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)

Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparaions from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5-fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase beta is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin-directed replication is differently arrested in non-dividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.
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PMID:Replicative activity of isolated chromatin from proliferating and quiescent early passage and aging cultured mouse cells. 51 52

DNA-dependent DNA polymerase has been extracted from the soluble cytoplasmic fraction of regenerating rat liver and purified using phosphocellulose and DEAE-cellulose chromatography. Glycerol gradient analysis showed that the enzyme was predominantly DNA polymerase alpha, having a sedimentation coefficient of 10.5 S at low ionic strength and of 6--8 S at higher salt concentrations. The fidelity of purified enzyme was assessed using the co-polymer poly(dA-dT).poly(dA-dT) as a template for DNA synthesis. For both the aggregated (10.5 S) and disaggregated (6--8 S) forms, fidelities in the range of 1 wrong base in 100,000--150,000 complementary bases were obtained.
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PMID:Molecular size and fidelity of DNA polymerase alpha from the regenerating liver of the rat. 62 56

Three patterns of activity were evident when the differential activation of the DNA polymerase associated with serum Dane particles by nonionic detergent and salt was investigated. The patterns were obtained by plotting the increase in enzyme activity mediated by the detergent Nonidet P-40 (NP-40) in increasing concentrations of KCl compared to the activity observed in the absence of detergent. The pattern of differential activity of hepatitis B (HB) DNA polymerase in detergent and salt was altered by subjecting the HBAg preparations to shearing forces. Hepatitis B DNA polymerase activity was stable even in NP-40 concentrations as high as 10%. In addition to hepatitis B DNA polymerase, DNA polymerase activated by calf thymus DNA was found in pellets containing Dane particles. The latter DNA polymerase activity was also activated by NP-40 and was not decreased by DNAse; this DNA polymerase coprecipitated with hepatitis B antigen (HBAg) upon addition of anti-HBs. However, the DNA polymerase activated by calf thymus DNA was inhibited by 0.4 M KCl. Electron microscopic observations of serum Dane particles in 0.4 M KCl showed no alterations of morphology of these particles when compared to particles in low-salt buffer. The data indicated that KCl activated HB DNA polymerase by a different mechanism from that of shear or NP-40, which removed the surface antigen coat from the Dane particles.
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PMID:Differential activation of hepatitis B DNA polymerase by detergent and salt. 68 13

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 low molecular weight DNA polymerase which sediments at 3.3 S on sucrose gradients has been purified from total cell homogenates of rapidly dividing embryos of the sea urchin Strongylocentrotus purpuratus. In the presence of 2 mM N-ethylmaleimide, it is the major polymerase activity in whole cell homogenates when assayed with an oligo(dT)10.poly(dA)200 template; a template which it uses about 200 times more efficiently than activated DNA. The requirement for N-ethylmaleimide exists only in crude cell fractions where it acts to inhibit a template digesting nuclease activity. The polymerase is highly stable if maintained in the presence of 20% glycerol, is completely dependent on added template, and shows no end addition activity. The physical and enzymatic properties of this enzyme clearly distinguish it from the DNA polymerase previously described by Loeb (Loeb, L. A. (1969) J. Biol. Chem. 244, 1672-1681) which sediments as a high moeluclar weight (5.6 to 6.6 S) enzyme and prefers the activated DNA template. In addition, these two DNA polymerase enzymes show distinctive chromatographic properties using DEAE-cellulose and phosphocellulose columns as well as their sensitivity to N-ethylmaleimide. The properties of the low molecular weight polymerase indicate close similarity to the beta-polymerase isolated from mammalian cells. These low molecular weight enzymes are both sensitive to phosphate salt and able to utilize the artificial ribohomopolymer template oligo(dT)10.poly(rA)200. A quantitative analysis of the low molecular weight DNA polymerase during early embryonic development indicates that the activity of this enzyme increases at least 2-fold immediately following fertilization and again during early blastula stage (hatching). Such quantitative changes in a beta enzyme activity are in contrast to findings with the alpha-polymerase which remains constant during early development.
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PMID:A low molecular weight DNA polymerase beta in the sea urchin Strongylocentrotus purpurantus. Partial purification, properties, and changes in development. 71 48

The DNA polymerase induced by an antimutator T4 phage has been purified to apparent homogeneity and has been compared to the wild type polymerase. The mutant enzyme resembles the wild type in thermal stability, pH optimum, salt activation, divalent metal ion requirement, inhibition by a sulfhydryl reagent, and apparent affinity for DNA. However, the mutant enzyme differs from the wild type in its 8-fold higher 3'-exonuclease activity and in its decreased apparent affinity for deoxyribonucleoside triphosphates. Inhibition studies indicate that the exonuclease of the mutant enzyme is more vulnerable to physical and chemical modification than its wild type counterpart.
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PMID:An antimutator deoxyribonucleic acid polymerase. I. Purification and properties of the enzyme. 77 Apr 66

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

DNA polymerase III from Bacillus subtilis has been purified about 4,500-fold. Disc gel electrophoresis of the purified fraction reveals a single major protein band which co-migrates with the polymerase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polymerase yields a single, 166,000 dalton band. The hydrodynamic properties of the enzyme are ionic strength-dependent. The average values from determinations in high and low salt are 7.6 S for the sedimentation coefficient and 52 A for the Stokes radius. These two parameters indicate a molecular weight for the native enzyme of 160,000. Therefore, the enzyme appears to contain a single, long, polypeptide chain. The enzyme has no endonuclease activity but does have single strand specific exonuclease activity. Hydrolysis is initiated exclusively from the 3' terminus yielding 5' mononucleotides, and a dinucleotide is the limit of digestion. The exonuclease activity has an ionic strength dependence of pH optimum similar to that of the polymerase but appears to be more fastidious in its divalent metal requirement. The mode of attack by the enzyme is strictly distributive. The activity of the exonuclease decreases markedly with increasing substrate size. Two opposing mechanisms account quantitatively for this effect--intrinsic competitive inhibition by interior substrate nucleotides and increasing accessibility of the substrate terminus to the enzyme with increasing chain length. The polymerase synthesizes DNA in the 5' leads to 3' direction and the apparent Km for each of the deoxyribonucleoside triphosphates is about 1 muM. The polymerase replicates RNA-primed, phiX174 DNA in the presence of Escherichia coli elongation Factors I and II. In contrast to polymerase III, B. subtilis DNA polymerase II has no detectable nuclease activity.
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PMID:Purification and characterization of DNA polymerase III from Bacillus subtilis. 81 56

A DNA-dependent DNA polymerase from rat liver mitochondria was partially purified and characterized. Mitochondrial DNA polymerase has been found to be quite different from other DNA-dependent DNA polymerases alpha and beta present in the rat liver in the following points: elution patterns in a DEAE-cellulose column chromatography, sedimentation coefficients determined by the glycerol gradient centrifugation in the presence of high salt, and sensitivities to N-ethylmaleimide, ethidium bromide and KCl.
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PMID:Mitochondrial DNA polymerase from rat liver. 85 77

Chromatin isolated from adult rat liver was fractionated into template active and inactive components by controlled shearing and glycerol gradient centrifugation. The fractionated chromatin was assayed for DNA-dependent DNA polymerase (DNA mucleotidyl transferase EC 2.7.7.7) activity with and without exogenous activated DNA serving as template. With endogenous chromatin as template, it was found that 90% of the endogenous chromatin bound DNA polymerase activity was located in the transcriptionally active fraction of chromatin, while the distribution of DNA polymerase assayed with exogenous activated DNA was found to be 65% in the transcriptionally active and 35% in the inactive fractions. However, when DNA polymerase was solubilized from these fractions by salt extraction, enzyme activity was found to be equally distributed, suggesting that the difference in endogenous DNA polymerase activity observed between eu- and heterochromatin is due to the restricted template found in repressed fractions.
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PMID:Endogenous DNA polymerase activity in fractionated rat lever chromatin. 90 88


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