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)

When HeLa cell nuclei, isolated 17 h after infection with adenovirus type 2 (Ad2), were extracted with 200 mM ammonium sulfate, Ad2 nucleoprotein complexes were selectively released. These complexes contained a DNA polymerase activity that corresponded to DNA polymerase molecules actively engaged in Ad2 DNA replication. Under our high-salt (200 mM ammonium sulfate) incubation conditions, where no reinitiation occurred, full-length Ad2 DNA chains were synthesized by elongation of chains that had been initiated in vivo. This conclusion was further supported by density labeling experiments indicating that the in vitro DNA synthesis was semiconservative. Evidence is presented suggesting that at least part of the DNA polymerase molecules engaged in Ad2 DNA replication belong to the gamma class.
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PMID:Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei. 91 22

The DNA replication system of S-phase HeLa nuclei has been dissociated by cautious extraction at 0 degrees C with 0.25 M NaCl. Replicase activity has been reestablished by recombination of the fractions and reduction of the salt concentration. The reconstituted system, like the starting nuclei, depended on ATP, 4dNTP, MgCl2, the proper ionic strength and the soluble cytoplasmic protein fraction. The activity of the nuclear extract showed a cell cycle dependency and was elevated in the nuclei of cells at the G1 leads to S boundary. In the presence of Mg2+ the major activity of the nuclear extract precipitated during dialysis to reduce the salt concentration; this precipitate exhibited DNA polymerase alpha activity. Chromatography of the active extracts over phosphocellulose separated the replicase supporting factors into three fractions. The major activity eluted in the fraction containing the DNA polymerase alpha activity; the other two active fractions were devoid of polymerase activity. The fraction containing DNA polymerase alpha from the nuclear extracts supported DNA replicase activity in salt-extracted nuclei whereas an equivalent level of DNA polymerase alpha from the cytoplasm was not effective. The data suggest that the DNA polymerase alpha of the salt extracts of S-phase nuclei is either different than the cytoplasmic enzyme or is associated with some essential replicase-supporting factor.
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PMID:Dissociation and reconstitution of the DNA replicase system of HeLa cell nuclei. 94 95

DNA synthesizing reactions catalyzed both large and small species of calf thymus DNA polymerase (DNA polymerase-alpha and -beta) [EC 2.7.7.7] were stimulated to comparable extents by the presence of spermidine or spermine, and it was not possible to differentiate these two species in terms of their sensitivities to polyamines. Optimal concentrations for stimulation were 0.5-1.0 mM for spermidine and 2-10 mjM for spermine. Excess polyamines strongly inhibited the reactions. The modes of stimulation were as follows: 1) Stimulation was observed with templates bearing long single-stranded sections when either natural DNA or synthetic homopolymer-oligomer duplex was used. 2) The nautral DNA-dependent reaction was stimulated by polyamines at suboptimal concentrations of Mg2+; the apparent Km value for Mg2+ was lowered on adding polyamines, while the Vmax value was unchanged. When synthetic homopolymer-oligomer duplex was used as a template, the reaction was stimulated spermidine even at the optimal concentration of Mn2+. 3) Polyamines markedly influenced the salt requirements of the reactions of DNA polymerase. Spermidine could replace salts such as KC1 or NaC1 at concentrations less than 1/100 of those of salts.
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PMID:Effects of polyamines on in vitro dna synthesis by DNA polymerases from calf thymus. 95 42

The possible relationship between the nuclear and cytoplasmic DNA polymerases of regenerating rat liver was studied by sucrose gradient analysis, salt dissociation, and with specific inhibitors. After aqueous subcellular fractionation and removal of the nuclear membranes, three species of DNA-dependent DNA polymerases were characterized: 1) a DNA polymerase-beta in the nuclei. 2) a DNA polymerase-alpha in the cytosol which was not dissociated at high salt concentrations; and 3) an intermediate form in the cytosol and in the Triton wash containing the nuclear membranes. The latter form behaved like DNA polymerase-alpha et low salt concentration but was dissociated at high salt concentrations to a low molecular weight species with properties like DNA polymerase-beta (resistance to inhibition by N-ethylmaleimide, heparin and KCL). In vitro reassociation experiments suggest that this intermediate form corresponds to the association of DNA polymerase-beta with a membrane component or cytoplasmic protein(s) which appear(s) in regenerating rat liver.
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PMID:Regenerating rat liver DNA polymerases: disimilitude or relationship between nuclear and cytoplasmic enzymes? 96 78

We have isolated a nuclear membrane fraction from KB cells infected with human adenovirus 2 that synthesizes exclusively small viral DNA chains (approx. 9 S) in vitro (Yamashita, T., Arens, M. and Green, M. (1975) J. Biol. Chem. 250, 3273-3279). The DNA polymerase activity present in the adenovirus 2 DNA-nuclear membrane complex was purified through chromatography on phosphocellulose and DEAE-cellulose, glycerol gradient centrifuation and DNA-cellulose chromatography. A single peak of enzymatic activity sedimented in glycerol gradients at about 6.7 S which corresponds to a molecular weight of 125000. The enzyme preparation in the step of glycerol gradient centrifugation utilized activated calf thymus, KB cell and adenovirus 2 DNA as template-primer in the presence of Mg2+; Km values for these DNAs were 5.5, 4.0, and 0.8 mug/ml, respectively. The partially purified enzyme preparation was characterized by several criteria which were compared to the properties of the three major mammalian DNA polymerases, alpha, beta, and psi. On the basis of template-primer preference, effect of salt, inhibition by N-ethylmaleimide and Km for dTTP, the DNA polymerase activity from the membrane complex can be distinguished from the alpha and beta DNA polymerases. The elution profile from DNA cellulose revealed a minor peak (I) and a major peak (II) of DNA polymerase activity utilizing poly(A) -(dT)10 as template-primer in the presence of Mn2+ - Peak II from DNA cellulose, which contained about 90% of the total DNA polymerase activity eluted from the column, was 2-3 times as active with poly(A) - (dT)10 as template-primer in the presence of Mn2+ than with activated calf thymus DNA in the presence of Mg2+. On the other hand, peak I had a low ratio of poly(A) - (dT)10 to activated calf thymus DNA activity. DNA polymerase was also purified from the nuclear membrane fraction of uninfected KB cells by the same procedures as those used in enzyme purification from the adenovirus 2 DNA-nuclear membrane complex. A minor peak and a major peak of DNA polymerase activity utilizing poly(A) - (dT)10 as template primer in the presence of Mn2+ were again observed that eluted from DNA cellulose at the same KCl concentrations as peak I and II from adenovirus 2-infected cells. The enzymes of the nuclear membrane fraction of uninfected KB cells could not be differentiated from the enzymes of the adenovirus 2 DNA-nuclear membrane complex through any of the purification steps nor by their template specificities. These results indicate that the predominant enzyme in the adenovirus 2 DNA-nuclear membrane complex and in the KB cell nuclear membrane complex belongs to the class of DNA polymerase psi.
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PMID:Characterization of DNA polymerase associated with nuclear membrane fractions from uninfected and adenovirus 2-infected KB cells. 97 29

The freshly prepared crude cytoplasmic fraction of aqueously extracted KB cells contains a single major species of DNA polymerase activity (DNA polymerase C) that sediments homogeneously in low ionic strength sucrose gradients with a peak at 10.8 S. The enzyme activity from frozen crude extracts sediments heterogeneously under these conditions with peaks at 8.4 and 10 S. In 0.45 M salt-containing gradients all of the polymerase activity is recovered as a single 6.4 S species. When purified to a specific activity of 7,300, DNA polymerase C sediments in low ionic strength gradients as a single species of 6.5 S. From combined sedimentation and gel filtration analysis, we estimate the molecular weight of the active protomeric species of the polymerase to be about 170,000. Under no conditions of ionic strength does the enzyme disaggregate to active species smaller than 6.4 to 6.5 S. Sodium dodecyl sulfate-polyacrylamide gel analysis of the most highly purified enzyme fractions reveals two major protein bands of 87,000 and 175,000 daltons, respectively. These data suggest that DNA polymerase C contains an 87,000-dalton component and permit the interpretation that the active protomer of Mr equal 170,000 may be a dimer. The purified enzyme shows maximal activity with gapped duplex DNA and has an absolute requirement for 3'-hydroxyl termini. It utilizes initiated polydeoxynucleotide templates poorly and initiated polyribonucleotide templates not at all. Although the polymerase is inhibited by PPi it has only minimal ability to promote PPi exchange (0.8% of the polymerase activity). The purified enzyme is free of endonuclease and exonuclease activities (less than or equal to 0.003% of the polymerase activity) and demonstrates no primer-template-dependent conversion of substrate dNTP to free dNMP during the polymerization reaction. Finally, DNA polymerase C does not excise misparied primer termini from a synthetic homopolymer primer-template but can utilize such termini as initiation sites, although at a very slow rate.
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PMID:"Cytoplasmic" deoxyribonucleic acid polymerase. Structure and properties of the highly purified enzyme from human KB cells. 109

At low ionic strength KB cell DNA polymerase N1 forms large aggregates of a size comparable to those of DNA polymerase C. However, in contrast to polymerase C, the polymerase N1 aggregate: (a) retains the distinctive features of the polymerase N1 monomer, specifically its relative insensitivity to salt and to p-hydroxymercuribenzoate, and its pI of 9.3; and (b) is quantitatively converted to the polymerase N1 monomer form at appropriate ionic strength. It is important to recognize that since both polymerase N1 and polymerase C undergo salt-dependent association-dissociation reactions, attempts to distinguish these clearly indedependent polymerase species on the basis of size criteria can be very misleading. This is particularly true in relatively impure enzyme fractions that are generally isolated from eukaryotic tissue sources in low ionic strength buffers. We had earlier reported (Wang, T. S.-F., Sedwick, W. D., and Korn, D. (1974) J. Biol. Chem. 249,841-850; Sedwick, W. D., Wang, T. S.-F., and Korn, D. (1972) J. Biol. Chem. 247,5026-5033; Sedwick, W. D., Wang, T. S.-F., and Korn, D. (1974) Methods Enzymol. 29, 89-102) that DNA polymerase N1 could not utilize homoribopolymer templates. We have re-examined this question with a modified and more stringent method of product assay, and we show here that a greater than or equal 95% homogeneous preparation of polymerase N1 can copy the primer-template (A)n-(dT)-/16 at about one-half the rate that it copies activated DNA under optimum incubation conditions.
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PMID:Nuclear deoxyribonucleic acid polymerase. Further observations on the structure and properties of the enzyme from human KB cells. 115 97

After aqueous subcellular fractionation and partial purification by phosphocellulose chromatography, murine cells are found to contain a low molecular weight DNA-dependent DNA polymerase (beta) in the nuclear fraction and two distinguishable DNA-dependent DNA polymerases (C-I and C-II) in the cytosol. Both C-I and C-II are found in testis, liver, and regenerating liver; the amount of C-I being several fold increased in the regenerating liver and in immature testis. C-I and C-II are distinguishable by the criteria of salt sensitivity, inhibition by single-stranded DNA, elution from phosphocellulose, inhibition by 0.3 mM N-ethylmaleimide, template preference, and sedimentation coefficient. C-II is dissociated by 0.25 M KC1 to an active form of DNA polymerase of sedimentation coefficient 3.5 S while C-I is not dissociated, maintaining its sedimentation coefficient of 7.2 S. Many similar chemical and physical properties of C-II and the low molecular weight nuclear DNA polymerase (beta) suggest that C-II may represent an aggregate state of beta monomers, The size, reaction properties and the increase in enzyme activity under conditions of rapid cellular proliferation suggest C-I is analogous to the alpha DNA polymerase.
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PMID:The relationship between two murine DNA-dependent DNA polymerases from the cytosol and the low molecular weight DNA polymerase. 116 3

Cytoplasmic (high-molecular-weight) DNA polymerase was partially purified from mouse myeloma. Upon chromatography on DEAE-Sephadex, following fractionation on phosphocellulose, the enzyme was resolved into three species named CI, CII, and CIII. The species CI and CII have equal sedimentation coefficients (10.5 S) in sucrose gradients without salt. In the presence of 125 mM ammonium sulfate the sedimentation coefficients are reduced to 8.6 S. The species CIII shows sedimentation coefficients of 5.7 S and 5.2 S without salt and in the presence of 125 mM ammonium sulfate, respectively. This species is assumed to be an artifact arising from either CI or to a minor extent from CII. The optima for pH, KCl and Mg2+ concentration, and the extent of inhibition by N-ethylmaleimide are the same. However, the enzymes differ in their responses to Mn2+ (substituting for Mg-2+), and to addition of ethanol, dimethylsulfoxide, and various phospholipids in the assay mixture. The enzymes prefer poly[d(A-T - d(A-T)] or partially degraded (activated) DNA as template rather than double-stranded or single-stranded DNA. The activity on activated DNA relative to that on poly[d(A-T) - D(A-T)] was found to be 93, 66, and 29% for DNA polymerases CI, CII, and CIII, respectively.
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PMID:High-molecular-weight DNA polymerases from mouse myeloma. Purification and properties of three enzymes. 116 71

A DNA polymerase from Ustilago maydis has been purified to apparent homogeneity. The native enzyme possesses a subunit structure consisting of 50000 and 55000-dalton monomers. The apparent sedimentation coefficient of the polymerase activity in the absence of salt is 8.4 S (Mr=180000-200000), that in its presence (0.6 M NaCl or 0.12 M KCl) being 6.3 S (Mr=80000-100000). Low concentrations of EDTA also converted the 8.4-S to a 6.3-S form, whereas magnesium ions catalysed the reverse association. The enzyme has an absolute requirement for both a DNA or RNA template and a DNA primer. For homopolymer templates the primer requirement was satisified by a short complementary oligodeoxynucleotide, but oligoribonucleotides were extremely inefficient primers. With the template-primer poly(dA) X (dT)12, the enzyme added an average of 50 dTMP nucleotides on to each primer molecule, whereas with poly(rA) X (dT)12, this figure was 300. The enzyme also possesses an associated deoxyribonuclease activity. No other DNA polymerase activity was detected in cell-free extracts of U. maydis.
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PMID:A DNA polymerase from Ustilago maydis. 1. Purification and properties of the polymerase activity. 124 75


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