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)

Three DNA-dependent DNA polymerase activities, A, B, and C, have been detected from the macronucleus of Paramecium. The enzymes were separated and partially purified by DEAE-cellulose and phosphocellulose chromatography. These three enzymes can be further distinguished from each other by the effect of KCl, araCTP and different DNA templates on their activities. All three enzymes have the same molecular weight (900 000-110 000), are dependent on Mg2+ and added DNA template and are inhibited by sodium pyrophosphate. The relative amounts of the three enzyme activities vary with the growth stage of the Paramecia from which they are isolated. In balanced growth, three polymerase activities (A, B AND C) are observed, whereas in late log or stationary phase polymerase C is absent, while the level of polymerase B decreases by a larger factor than polymerase A. These results have been related to the possible functional roles of the three activities and their relationship to activities in other organisms studies is discussed.
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PMID:DNA-dependent DNA polymerase activities from Paramecia macronuclei. 112 32

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

DNA polymerases from Bacillus stearothermophilus, Bacillus caldotenax, and Bacillus caldovelox were purified by chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose and obtained in high yield. The enzyme preparations are free of exo- and endonuclease activities. Additional purification steps, e.g., hydrophobic interaction chromatography and chromatography on a Mono Q column or sucrose density gradient centrifugation, are needed to obtain the enzymes in the form of homogeneous 95-kDa proteins. Each of the three organisms possesses a major DNA polymerase activity comparable to DNA polymerase I. The enzymes require Mg2+ (10 to 30 mM) for optimal activity, although 0.4 mM Mn2+ could substitute for magnesium. The optimal reaction temperatures were lowest in B. stearothermophilus (60 to 65 degrees C) and about equal in B. caldovelox and B. caldotenax (65 to 70 degrees C). The thermal stabilities of the enzymes increased in the same order. The DNA polymerase from Thermus thermophilus was isolated for comparison by using a similar procedure. The enzyme was obtained as a homogeneous 85-kDa protein that was also free of exo- and endonucleolytic activities.
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PMID:Purification and characterization of DNA polymerases from Bacillus species. 132 Jun 8

In spite of the fact that a DNA helicase is clearly required for the predominantly leading-strand synthesis occurring during mammalian mtDNA replication, no such activity has heretofore been identified. We report the characterization of a mammalian mitochondrial DNA helicase isolated from bovine brain tissue. The sucrose gradient-purified mitochondria in which the activity was detected had less than 1 part in 2500 nuclear contamination according to Western blot analysis using nuclear- and mitochondrial-specific probes. Mitochondrial protein fractionation by DEAE-Sephacel chromatography yielded a DNA helicase activity dependent upon hydrolysis of ATP or dATP but not other NTPs or dNTPs. The mitochondrial helicase unwound 15- and 20-base oligonucleotides but was unable to unwind 32-base or longer oligonucleotides, and the polarity of the unwinding is 3'-to-5' with respect to the single-stranded portion of the partial duplex DNA substrate. This direction of unwinding would place the bovine mitochondrial helicase on the template strand ahead of DNA polymerase gamma during mtDNA replication, a situation analogous to that of the Rep helicase of Escherichia coli during leading-strand DNA synthesis of certain bacteriophages.
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PMID:DNA helicase from mammalian mitochondria. 132 59

A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE-cellulose and heparin--avidgel-F, followed by FPLC chromatography on mono S, Superose 12 and a second mono S column. This enzyme digests double-stranded DNA more efficiently than heat-denatured DNA. The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel-filtration Superose 12 column. The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein. The enzyme has an absolute requirement for divalent cations, preferring Mg2+ over Mn2+. No activity could be detected when these cations were replaced by Ca2+ or Zn2+. The pH optimum for this enzyme activity is 6.5-7.4 and its isoelectric point is 4.9. Both single-strand and double-strand breaks are introduced simultaneously into a supercoiled substrate in the presence of MgCl2 or MnCl2. Endonuclease-treated DNA serves as a substrate for DNA polymerase I from Escherichia coli, suggesting that 3'-OH termini are generated during cleavage. The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity. Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharomyces cerevisiae have revealed a potential structural homology between these nucleases.
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PMID:Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos. 133 52

Proliferating-cell nuclear antigen (PCNA), also known as cyclin, is synthesized in proliferative cells and recently was identified as DNA polymerase-delta auxiliary protein. In this paper, the association of PCNA to the proliferative cells of plants was analysed using both autoantibodies to PCNA obtained from a patient with systemic lupus erythematosus (SLE) and murine monoclonal antibodies. By immunohistochemical analysis, nuclei of cells around the growing point in soybean root tips reacted strongly with autoantibodies to PCNA in the serum from a patient with SLE. The plant PCNA in root tip cells was purified by ammonium sulfate fractionation, DEAE chromatography, and affinity chromatography. The partially purified plant PCNA was tested by immunoblotting and a 34 kD polypeptide reacted with both the human anti-PCNA autoantibody and a mouse monoclonal antibody against human PCNA (TOB 7). In addition, the purified plant PCNA reacted with both antibodies in enzyme-linked immunosorbent assay (ELISA). The binding of anti-PCNA serum to the animal PCNA was blocked by the plant PCNA in this ELISA. The association of PCNA with growing cells in plants was further confirmed by quantitative sandwich type ELISA using two murine monoclonal antibodies to PCNA, TOB7 and TO17. Those results suggested that PCNA in both plant and animal cells had the same immunological and biochemical characteristics and the plant PCNA might play an important role in cell growth, existing as it does in proliferating plant cells. The concentration of PCNA in soybean germ extract before germination was less than 5 ng ml-1 (protein concentration, 6.8 mg ml-1), but that of the root tip stem including the growing point increased to 887 ng ml-1 (protein concentration 3.8 mg ml-1) in the second day after germination.
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PMID:Proliferating cell nuclear antigen (PCNA/cyclin) in plant proliferating cells: immunohistochemical and quantitative analysis using autoantibody and murine monoclonal antibodies to PCNA. 135 40

Three different DNA polymerase activities can be resolved by passing a protein extract from 24 h imbibed maize axes through DEAE-cellulose. These activities have been numbered 1, 2 and 3, according to their elution order. One of them, DNA polymerase 2, elutes at 100-120 mM phosphates. This enzyme was further purified by passing it through Heparin-Sepharose, Sephacryl S-300 and DNA cellulose. Purification was nearly 5000-fold. The enzyme needs Mg2+, is stimulated by K+, has an optimum pH of 7.0 and its optimum temperature is 30-37 degrees C. Specific inhibitors for different types of polymerases, such as aphidicolin, dideoxythymidine triphosphate and N-ethyl maleimide, gave intermediate values of inhibition, making impossible the definition of the type of enzyme purified by its inhibitory pattern. SDS-PAGE indicated the presence of several bands of molecular masses of 28-40, 56 and 15 kDa. Most of these bands could be visualized when proteins from crude extracts were analyzed by western blot, using an antibody against calf thymus DNA polymerase alpha. A high molecular mass (around 500 kDa) was calculated by western blot of native gels using the same antibody. Finally, specific activity of this enzyme increased 100-fold during maize germination whereas polymerase 3 virtually did not increase. Furthermore, immunoprecipitation experiments with the antipolymerase alpha-antibody showed a decrease in DNA polymerase activity by 70%. The possibility that polymerase 2 is a replicative enzyme is discussed.
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PMID:A DNA polymerase from maize axes: its purification and possible role. 146 49

A modified method for the detection of DNA polymerases in cell extracts and purified enzyme preparations after electrophoresis in polyacrylamide gradient cylindrical gels is described. The technique, which is based on direct assay of activity in a reaction mixture during elution of DNA polymerases from gel slices, was applied to the pursuit of enzyme forms of Streptomyces aureofaciens DNA polymerase during purification procedure. In a crude extract of S. aureofaciens mycelium many catalytically active forms of DNA polymerase ranging from 35 to 860 kDa were detected. After purification, that included mycelium homogenization, precipitation of nucleic acids by polyethylene glycol, DEAE-Sephadex, QAE-Sephadex and DNA-Sepharose chromatography, higher molecular mass forms of more than 172 kDa have not been found. The lower molecular mass forms were separated into two groups by DNA-Sepharose chromatography. On the basis of their characterization, it is assumed that the lower molecular mass forms are produced by proteolysis and the major form found after purification of S. aureofaciens DNA polymerase in the presence of suitable proteinase inhibitors should be a protein of 172 kDa.
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PMID:Production of multiple forms during purification of Streptomyces aureofaciens DNA polymerase: a study using nondenaturing polyacrylamide gradient gel electrophoresis. 153 80

DNA polymerase alpha from germinated wheat embryos was purified by ammonium sulphate fractionation, chromatography on DEAE-Toyopearl, followed by phosphocellulose and heparin Sepharose columns. The specific activity of the purified enzyme was more than 60,000 units/mg. It belongs to the alpha-type according to the large molecular mass, high sensitivity to NEM, aphidicoline, 200 mM KCl, low sensitivity to ethidium bromide and the absence of inhibition by ddTTP. DNA polymerase alpha consists of four subunits as shown by SDS-PAGE and seems to be homogeneous under non-denaturing conditions.
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PMID:Isolation of DNA polymerase alpha from germinated wheat embryos. 154 80

The DNA-dependent DNA-polymerase (DNA polymerase I which is not sorbed on the column with DEAE-cellulose, and DNA-polymerase II, which is absorbed by this column and is eluted from it by 0.3 M of NaCl), have been isolated from Acholeplasma laidlawii PG-8. DNA-polymerase I in homogeneous state was obtained as a result of the stepwise treatment by heparin-sepharose (elution at 0.35 M of NaCl) and poly-U-sepharose (elution at 0.3 M of NaCl). It was presented on the electrophoregram by one polypeptide with molecular weight of 72 kDalton. The second form of DNA polymerase was also obtained in homogeneous state as a result of sequential treatment on heparin-sepharose (elution at 0.3 M of NaCl) and on poly-A-sepharose (elution at 0.25 M of NaCl): the protein which had manifested polymerase activity was a polypeptide with molecular weight of 45 kDalton.
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PMID:[The chromatographic properties of the DNA-dependent DNA polymerases from Acholeplasma laidlawii PG-8]. 158 84


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