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)

The fidelity of DNA synthesis with purified DNA polymerase alpha and beta from human placenta has been studied. With poly[d(A-T)] as the template-primer and Mg2+ as the metal activator, DNA polymerase alpha incorporates 1 mol of dGMP for every 6,000 to 12,000 mol of complementary nucleotides polymerized. Under the same conditions, DNA polymerase beta is more accurate, the error rate being 1/20,000 to 1/60,000. This greater accuracy of DNA polymerase beta is observed with a variety of homopolymer templates. With both enzymes, substitution of Mg2+ with activating concentrations of Mn2+ or Co2+ enhances the frequency of misincorporation. At greater than activating concentrations of Mn2+ and Co2+, there is an inhibition of complementary nucleotide incorporation, further increasing the frequency of misincorporation. Nearest neighbor analysis of the products synthesized with both enzymes indicates that the noncomplementary nucleotides are incorporated predominantly as single base substitutions. The greater accuracy of DNA polymerase beta over DNA polymerase alpha should be considered in relationship to their possible roles in DNA replication and repair.
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PMID:On the fidelity of DNA replication. Studies with human placenta DNA polymerases. 44 44

This report describes the results of our initial enzymological characterization of a homogeneous preparation of DNA polymerase alpha that we have purified from cultured human KB cells. Although the enzyme is most reactive with duplex DNA substrates that contain short gaps (optimally activated) in incubations that require Mg2+, the polymerase possesses the intrinsic capacity to copy the initiated ribohomopolymer template, (A)-n, (dT)-200, at low rates in the presence of Mn2+. Because of the preponderance of DNA polymerase alpha in actively multiplying vertebrate cells, it is probable that this low level of activity comprises the majority of the ribopolymer copying activity that can be detected in crude tissue extracts. The presence of contaminating or associated deoxyribonuclease activities can be excluded from the purified enzyme to levels of 10(-4) to 10(-7) of the polymerase activity. The mechanism of polymerization on activated DNA under optimum conditions is moderately processive, with 11 +/- 5 nucleotides incorporated per polymerization cycle. The polymerase is unable to work at nicks or at short gaps of approximately 20 to 30 nucleotides in length, and it measures a surprisingly invariant effective template length on optimally activated DNA and on DNA molecules that have been gapped to varying extents with Escherichia coli exonuclease III. In the "Appendix" we present an amplification of the theoretical formulation of Bambara et al. (Bambara, R. A., Uyemura, D., and Choi, T. (1978) J. Biol. Chem. 253, 413--423) that permits the use of DNA polymerases with significant associated 3' leads to 5'-exonuclease activities for the accurate measurement of average template lengths (gap sizes) and titration of usable 3'-hydroxyl primer termini in gapped, duplex DNA substrates.
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PMID:Enzymological characterization of DNA polymerase alpha. Basic catalytic properties processivity, and gap utilization of the homogeneous enzyme from human KB cells. 44 99

DNA polymerase gamma and mitochondrial DNA polymerase were isolated from brain nuclei and synaptosomes respectively. The presence of a single DNA polymerase in synaptosomal mitochondria was established by chromatography on DEAE-cellulose, phosphocellulose and DNA-cellulose, as well as by sedimentation analysis and isoelectric focusing. A great similarity between the purified nuclear DNA polymerase gamma and the mitochondrial enzyme was found by the following criteria: chromatographic behaviour in three column systems; essentially complete inhibition by N-ethyl-maleimide (2 mM); optimal requirements of Mn2+ (0.1 mM), Mg2+ (5 mM) and pH (8.0); template preferences, poly(A) - (dT)20-25 larger than activated DNA larger than poly(dA) - (dT)12-18; lack of activity on single-stranded polynucleotides and (dT)12-primed mRNA; molecular weight (180000), sedimentation (9.2 S) and isoelectric point (pI 5.4). We therefore conclude that brain nuclear DNA polymerase gamma and synaptosomal mitochondrial DNA polymerase are closely related and may even be identical.
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PMID:Identity of DNA polymerase gamma from synaptosomal mitochondria and rat-brain nuclei. 59 67

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) has been purified over 100 000-fold from a whole cell extract of guinea pig liver. The enzyme yields a single stainable band when subjected to non-denaturing polyacrylamide gel electrophoresis, and this band corresponds to the DNA polymerase activity when a sister gel is sliced and assayed. The final fraction has a specific activity of 21 000 units/mg; this value can be increased significantly by addition of various components, including glycols, polyamines or any of several protein factors which can be purified from the crude extract. The DNA polymerase-beta lacks detectable exonuclease or endonuclease activity, has an alkaline pH optimum and has a requirement for all four deoxyribonucleoside triphosphates, a divalent cation and a primer-template for maximal activity. While activated DNA is the preferred primer-template, the enzyme is capable of utilizing native and denatured DNA as well as several synthetic polynucleotides as primer-templates. The latter are especially effective when manganese is the divalent cation. Magnesium, at 10 mM, is the preferred divalent cation when activated DNA is used. Manganese, and to a lesser extent cobalt, can substitute for magnesium while zinc and calcium cannot. The beta-polymerase has a half-life of 10 min at 40 degrees C and this is increased in the presence of either DNA or NaCl. The enzyme is stimulated by glycols, polyamines and NaCal or KCl, and is inhibited by several known inhibitors of DNA polymerase activity including o-phenanthroline, heparin, organic solvents and sulfhydryl blocking agents. Guinea pig liver DNA polymerase-beta is remarkably similar to the rat Novikoff hepatoma beta-polymerase with respect to its isoelectric point of 8.4 and its molecular weight of 32 000 as determined by sucrose gradient centrifugation under high or low salt conditions or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This similarity is further extended to the removal, at the final step in purification, of a protein capable of stimulating the homogeneous enzyme. Removal of this protein could explain the lower molecular weight of the guinea pig and other rodent-derived beta-polymerases, when compared to the beta-polymerases from other systems.
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PMID:Purification and properties of DNA polymerase-beta from guinea pig liver. 70 39

A novel DNA polymerase, which could use both poly(rA) . oligo(dT) and activated calf thymus DNA efficiently as template-primers, was purified 20 000-fold from calf thymus extract. These activities were co-purified throughout successive column chromatographies and banded at the same position in either electrofocussing (pI = 6.5--7.0) or sucrose rate-zonal centrifugation (10--10.5 S). The most purified fraction (DNA-cellulose fraction) possessed specific activities of 3900 units/mg of protein with poly(rA) . oligo(dT) and 32 000 units/mg of protein with activated DNA. The poly(rA) . oligo(dT)-dependent activity differed from the previously described DNA polymerase gamma from other sources in the following ways: 1. The activity was inhibited by 100--300 mM KCl and and 80 mM potassium phosphate buffer. 2. The activity was 4-fold higher at 26 degrees C than at 37 degrees C. 3. The Km value for dTTP was 2.6--3.0 . 10(-4) M, which is several hundred-fold greater than that of DNA polymerase gamma. 4. Mn2+ was essential for the reaction and could not be replaced by Mg2+. The activated DNA-dependent activity shared many properties with DNA polymerase alpha, except that it was less sensitive to N-ethylmaleimide and anti-alpha polymerase immunoglobulin G. The 10-S DNA polymerase was dissociated into 8.5-S and 3.3-S by treatment with Triton X-100.
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PMID:10-S DNA polymerase from calf thymus which copies both poly(rA) . oligo(dT) and activated DNA. 71 38

Bull spermatozoa heads were separated from cytoplasmic contaminants, especially mitochondria-rich middle pieces, by centrifugation through 2.4M-sucrose. DNA polymerase activity was demonstrated by incubating nuclear heads for 1 h at 37 degrees C or for 20 h at room temperature in a medium containing detergent and dithiothreitol or 2-mercaptoethanol. Optimal DNA polymerase activity was detected after extraction in a medium containing 50 mM-borate, pH9, 1 mg of soya-bean trypsin inhibitor/ml and supplemented with either 20 mM-dithiothreitol and 4% Tween 80 or 100mM-2-mercaptoethanol and 10% Tween 80. The DNA polymerase reaction was Mg2+-dependent; Mn2+ or Ca2+ could not replace Mg2+ and all four deoxynucleoside triphosphates were required for optimal activity. The polymerase activity was pH-dependent (optimum between 8.2 and 10.5) and was a function of buffer composition and also of pH values. Optimal activity was obtained with 50 mM-Na+ or 150mM-K+ and was partially lowered by N-ethylmaleimide; it was inhibited by spermidine and by salmon protamines, but was greatly stimulated by calf thymus histones. It was also resistant to actinomycin D, netropsin and ethidium bromide. The present results suggest that bull spermatozoa heads contain a beta-type DNA polymerase activity.
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PMID:Extraction and biochemical characterization of a nuclear deoxyribonucleic acid polymerase activity in bull spermatozoa. 74 11

The fidelity of DNA synthesis with synthetic homopolymer templates by two ribodeoxyvirus DNA polymerases and E. coli DNA polymerase I was examined by nearest neighbor frequency analyses. The experiments were designed to favor the incorporation of noncomplementary nucleotides, and the reactions were carried out only for short periods of time. All incorporations were template dependent. The frequency of incorporation of noncomplementary nucleotides next to complementary nucleotides by all three of these DNA polymerases significantly varied, depending on the conditions, from almost none (less than 0.2%) to 100% of the incorporation of the complementary nucleotide. In general, there was more incorporation of noncomplementary nucleotides in the presence of Mn2+ than in the presence of Mg2+ and with ribohomopolymer templates than with deoxyribohomopolymer templates. With a heteropolymer RNA template, the infidelity of DNA synthesis by one of the ribodeoxyvirus DNA polymerases was also increased by the presence of Mn2+ in the DNA synthesizing reaction. These findings indicate that, under appropriate conditions, any DNA polymerase has the potential to make a significatn number of errors in DNA synthesis and that altered conditions for DNA polymerase action might play a role in spontaneous mutation.
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PMID:Incorporation of noncomplementary nucleotides at high frequencies by ribodeoxyvirus DNA polymerases and Escherichia coli DNA polymerase I. 76 23

An aqueous solution of 5'-amino-5'-deoxythymidine 5'-triphosphate, prepared by incubation of equimolar solutions of 5'-amino-5'-deoxythymidine and sodium trimetaphosphate, stimulates synthesis of acid-precipitable polynucleotides in a system containing single-strand phiX174 DNA template, random oligonucleotide primers, dATP, dCTP,dGTP, Escherichia coli DNA polymerase I, and either magnesium or manganese ion. Approximately onefold synthesis on the template can be achieved and each of the indicated reagents is essential for extensive synthesis. The reaction is slower than the corresponding reaction of dTTP as a consequence of a lower V max and a higher Km for the amino analogue. That aminodeoxythymidine phosphate is incorporated into the synthetic polynucleotides was shown by a double-labeling experiment with [14C]dATP and [32P]-5'-amino-5'-deoxythymidine 5'-triphosphate and by the unusually high lability of the phosphoramidate polynucleotides toward acid. The phosphoramidate polynucleotides range in size from about 100 nucleotide units to well over a thousand nucleotide units, and the size is increased by addition of DNA ligase to the system. These experiments indicate that synthetic polynucleotides in which oligonucleotide blocks have been joined by means of phosphoramidate bonds should prove useful as primers for enzymatic syntheses with DNA polymerase I.
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PMID:Incorporation of 5'-amino-5'-deoxythymidine5'-phosphate in polynucleotides by use of DNA polymerase I and a phiX174 DNA template. 77 32

The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on the carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide. Mitochondria DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacylamide gel electrophoresis revealed the following structure: a monomer of molecular weight around 60 000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120 000 which under low salt concentration (0.2 M Tris-HCl buffer) formed higher aggregates. For optimal activity an Mg2+ ion concentration of 50 mM was found necessary, Mn ions did not promote activity at any concentration tested (0.5--50 mM). Indeed, if added to Mg2+-containing assays, Mn2+ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn2+ ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the -SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50% decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the Km concentration. The enzyme preferred a gapped template primer, poly(dA) - (dT)10, over nicked DNA and was unable to use a polyribonucleotide template, poly(rA) - (dT)10. In the purest preparations no exonuclease activity could be detected.
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PMID:DNA-dependent DNA polymerase from yeast mitochondria. Dependence of enzyme activity on conditions of cell growth, and properties of the highly purified polymerase. 78 35

Escherichia coli DNA polymerases II and III have been extensively studied in vitro when activated with Mg2+. The Mn2+-activated polymerization reactions are considered here, and shown to differ from the Mg2+-activated reactions. The Mn2+-activated DNA polymerase II reaction requires K+ or spermidine, and the effects of monovalent cation and polyamine are additive. In contrast, the Mg2+-activated reaction does not require, but is stimulated by, K+ or spermidine, in a non-additive manner. Under optimal conditions, DNA polymerase II is activated better with Mn2+ than it is with Mg2+, suggesting a physiological role for the Mn2+-activated enzyme. The observed preference for Mn2+ over Mg2+ in reaction kinetics and at high DNA template concentrations suggest that Mg2+ may preferentially activate the associated exonuclease activity. At 29 degrees C, the Mn2+-activated DNA polymerase III reaction is stimulated by K+ and inhibited by ethanol or phosphatidylethanolamine. In contrast, the latter compounds and Triton X-100 increase the initial rate of the Mg2+-activated reaction, whereas K+ inhibits this reaction at all concentrations. The K+ inhibition is reduced at low Mg concentrations when Mn2+ is also present. After stimulating the initial reaction rate, ethanol causes a rapid decrease in the rate of the Mg2+-activated reaction during incubation at 20 degrees C. At 27 degrees C, all surface-active compounds inhibit the Mg2+-activated reaction. Preincubation of the enzyme at 30 degrees C or below with DNA template and divalent cation increases the initial reaction rate, suggesting that formation of an enzyme-divalent cation-DNA template complex occurs as the first step in DNA polymerase III catalysis. The apparent Km at 21 degrees C for gapped calf thymus DNA was 25 muM with Mn2+ and 125 muM with Mg2+ for DNA polymerase III, and 18 muM at 30 degrees C for DNA polymerase II with either Mn2+ or Mg2+. Reactions with poly[d(A-T)] were enhanced by Mn2+ relative to Mg2+, and activity with poly(rA)-poly(dT) was Mn2+ dependent for both enzymes.
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PMID:Escherichia coli DNA polymerases II and III: activation by magnesium or by manganous ions. 78 84


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