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)

Confirmatory evidence for the existence of a multienzyme complex of DNA precursor pathways in mammalian cells was obtained. Using neutral sucrose gradient centrifugation of cell lysates we found that at least five enzymes involved in DNA precursor metabolism in uninfected. S-phase BHK-cell fibroblasts cosediment at a common rate, indicative of a multienzyme complex. The enzymes include DNA polymerase thymidine kinase, ribonucleotide reductase, dihydrofolate reductase, and NDP-kinase. This complex was partially, but not completely, disrupted when lysates from GO-phase cells were centrifuged. Using lysates from cells infected with herpes simplex virus (HSV) type I some of the virus-induced ribonucleotide reductase and a minor proportion of the HSV-thymidine kinase cosedimented rapidly. The virus-induced DNA polymerase sedimented independently near the middle of the gradient, in contrast to the behaviour of the host polymerase. The enzyme associations observed were disrupted by NaCl or by inclusion of ethylenediamine tetraacetic acid during the cell lysis procedure, instead of the usual EGTA. These results indicate the importance of ionic forces in maintaining the enzyme complexes. The bulk of the DNA and the RNA present in the lysates did not sediment at the same rate as the complexes, showing that the enzymes were not simply adhering nonspecifically to these polyanions. Newly synthesised radiolabeled DNA (15 min pulse with [3H]thymidine) was not preferentially associated with the enzymes, but some functional DNA was evident in the enzyme complex fraction from the uninfected S-phase cells. DNA polymerase activity in this fraction did not require, nor was it stimulated by, exogenous "activated" DNA. Added DNA primer-template was required, however, for maximal activity of the polymerase in gradient fractions derived from GO-phase cells and from HSV-infected cells. No evidence for channeling of ribonucleotide precursors into DNA of permeabilized cells (uninfected or HSV-infected) was detected. Most rCDP was incorporated into RNA. In the uninfected, S-phase cells about 10 pmol/10(6) cells/90 min of rCDP residues was incorporated into DNA compared with 120 pmol/10(6) cells/90 min when radiolabeled dCTP was used. Nonradioactive dCTP present in equimolar concentration in the incubation with labeled rCDP did not, however, diminish the incorporation of label from the ribonucleotide. In permeabilized HSV-infected cells incorporation of radiolabel from rCDP into DNA was barely detectable.
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PMID:Search for multienzyme complexes of DNA precursor pathways in uninfected mammalian cells and in cells infected with herpes simplex virus type I. 244 4

The duck hepatitis B virus (DHBV)-associated activities of reverse transcriptase and DNA polymerase and their inhibition in vitro were studied. Replicative complexes (RCs) were isolated from DHBV-infected liver by gel chromatography followed by sucrose gradient centrifugation. The RCs were detected by dot blot hybridization, using radiolabeled cloned DHBV DNA as a probe, and by the incorporation of 32P-TTP in the presence of dATP, dCTP, dGTP, and Mg2+ (endogenous DNA polymerase activity). The endogenous DNA polymerase activity associated with RCs was further studied using exogenous templates: reverse transcriptase and DNA polymerase activities were demonstrated using as substrates 32P-TTP and poly(rA) p(dT)12 or poly(dA) p(dT)12-18, respectively. Both activities were biochemically characterized. Their inhibition by various antiviral agents was studied in vitro: actinomycin D, ara-ATP, aphidicolin, suramin, chloroquin, and phosphonoformate. Among these, suramin, chloroquin, phosphonoformate, and ara-ATP were shown to be potent inhibitors of viral reverse transcriptase and DNA polymerase. Studies are now in progress to establish their antiviral activity in vivo.
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PMID:Duck hepatitis B virus: DNA polymerase and reverse transcriptase activities of replicative complexes isolated from liver and their inhibition in vitro. 245 18

We have developed a modified primer extension procedure for specific detection of mRNA. Alkali-fragmented total cellular RNA or some RNA fraction is hybridized to single-stranded or double-stranded M13 DNA containing the insert of interest which is immobilized on nylon membranes. Hybridized RNA is then detected by incubation of membranes with Escherichia coli RNase H and DNA polymerase I. RNase H is used for nicking the RNA in the hybrids. The resulting 3'-OH groups can subsequently be used by DNA polymerase I to synthesize a labeled complementary strand. The method described is both relatively fast and sensitive and particularly useful for screening large numbers of DNA clones for their representation in RNA populations. Using total cellular RNA as hybridization probe and single-stranded M13 DNA as template as low as 0.25 ng of a specific mRNA was detected (2.5-fold background) when adding 1 microCi [3H]dCTP or 2.5 microCi [32P]d-CTP alternatively as radioactive precursor for the labeling reaction. The detection limit increased to 1 ng (2-fold background) with denatured replicative form double-stranded M13 DNA as template.
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PMID:A modified primer extension procedure for specific detection of DNA-RNA hybrids on nylon membranes. 247 44

To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific genes revealed a number of exceptions. Approximately 25% of total poly(A) RNA was transcribed from the late-replicating DNA.
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PMID:Temporal order of gene replication in Chinese hamster ovary cells. 247 59

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.
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PMID:DNA polymerase alpha from normal rat liver is different than DNA polymerases alpha from Morris hepatoma strains. 250 1

Aphidicolin is a specific inhibitor of DNA polymerase-alpha and -delta from eukaryotic cells. Because of the specificity of this inhibitor, it is potentially a useful probe for the detailed studies of the function of these polymerases. DNA polymerase-alpha mutants isolated on the basis of resistance to aphidicolin have been described. We have isolated four variants that exhibit hypersensitivities to aphidicolin (Aphhs) from Chinese hamster V79/743X fibroblasts. These variants are designated aphhs-1, aphhs-2, aphhs-3 and aphhs-4. We reported here results of studies involving immunochemical characterization. The Aphhs phenotype in all mutants was stable for at least 30 days in the absence of selection pressure. The dCTP pools in the 743X and Aphhs cell lines were not significantly different. The level of total DNA polymerase activity in the crude extract from aphhs-2 cells was 30% of that observed in the parental 743X clone. We developed a method to quantitate DNA polymerase-alpha antigen at single cells in situ using monoclonal antibody SJK 132-20 and fluorescence pseudocolor image. We found that the antigen of DNA polymerase-alpha in aphhs-2 was 30-50% of that in the parental 743X cells. The underproduction of the antigen of DNA polymerase-alpha provides a basis for the observed Aphhs phenotype. Possible mechanisms for the underproduction of DNA polymerase-alpha in aphhs-2 clone are presented.
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PMID:Aphidicolin hypersensitive mutant of Chinese hamster V79 fibroblasts that underproduces DNA polymerase-alpha antigen. 250 94

Aphidicolin and 17 derivatives that have been structurally modified in the A- and D-rings were assessed for their ability to inhibit DNA polymerase alpha. No derivative surpassed the activity of aphidicolin; derivatives with structural alterations in the A-ring exhibited significantly greater loss of activity relative to derivatives with structural alterations in the D-ring. The conclusions of these studies indicate a critical role for the C-18 function in the interaction of aphidicolin with polymerase alpha. Molecular modelling studies could not identify structural features of the aphidicolin-dCTP "overlap" that is unique to dCTP, relative to the remaining dNTPs, and that is consistent with the extant structure-activity data.
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PMID:Structure-activity relationships for the inhibition of DNA polymerase alpha by aphidicolin derivatives. 250 32

Acyclovir triphosphate (ACVTP) was a substrate for herpes simplex virus type 1 (HSV-1) DNA polymerase and was rapidly incorporated into a synthetic template-primer designed to accept either dGTP or ACVTP followed by dCTP. HSV-1 DNA polymerase was not inactivated by ACVTP, nor was the template-primer with a 3'-terminal acyclovir monophosphate moiety a potent inhibitor. Potent inhibition of HSV-1 DNA polymerase was observed upon binding of the next deoxynucleoside 5'-triphosphate coded by the template subsequent to the incorporation of acyclovir monophosphate into the 3'-end of the primer. The Ki for the dissociation of dCTP (the "next nucleotide") from this dead-end complex was 76 nM. In contrast, the Km for dCTP as a substrate for incorporation into a template-primer containing dGMP in place of acyclovir monophosphate at the 3'-primer terminus was 2.6 microM. The structural requirements for effective binding of the next nucleotide revealed that the order of potency of inhibition of a series of analogs was: dCTP much greater than arabinosyl-CTP greater than 2'-3'-dideoxy-CTP much greater than CTP, dCMP, dCMP + PPi. In the presence of the next required deoxynucleotide (dCTP), high concentrations of dGTP compete with ACVTP for binding and thus retard the formation of the dead-end complex. This results in a first-order loss of enzyme activity indistinguishable from that expected for a mechanism-based inactivator. The reversibility of the dead-end complex was demonstrated by steady-state kinetic analysis, analytical gel filtration, and by rapid gel filtration through Sephadex G-25. Studies indicated that potent, reversible inhibition by ACVTP and the next required deoxynucleoside 5'-triphosphate also occurred when poly(dC)-oligo(dG) or activated calf thymus DNA were used as the template-primer.
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PMID:Herpes simplex virus type 1 DNA polymerase. Mechanism of inhibition by acyclovir triphosphate. 254 Jan 93

The nuclear distribution of DNA polymerase alpha and PCNA/cyclin in embryonic nuclei has been investigated, in a cell-free extract of Xenopus eggs that recapitulates a basic cell-cycle in vitro, by indirect immunofluorescence microscopy. Both antigens co-distribute with the chromatin in S-phase nuclei; however, as DNA replication is completed and nuclei progress into a G2 state anti-PCNA fluorescence disappears and anti-DNA polymerase alpha fluorescence becomes resolved into bright spots. These spots are initially associated with the chromatin strands and can be seen to share both anti-PCNA and anti-DNA polymerase alpha fluorescence, but as anti-PCNA fluorescence fades the spots become dissociated from the chromatin and are redistributed throughout the nucleus until they are dispersed during nuclear envelope breakdown. The loss of anti-PCNA fluorescence and displacement of anti-DNA polymerase alpha fluorescence from the chromatin can be prevented by inhibiting DNA synthesis with aphidicolin. Under these conditions both antigens remain associated with the chromatin even after nuclear envelope breakdown and lamin dispersal. The association of these antigens with mitotic figures appears to be functional, as both biotin-11-dUTP and [32P]dCTP can be incorporated efficiently into DNA during the mitotic period.
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PMID:Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs. 257 94

The ability of the analog 2'-fluoro-2'-deoxycytidine triphosphate (dCflTP) to be used as a substrate in the reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha and AMV reverse transcriptase has been studied. The apparent Km values for dCTP and dCflTP, using activated DNA as templates, were 0.6 microM and 7 mM with DNA polymerase alpha and 0.14 microM and 7 microM with AMV reverse transcriptase, respectively. As observed with dCTP, aphidicolin was a noncompetitive inhibitor in the DNA polymerase alpha-catalyzed DNA synthesis; the Ki values were about 2 microM for both substrates. dCflTP can also be incorporated into DNA synthetized by other eukaryotic DNA polymerases and by reverse transcriptase with RNA as a template, both in the presence or absence of (dT)12 primer.
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PMID:2'-Fluoro-2'-deoxycytidine triphosphate as a substrate for RNA- and DNA-dependent DNA polymerases. 257 19


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