyuridine-substituted chromatids are flanking outside regardless of the presence of sister chromatid exchange or intradiplochromatid interchange.
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PMID:Aphidicolin-induced endoreduplication in Chinese hamster cells. 640 98

Of several phytotoxins isolated from culture filtrates of Phoma betae Frank PS-13, an incitant of leaf spot disease of sugar beet, three have been identified as aphidicolin, 3-deoxyaphidicolin and aphidicolin-17-monoacetate. Aphidicolin is a selective inhibitor of eukaryotic DNA polymerase alpha (Ikegami et al. (1978) Nature 275, 458-460). Consequently, we studied the action mechanism of 3-deoxyaphidicolin and aphidicolin-17-monoacetate. These aphidicolin analogues markedly inhibited the in vivo DNA synthesis of sea urchin embryos and HeLa cells but not RNA and protein syntheses. Only DNA polymerase alpha, not DNA polymerase beta and gamma, was inhibited by these drugs. The mode of action of these analogues on DNA polymerase alpha from the sea urchin was competitive inhibition with respect to dCTP with Ki values of 0.44 micrograms/ml for deoxyaphidicolin and 0.89 micrograms/ml for aphidicolin monoacetate, respectively. None of the other three dNTPs competed with these drugs. A similar inhibitory mode was observed using the enzyme from HeLa cells and toad oocytes. These drugs at a concentration of 2 micrograms/ml caused a delay in the cleavage of fertilized eggs of the sea urchin and decomposition before blastulation, indicating the possibility of achromosomal cleavage because of the absence of DNA synthesis. Based on the above, it is concluded that these analogues can be used as other inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha.
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PMID:Specific inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha, 3-deoxyaphidicolin and aphidicolin-17-monoacetate. 640 59


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