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)

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) FROM THE Novikoff hepatoma has been purified over 200 000-fold (based on the increase in specific activity), by ammonium sulfate fractionation and chromatography on DEAE-Sephadex, phosphocellulose, hydroxylapatite, and DNA-cellulose. The enzyme is remarkably stable through all stages of purification until DNA-cellulose chromatography when it must be kept in buffers containing 0.5 M NaCl and 1 mg/ml bovine serum albumin for stability. The enzyme appears to be homogeneous as evidenced by a single stainable band when subjected to electrophoresis in polyacrylamide gels of different porosity. The stainable band corresponds to the DNA polymerase as determined by slicing sister gels and assaying for enzyme activity. The specific activity of the homogeneous preparation is about 60 000 units/mg. The enzyme lacks detectable exonuclease or endonuclease activity. It has a molecular weight of 32 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. In sucrose gradients, the molecular weight is estimated at 31 000. The isoelectric point of the hydroxylapatite fraction enzyme is 8.5. The Novikoff beta-polymerase requires all four deoxyribonucleoside triphosphates, primer-template, and a divalent cation for maximal activity. The apparent Km for total deoxyribonucleoside triphosphate is 7-8 muM and for DNA 125 mug/ml. Activated DNA, rendered 7% acid soluble by DNase I, is the preferred primer-template, although a number of synthetic polynucleotides can by efficiently utilized, particularly in the presence of Mm2+ optimum is 7 mM; the Mn2+ optimum is 1 mM. The pH optimum is 8.4 in Tris-HCl or 9.2 in glycine buffer. The beta-polymerase is sstimulated about twofold by NaCl or KCl at an optimum of 50-100 MM, and the enzyme maintains considerable activity at high ionic strengths. The DNA polymerase is inhibited by ethanol, acetone, and a variety of known polymerase inhibitors. Glycols stimulate the enzyme as does spermine or spermidine. Unlike most beta-polymerases, the Novikoff enzyme is moderately sensitive to N-ethylmaleimide.
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PMID:Novikoff hepatoma deoxyribonucleic acid polymerase. Purification and properties of a homogeneous beta polymerase. 18 3

An RNA-directed DNA polymerase was purified from baboon endogenous type-C virus by successive column chromatography on DEAE cellulose, phosphocellulose and hydroxyapatite. The purified DNA polymerase has a molecular weight of 68 000, a pH optimum of 8.0, a Mn2+ optimum of 1 mM, and a KCl optimum of 40 mM. The purified enzyme transcribes heteropolymeric regions of viral 60--70 S RNA isolated from different type-C viruses. The purified enzyme is immunologically related to a similarly purified polymerase from the cat endogenous type-C virus RD114.
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PMID:Purification and characterization of baboon endogenous virus DNA polymerase. 20 Feb 68

A DNA endonuclease, Endo-I, which cleaves superhelical DNAs, has been isolated from avian myeloblastosis virions stripped of their coats by mild detergent treatment. The enzyme has a broad pH optimum around 7.5-8.0 and requires Mg2+ for activity. A second endonuclease, Endo-II, with a requirement for Mn2+, also present in viral cores, copurified with avian myeloblastosis virus alpha beta DNA polymerase (reverse transcriptase, RNA-dependent DNA nucleotidyltransferase) and similarly cleaved superhelical DNAs. Heat denaturation and sodium fluoride and N-ethylmaleimide inhibition studies were carried out to demonstrate a possible relationship between the two endonucleases and the viral DNA polymerase and RNase H activities. It appears that Endo-II may be an intrinsic activity of the polymerase.
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PMID:DNA endonucleases associated with the avian myeloblastosis virus DNA polymerase. 22 53

An RNA-directed DNA polymerase was purified from bovine leukemia virus (BLV) by successive glycerol gradient centrifugation, column chromatography on phosphocellulose and gel filtration on Sephadex G-200. The purified DNA polymerase transcribes heteropolymeric regions of 30--40 S RNA isolated from avian myeloblastosis virus. The enzyme differs from other known DNA polymerases of mammalian type-C RNA tumor viruses by the following properties: 1. Its apparent molecular weight as estimated by velocity sedimentation data is 58,000 at 0.12 M KCl and 43,000 in the presence of 0.50 M KCl. 2. It has a Mg2+ optimum of 10 mM, and a Mn2+ optimum of 0.25 mM with (rA)n-(dT)10 as template. 3. At 50 mM KCl it is inhibited more than 70%, but it is not inhibited by phosphate ions at 2 mM. These properties confirm the peculiar position of BLV within the family Retraviridae.
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PMID:Purification and characterization of bovine leukemia virus DNA polymerase. 23 43

A high molecular weight membrane-bound DNA polymerase from the mouse myeloma, MOPC-104E, has been purified extensively, and characterized with regard to physical and reaction properties. This enzyme, which is readily distinguishable from other myeloma enzymes that are analogous to the recognized forms of cellular DNA polymerase, is ddesignated DNA polymerase III. DNA polymerase III activity in whole homogenates from MOPC-104E was solubilized and then prurifed using a series of ion-exchange chromatographic procedures followed by DNA-cellulose chromatography and glycerol gradient centrifugation; the enzyme activity as measured with poly(rA)-(dT)12-18 as template-primer and Mn2+ as divalent cation, was purified as much as 18,000-fold. In the final stages of the pruification, DNA polymerase III possessed no detectable RNA polymerase activity, nucleoside diphosphokinase activity, or nucease activity toward DNA or single- and double-stranded RNA...
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PMID:On the DNA polymerase III of mouse myeloma: partial purification and characterization. 23 42

DNA polymerase from BHK-21/C13 cells were separated into two species, DNA polymerase I corresponding to the heterogeneous enzyme with sedimentation coefficient of 6-8S, and DNA polymerase II, corresponding to the enzyme with sedimentation coefficient of 3.3S. DNA polymerase I was purified 114-fold and DNA polymerase II 154-fold by a simple extraction procedure followed by column chromatography on phosphocellulose and gel filtration through Sephadex G-100. The purified enzymes differed markedly in respect of pH optimum, stimulation and inhibition by K+, Km for the deoxyribonucleoside 5'-triphosphates, stability to heating at 45 degrees C, and inhibition by N-ethylmaleimide. The preferred primer-template for both enzymes was "activated" DNA (DNA submitted to limited degradation by pancreatic deoxyribonuclease); native or thermally denatured DNA templates were relatively very poorly copied. When certain synthetic templates were tested, substantial differences were revealed between the two enzymes. Poly[d(A-T)] was poorly used by polymerase I but was superior to "activated" DNA for polymerase II. Poly[d(A)]-oligo[d(pT)10] was used efficiently by polymerase I but not by polymerase II. Poly(A)-oligo[d(pT)10] was not an effective primer-template although polymerase I could use it to a limited extent when Mn2+ replaced Mg2+ in the polymerase reaction and when the temperature of incubation was lowered from 37 degrees to 30 degrees C. When only one or two or three triphosphates were supplied in the reaction mixture, the activity of polymerase I was more severly diminished than that of polymerase II.
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PMID:Deoxyribonucleic acid polymerases of BHK-21/C13 cells. Partial purification and characterization of the enzymes. 23 80

The more than 2,300-fold purification of a DNA polymerase from the embryos of Drosophila melanogaster is described. The enzyme, which forms a single band on gel electrophoresis, has a molecular weight of about 87,000 and a pH optimum of 8.5. A divalent metal is required for activity, Mg2+ being preferred with activated DNA, Mn2+ with homopolymer template-primers. The enzyme is inactivated completely by mercurials; polyamines are also inhibitory with certain templates. The most efficient template-primer is activated DNA, but homopolymers such as poly(dA)-oligo(dT), poly(A)-oligo(dT), and poly(A)-oligo(U) are also utilized with high efficiency. The purified enzyme preparations appear to be devoid of nuclease activity when assayed directly with suitable substrates. In addition, neither primer nor product is degraded after prolonged incubation with the enzyme. In accordance with previous observations on other DNA polymerases, the Drosophila enzyme can replicate single-stranded DNA only under conditions of simultaneous transcription by RNA polymerase.
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PMID:A DNA polymerase from embryos of Drosophila melanogaster. Purification and properties. 24 52

Purified yeast DNA was transcribed by homologous RNA polymerases I and II and Escherichia coli RNA polymerase. Transcripts synthesized in vitro were analyzed by molecular hybridization with complementary DNA (cDNA) synthesized from yeast poly(A)-containing mRNA with viral reverse transcriptase and ribosomal DNA labeled in vitro by nick translation with E. coli DNA polymerase I. RNA synthesized by polymerase I and II in the presence of Mn2+ contained sequences complementary to cDNA and rDNA at a frequency consistent with random transcription of the template. Similarly, E. coli RNA polymerase synthesized an apparently random transcript in the presence of either Mn2+ or Mg2+. In contrast to these results, RNA polymerase I but not polymerase II transcripts were markedly enriched in sequences complementary to rDNA when transcription was carried out in the presence of Mg2+. The observed enrichment was 15-30-fold higher than observed for polymerase II or E. coli polymerase transcripts and is consistent with the transcript being comprised of 6-10% ribosomal sequences. These data strongly suggest that RNA polymerase I plays a critical role in selective transcription of ribosomal cistrons.
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PMID:Transcription of yeast DNA by homologous RNA polymerases I and II: selective transcription of ribosomal genes by RNA polymerase I. 31 52

The error frequency of in vitro DNA synthesis using a natural DNA template has been measured with a biological assay for nucleotide substitutions. phiX174 DNA containing an amber mutation was copied in vitro by Escherichia coli DNA polymerase I, and the reversion frequency of the progeny DNA was determined by transfection of E. coli spheroplasts. E. coli polymerase I makes less than 1 mistake at the am3 locus for every 7700 nucleotides incorporated under standard reaction conditions. Substitution of Mn2+ for Mg2+ and unequal concentrations of deoxynucleoside triphosphate substrates raises this mutation frequency to greater than 1 in 1000. Thus, E. coli DNA polymerase I can copy natural DNA templates with high fidelity and its accuracy can be affected by alterations in reaction conditions.
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PMID:Mutagenesis during in vitro DNA synthesis. 34 50

Pancreatic DNase requires both Ca2+ and Mg2+ for its activity as measured by formation of an activated DNA template for in vitro DNA polymerase alpha assay and by the hyperchromic shift. Mn2+ can partially satisfy the Mg2+ requirement of the DNase for activation of DNA but the resulting template is only 50% as active in the DNA polymerase assay. When precautions are taken to avoid divalent ion contamination, pancreatic DNase is not active in the presence of Ca2+ or Mg2+ alone. analysis of the DNA by sucrose gradient centrifugation shows that only in the presence of Ca2+ plus Mg2+ or Mn2+ does pancreatic DNase produce extensive strand breaks in the DNA. The activated DNA template that yields maximal DNA polymerase activity is low molecular weight material of 30,000 to 50,000 daltons.
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PMID:Action of pancreatic DNase: requirements for activation of DNA as a template-primer for DNA polymerase. 41


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