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)

To establish an in vitro system for studying DNA repair, bleomycin-induced unscheduled DNA synthesis in permeable HeLa cells was investigated. Permeable HeLa cells were incubated at 0 degree C for 60 min with 0.11 mM bleomycin, washed to remove free bleomycin and assayed for DNA synthesis. Optimum [3H]deoxythymidine monophosphate incorporation occurred at pH 7.6-8.0 (adjusted at 20 degrees C with Tris-HCl buffer), 3-6 mM MgCl2, 40-60 mM NaCl, and 2.5-5 mM ATP in the presence of four deoxynucleoside triphosphates. The unscheduled nature of DNA synthesis in bleomycin-pretreated permeable cells was confirmed by the BrdUMP density shift technique. Exonuclease III sensitivity of repaired DNA was measured to determine whether or not the completion of repair patches and ligation occurred in bleomycin-pretreated permeable cells. Gap-filling and ligation were suggested to occur in the presence of ATP. Studies using the selective inhibitors (aphidicolin, 2',3'-dideoxythymidine 5'-triphosphate and N-ethylmaleimide) for DNA synthesis showed that DNA polymerases alpha and beta were involved in the repair process. Inhibitor studies suggested that DNA polymerase alpha plays a preferential role in repair label in the intranucleosomal region of nuclear chromatin and DNA polymerase beta in the completion of repair patches in bleomycin-pretreated permeable cells.
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PMID:DNA repair synthesis in bleomycin-pretreated permeable HeLa cells. 241 38

Reverse transcriptase was purified from human immunodeficiency virus (HIV). It utilized the artificial primer-template poly(rA)-oligo(dT)12-18 more efficiently than activated calf thymus DNA, poly(rI)-oligo(dC)12-18, poly(rC)-oligo(dG)12-18, or poly(rCm)-oligo(dG)12-18. Maximum activity was observed at pH 7.0 to 7.6 in the presence of 5 mM MgCl2 and 100 mM KCl. 3'-Azido-3'-deoxythymidine triphosphate competed with dTTP for binding to HIV reverse transcriptase. Different kinetic constants were obtained with different primer-templates. Km and Ki values of 2.8 and 0.04 microM, respectively, were obtained with poly(rA)-oligo(dT)12-18. The corresponding values were 1.2 and 0.3 microM, respectively, with activated calf thymus DNA and 0.3 and 0.01 microM, respectively, with extracted virus and native template. Inhibition of the host cell DNA polymerases alpha and beta was considerably weaker. The Km and Ki values obtained with activated calf thymus DNA as the primer-template were 2.4 and 230 microM, respectively, for DNA polymerase alpha and 6.0 and 73 microM, respectively, for DNA polymerase beta. 3'-Azido-3'-deoxythymidine triphosphate could also serve as an alternate substrate for HIV reverse transcriptase. The resulting incorporation of 3'-azido-3'-deoxythymidine triphosphate into poly(rA)-oligo(dT)12-18 caused chain termination and premature deceleration of the reaction. The terminated primer could not be elongated when incubated with dTTP and HIV reverse transcriptase.
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PMID:3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. 244 66

Various galloyl derivatives of quinic acid were found to be inhibitors of human DNA polymerases. Among them, 3,4,5-tri-O-galloylquinic acid (TGQA) was the most potent inhibitor of DNA polymerase alpha. Under identical conditions, this compound was 60-fold more potent than aphidicolin as an inhibitor of DNA polymerase alpha. The inhibition of DNA polymerase alpha by this compound was not competitive with either the template or any of the deoxynucleoside triphosphates with a Ki of 0.28 microM. Under similar reaction conditions, DNA polymerases beta and gamma were much less sensitive to the effects of these compounds and, in contrast to the effect seen with DNA polymerase alpha, the inhibition of DNA polymerases beta and gamma by TGQA was competitive with respect to the template with Ki values of 44.4 and 7.5 microM respectively. The potency of these compounds against DNA polymerase gamma varied according to the assay conditions used. The inhibition of DNA polymerase gamma by TGQA could be increased substantially by using MnCl2 in place of MgCl2 and by including 50 mM potassium phosphate, pH 7.5, in the assay mixture. DNA polymerase beta was also more sensitive to TGQA when measured with MnCl2. However, potassium phosphate had little, if any, effect on the inhibition by TGQA of either DNA polymerase alpha or beta. DNA polymerase alpha was less sensitive to TGQA when assayed with MnCl2. TGQA was not a potent inhibitor of human KB cell growth in culture, which could be due to its degradation or poor uptake. Nevertheless, this compound could serve as a model for developing antitumor drugs targeted at DNA polymerases.
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PMID:Characterization of a novel inhibitor of human DNA polymerases: 3,4,5-tri-O-galloylquinic acid. 248 Jul 88

We have isolated a highly enriched preparation of the multienzyme complex which synthesizes deoxyribonucleoside triphosphates (dNTPs) from bacteriophage T4-infected bacteria. By a combination of SDS polyacrylamide gel electrophoresis and assays for specific enzyme activities, we have been able to identify in our final preparation ten different gene products which were previously identified as constituents of this complex, based upon studies with crude preparations. The complex dissociates at high concentrations of NaCl and MgCl2 but is stable under ionic conditions thought to exist in vivo. The purified complex catalyzes the efficient five-step conversion of dCTP to dTTP. Experiments with several T4 mutants have demonstrated that gene products encoded by cd, regA, nrdA, and nrdB are necessary to retain physical integrity of the complex throughout the preparative procedure, while gp44, gp55, and gppseT are not required. We conclude from this evidence that the T4 early gene products which function in dNTP biosynthesis are, in fact, physically linked as a multienzyme complex, and that regA contributes to the integrity of this complex. However, the dNTP-synthesizing complex as we isolate it contains no detectable DNA polymerase, nor have other known replication proteins been detected.
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PMID:T4 phage deoxyribonucleoside triphosphate synthetase: purification of an enzyme complex and identification of gene products required for integrity. 307 39

Three proteins catalyze RNA-primed DNA synthesis on the lagging strand side of the replication fork of bacteriophage T7. Oligoribonucleotides are synthesized by T7 gene 4 protein, which also provides helicase activity. DNA synthesis is catalyzed by gene 5 protein of the phage, and processivity of DNA synthesis is conferred by Escherichia coli thioredoxin, a protein that is tightly associated with gene 5 protein. T7 DNA polymerase and gene 4 protein associate to form a complex that can be isolated by filtration through a molecular sieve. The complex is stable in 50 mM NaCl but is dissociated by 100 mM NaCl, a salt concentration that does not inhibit RNA-primed DNA synthesis. T7 DNA polymerase forms a stable complex with single-stranded M13 DNA at 50 mM NaCl as measured by gel filtration, and this complex requires 200 mM NaCl for dissociation, a salt concentration that inhibits RNA-primed DNA synthesis. Gene 4 protein alone does not bind to single-stranded DNA. In the presence of MgCl2 and dTTP or beta, gamma-methylene dTTP, a gene 4 protein-M13 DNA complex that is stable at 200 mM NaCl is formed. The affinity of DNA polymerase for both gene 4 protein and single-stranded DNA leads to the formation of a gene 4 protein-DNA polymerase-M13 DNA complex even in the absence of nucleoside triphosphates. However, the binding of each protein to DNA plays an important role in mediating the interaction of the proteins with each other. High concentrations of single-stranded DNA inhibit RNA-primed DNA synthesis by diluting the amount of proteins bound to each template and reducing the frequency of protein-protein interactions. Preincubation of gene 4 protein, DNA polymerase, and M13 DNA in the presence of dTTP forms protein-DNA complexes that most efficiently catalyze RNA-primed DNA synthesis in the presence of excess single-stranded competitor DNA.
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PMID:Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis. 353 39

An immunoaffinity chromatographic procedure was developed to purify DNA polymerase-DNA primase complex from crude soluble extracts of yeast cells. The immunoabsorbent column is made of mouse monoclonal antibody to yeast DNA polymerase I covalently linked to Protein A-Sepharose. Purification of the complex involves binding of the complex to the immunoabsorbent column and elution with concentrated MgCl2 solutions. After rebinding to the monoclonal antibody column free primase activity is selectively eluted with a lower concentration of MgCl2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of five major peptides, p180, p140, p74, p58, and p48 in the immunoaffinity-purified DNA polymerase-DNA primase complex. Free primase and free polymerase fractions obtained by fractionation on the immunoabsorbent column were analyzed on activity gels and immunoblots. These analyses showed that p180 and p140 are DNA polymerase peptides. Two polypeptides of 58 and 48 kDa co-fractionated with the free yeast DNA primase. From sucrose gradient analysis we estimate a molecular weight of 110 kDa for the native DNA primase.
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PMID:Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex. 388 95

A DNA polymerase with properties similar to mammalian polymerase beta has been isolated to near homogeneity from embryos of Drosophila melanogaster. A combination of exclusion chromatography and sodium dodecyl sulfate-gel electrophoresis indicates that this enzyme is composed of a single polypeptide of molecular weight-110,000. Optimum activity on a nicked template occurs at pH 8.4 in the presence of 15 mM MgCl2 and 250 mM NaCl. Enzyme activity is strongly inhibited by dideoxythymidine triphosphate but is relatively insensitive to aphidicolin and N-ethylmalemide. These properties clearly distinguish this enzyme from polymerase alpha, which has previously been characterized from this tissue. This report represents the first extensive purification of a beta-like polymerase from the Protostomic branch of the animal phylogenetic tree. It furthermore generates the potential for a genetic analysis of the function of polymerase beta in DNA recombination, repair, and synthesis.
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PMID:Purification and characterization of a DNA polymerase beta from Drosophila*. 392 21

An in vitro system capable of synthesizing infectious phi X174 phage particles was reconstituted from purified components. The synthesis required phi X174 supercoiled replicative form DNA, phi X174-encoded proteins A, C, J, and prohead, Escherichia coli DNA polymerase III holoenzyme, rep protein, and deoxyuridinetriphosphatase (dUTPase, dUTP nucleotidohydrolase, EC 3.6.1.23) as well as MgCl2, four deoxyribonucleoside triphosphates, and ATP. Phage production was coupled to the synthesis of viral single-stranded DNA. More than 70% of the synthesized particles sedimented at the position of mature phage in a sucrose gradient and associated with the infectivity. The simple requirement of the host proteins suggests that the mechanism of viral strand synthesis in the phage-synthesizing reaction resembles that of viral strand synthesis during the replication of replicative form DNA.
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PMID:In vitro synthesis of bacteriophage phi X174 by purified components. 622 17

The principal properties of the DNA polymerases of woodchuck hepatitis virus and human hepatitis B virus were compared. The enzymes of both viruses exhibited optimal activities in the same range of pH, ionic strength, and MgCl2 concentration. Like human hepatitis B virus DNA polymerase, the woodchuck hepatitis virus DNA polymerase was strongly inhibited by phosphonoformic acid but not by phosphonoacetic acid and aphidicolin. Similar inhibition patterns for both enzymes were observed with arabinofuranosyl nucleotides (9-beta-D-arabinofuranosyladenine-5'-triphosphate, 1-beta-D-arabinofuranosylcytosine-5'-triphosphate, 1-beta-D-arabinofuranosylthymine-5'-triphosphate) and dideoxythymidine triphosphate, whereas no effect was obtained with corresponding nucleosides. The therapeutic significance of these results and the relevance of the woodchuck as an experimental animal model for the study of human hepatitis B virus infections are discussed.
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PMID:Comparison of properties of woodchuck hepatitis virus and human hepatitis B virus endogenous DNA polymerases. 623 85

Human cytomegalovirus (HCMV), purified exclusively from the extracellular media, contained a DNA polymerase activity in addition to a protein kinase activity. The DNA polymerase expressed its maximum activity in the presence of 5 to 10 mM-MgCl2. The enzyme was able to use effectively activated calf thymus DNA, poly(dA).oligo(dT)12--18 and poly(dC).oligo(dG)12--18 as the template primers. The DNA polymerizing activity was eluted with 0.18 to 0.2 M-KCl from a phosphocellulose column. It was relatively resistant to phosphonoacetic acid inhibition even at a high concentration of 100 micrograms/ml with activated calf thymus DNA as the template primer, but the DNA polymerase activity was totally suppressed at this concentration when poly(dA).oligo(dT)12--18 was used as the template primer. The enzyme activity was inhibited by ammonium sulphate at 0.01 to 0.3 M with either activated calf thymus DNA or poly(dA).oligo(dT)12--18 as the template primer. The protein kinase has maximum activity in the presence of 10 to 20 mM-MgCl2, and preferred virion proteins as phospho-acceptor to protamine sulphate. Histone, caesin and bovine serum albumin (BSA) were found to be poor substrates. The phosphorylated protein pattern of the in vivo [32P]orthophosphate-labelled virions was not identical to that of the in vitro phosphorylated Nonidet P40-dissociated virions, although seven phosphorylated polypeptides did co-migrate in SDS--polyacrylamide gel electrophoresis (SDS--PAGE). Procedures known to solubilize virions showed that the DNA polymerase and protein kinase were internal components of the virion.
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PMID:Human cytomegalovirus-associated DNA polymerase and protein kinase activities. 627 14


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