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)

A high molecular weight (6 S) plant DNA polymerase from axenic Vinca rosea tissue culture cells has been purified 2200-fold and characterized. The enzyme has a molecular weight of 105 000 (+/-5000). Sodium dodecyl sulfate-acrylamide gel electrophoresis of the purified enzyme yields polypeptide subunits having molecular weights of 70 000 and 34 000. The purified enzyme has a pH optimum of 7.5; a cation requirement optimum of 6 mM Mg2+ or 0.5 mM Mn2+; an apparent requirement for Zn2+; a Km of 1 muM for dTTP; and a 3.5-fold stimulation by 50 mM KCl. The enzyme is sensitive to N-ethylmaleimide (1 mM), heparin (0.1 muM), ethanol (5%), pyrophosphate (0.05 muM), and o-phenanthroline (0.1 mM) but is insensitive to rifamycin. Denatured DNA is found to be the best natural template, and only negligible activity can be demonstrated with the ribopolymer templates poly(dT)n-poly(rA)n and p(dT)10-poly(rA)n. In addition to the polymerization reaction, the enzyme catalyzes a pyrophosphate exchange reaction. Antibody to calf thymus 6-8S DNA polymerase does not inhibit DNA polymerase from Vinca rosea, suggesting no antigenic relationships between the mammalian and plant enzymes.
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PMID:High molecular weight deoxyribonucleic acid polymerase from crown gall tumor cells of periwinkle (Vinca rosea). 0 5

A stable deoxyribonucleic acid (DNA) polymerase (EC 2.7.7.7) with a temperature optimum of 80 degrees C has been purified from the extreme thermophile Thermus aquaticus. The enzyme is free from phosphomonoesterase, phosphodiesterase and single-stranded exonuclease activities. Maximal activity of the enzyme requires all four deoxyribonucleotides and activated calf thymus DNA. An absolute requirement for divalent cation cofactor was satisfied by Mg2+ or to a lesser extent by Mn2+. Monovalent cations at concentrations as high as 0.1 M did not show a significant inhibitory effect. The pH optimum was 8.0 in tris(hydroxymethyl)aminomethane-hydrochloride buffer. The molecular weight of the enzyme was estimated by sucrose gradient centrifugation and gel filtrations on Sephadex G-100 to be approximately 63,000 to 68,000. The elevated temperature requirement, small size, and lack of nuclease activity distinguish this polymerase from the DNA polymerase of Escherichia coli.
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PMID:Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. 0 32

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.
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PMID:Further characterization of a DNA polymerase activity in mouse sperm nuclei. 1 3

Three DNA polymerase activities, A, B and C, were identified in extracts of exponentially growing synchronous cultures of Chlamydomonas reinhardii, and DNA polymerases A and B were characterized in detail. Both enzymes have the same binding affinity for DEAE-cellulose at pH 7.8, but can be distinguished from each other by their behaviour on phosphocellulose and DNA-agarose. 'Activated' calf thymus DNA was used as template, and the pH, K+ and bivalent-cation optima were measured. DNA polymerase A sediments at 5.3 S in glycerol gradients, with an apparent mol.wt. of 90000-100000. Polymerase B sediments between 8S and 10S in 100mM-KCl, the predominant species having an apparent mol.wt. of 200000. In 200mM-KCl, polymerase B dissociates to a single species, which sediments at 5.8S. A 3S species was found in aged preparations of both enzymes. The activity of polymerase B from cells harvested during nuclear DNA synthesis is twice that found in Chlamydomonas at other times during the cell cycle.
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PMID:DNA polymerases from Chlamydomonas reinhardii. Purification and properties. 2 59

The heterogeneity of calf thymus DNA polymerase-alpha has been further investigated. In particular, an enzyme (enzyme D) which exhibits higher activity on poly(dA) . (dT)10 (A:T = 20:1) compared with that on activated DNA, has been further purified and its properties compared with two other activities of the DNA polymerase-alpha fraction (enzymes A1 and C) which do not show a preference for poly(dA) . (dT)10 over activated DNA. As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase-alpha in that it is an acidic protein as judged by its binding to DEAE-cellulose, has a molecular weight of about 140000, does not use a poly (A) . (dT)10 template-initiator complex and is inhibited by N-ethylmaleimide. It exhibits anomalous gel filtration behaviour on Sepharose 6B and it binds relatively weakly to DNA-cellulose compared with DNA polymerase-beta. The extreme sensitivity of enzyme D to inhibtion by N-ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE-cellulose column. On the other hand enzymes C and D are readily inactivated by heating at 45 degrees C unlike enzyme A1. The possible interrelationships of the multiple activities of calf thymus DNA polymerase-alpha are discussed.
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PMID:Studies on the purification and properties of a 6.8-S DNA polymerase activity found in calf-thymus DNA polymerase-alpha fraction. 2 65

A new method for the analysis and purification of the RNA-directed DNA polymerase of RNA tumor viruses has been developed. This nucleic acid affinity chromatography system utilizes an immobilized oligo (dT) moiety annealed with poly (A). The alpha and alphabeta DNA polymerases of avain myeloblastosis virus bound effectively to poly (A) oligo (dT)-cellulose. Alpha DNA polymerase did not bind effectively to poly (A) oligo (dT)-cellulose, poly (A)-cellulose, or to cellulose. Alphabeta bound to oligo (dT)-cellulose and cellulose at the same extent (approximately 30%), indicating that this enzyme did not bind specifically to the oligo (DT) moiety only. However, alphabeta bound to poly (A)-cellulose two to three times better than to cellulose itself, showing that alphabeta could bind to poly (A) without a primer. Alphabeta DNA polymerase also bound to poly (C)-cellulose, whereas alpha did not. These data show that the alpha DNA polymerase is defective in binding to nucleic acids if the beta subunit is not present. Data is presented which demonstrates that the alphabeta DNA polymerase bound tighter to poly (A). oligo (DT)-cellulose and to calf thymus DNA-cellulose than the alpha DNA polymerase, suggesting that the beta subunit or, at least part of it is responsible for this tighter binding. In addition, alphabeta DNA polymerase is able to reversibly transcribe avian myeloblastosis virus 70S RNA approximately fivefold faster than alpha DNA polymerase in the presence of Mg2+ and equally efficient in the presence of Mn2+. alpha DNA polymerase transcribed 9S globin m RNA slightly better than alphabeta with either metal ion.
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PMID:Binding properties of avian myeloblastosis virus DNA polymerases to nucleic acid affinity columns. 4 87

Inhibition of DNA polymerase from oncorna viruses by a new class of macromolecular inhibitors is reported. The macromolecule, designated as mercaptopolycytidylic acid (MPC), is a chemically modified polycytidylic acid containing 5-SH cytidylic bases in the polymerase. Partially thiolated polycytidylic acids (MPC I-III, containing 1.7%, 3.5%, and 8.6% 5-mercaptocytidylate units, respectively) inhibited the DNA-polymerase of Friend leukemia virus (FVL) in the endogenic reaction as well as in the presence of poly rA-(dT)14 or poly (dA-dT) templates; the inhibitory activities were directly related to the percent of tholation. In a bacterial DNA polymerase (E coli-K12 with denatured calf thymus DNA as template) MPCI-III showed no activity. Biological experiments showed that MPC III inhibits the leukemogenic potential of cell-free spleen extracts from FVL-infected mice to about 60%, measured on the basis of spleen weight. The enzymatic and animal experiments have led us to carry out preliminary clinical trials in some cases of Children leukemia. These cases, resistent to the known therapeutic regimes (combination chemotherapy), responded well when treated with MPC along, or in combination with poly I. The experiments indicate that the development of modified polynucleotids with structural similarities to functional templates may be of potential use in the future chemotherapy of leukemia.
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PMID:[Inhibition of viral reverse transcriptase and leukemogenesis by modified nucleic acids (author's transl)]. 4 86

At concentrations of 7 times 10(-6) to 7 times 10(-5) M, derivatives consisting of the polycylic ring structures fluoranthene, fluorenone, fluorene, anthraquinone, xanthenone, and dibenzofuran with appropriate amine side chains inhibited by over 90% the purified RNA-directed DNA polymerase of avian myeloblastosis virus acting on poly(deoxyadenylate-deoxythymidylate) [poly(dA-dT)]. Of these, only the fluoranthene derivatives were strong inhibitors of the viral DNA polymerase directed by polyadenylate-oligodeoxythymidylate [poly(A)-(dT)12-18]. Low levels of fluoranthene derivatives (1 times 10(-5) M) also strongly inhibited polymerase with polyinosinate-oligodeoxycytidylate [poly(I)-(dC)12-18], activated calf thymus DNA, and viral 70S RNA as templates, but not with polycytidylate-oligodeoxyguanylate as template. A comparison of the activity of 11 fluoranthene derivatives with different side chains showed that the structure of the amine side chain influenced both the extent of antipolymerase activity with a given template and the relative inhibition with different synthetic DNA and RNA templates. The naturally occurring polyamines, spermine, spermidine, and putrescine, did not inhibit the activity of the viral DNA polymerase. Studies on the mechanism of action indicated that the synthetic derivatives inhibited polymerase activity by binding to the template and not to the enzyme: 1) inhibition by fluoranthene derivatives was overcome by the addition of excess template including poly(dA-dT), poly(A)-(dT)12-18, poly(I)-(dC)12-18, viral 70S RNA, and activated calf thymus DNA; 2) the degree of inhibition by fluoranthene derivatives was unaffected by the addition of the creased viral DNA polymerase; 3) with the same template, Escherichia coli DNA-directed RNA polymerase and the viral RNA-directed DNA polymerase were inhibited to about the same extent; and 4) the derivatives formed a complex with DNA, poly(I), and poly(A) that was stable to exclusion chromatography on Sephadex G-100. Several derivatives also had biologic activity, since they blocked the ability of the murine sarcoma virus to transform cells.
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PMID:Inhibition of purified DNA polymerase of RNA tumor viruses by fluoranthene derivatives and analogues of tilorone hydrochloride. 5 Oct 87

The effect of bleomycin on [3H]thymidine 5'-triphosphate ([3H]TTP) incorporation into isolated sucrose nuclei from host liver and Morris hepatomas has been compared. Bleomycin stimulates [3H]TTP incorporation 13-fold in host liver and hepatoma 16 nuclei, 8-fold in hepatoma 7800 nuclei, and 3-fold in hepatoma 7777 nuclei. Differences in the nuclear membranes are not responsible for the different response of the nuclei. Nuclei, denuded of their membranes by Triton X-100 treatment, give similar results to sucrose nuclei. Analysis of DNA extracted from liver or hepatoma nuclei incubated with bleomycin indicates that bleomycin produces scissions in the nuclear DNA and that some repair synthesis takes place. Incubation of nuclei with 111indium-labeled bleomycin shows an equal binding capacity of liver and hepatoma nuclei for bleomycin. Bleomycin also stimulates incorporation of [3H]TTP in a system using chromatin or calf thymus DNA as primer. Host liver or hepatoma chromatin incubated with a DNA polymerase extracted from normal rat liver nuclei is stimulated approximately to the same extent by bleomycin. When DNA polymerase extracts from host liver and hepatoma nuclei are assayed with calf thymus DNA as primer, bleomycin has a greater stimulatory effect on [3H]TTP incorporation with host liver DNA polymerase than with hepatoma DNA polymerase in the system. We suggest that a defect in the repair system in hepatoma nuclei is responsible for the relatively lower response to bleomycin.
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PMID:Effect of bleomycin on [3H]Thymidine 5'-Triphosphate incorporation into host liver and hepatoma nuclei. 5 97

Terminal deoxynucleotidyl transferase (TDT) is an unusual DNA polymerase that does not use template information to synthesize new strands of DNA. It is normally found in high concentration in thymus (50 u/10(8) cells) and in low concentration in bone marrow (less than 5 u/10(8)). We report TDT measurements in the marrow and/or peripheral blood of 51 adult patients, 28 of whom had leukaemia. TDT is present in very high levels (greater than 50 u/10(8) cells) in leukaemic lymphoblasts and in low levels in leukaemic myeloblasts (less than 9 u/10(8) cells). Of two patients who developed lymphosarcoma-cell leukaemia following treatment of poorly differentiated lymphocytic lymphoma, one had high and one low levels of TDT in the leukaemic blast cells. Leukaemic cells from three of seven patients with chronic myeloid leukaemia in blast crisis had TDT levels within the range expected of acute lymphoblastic rather than acute myeloid leukaemia. High TDT in leukaemic cells probably marks them as derivatives of lymphoid progenitor, thymic or pluripotential stem cells. Quantitative assay of TDT may provide information useful in classifying haematological neoplasms.
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PMID:Terminal deoxynucleotidyl transferase measurements in the differential diagnosis of adult leukaemias. 6 84


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