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)

DNA polymerase activities in Micrococcus radiodurans were separated into two fractions after purification more than 2000 fold. They differ in pH optimum and residual activities in the absence of a full deoxyribonucleoside triphosphates complement. NAD partly inhibited one of the activities. Both activities were eluted as a single peak on gel filtration and sedimented at the same rate on glycerol gradient centrifugation. Molecular weight 140000 was calculated from Stokes radius and sedimentation constant. Deoxyribonuclease activity was detected on one of the polymerase activities which preferentially degraded double-stranded DNA. Priming activity of nicked DNA was reduced by gamma-irradiation. These results have been related to the possible rolls in repair synthesis in vivo or DNA synthesis in permeable cells of M. radiodurans.
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PMID:Separation of DNA-dependent DNA polymerase activities in Micrococcus radiodurans. 1 86

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

DNA polymerase was purified from a cloned isolate of Moloney murine leukemia virus (M-MuLV). Purified M-MuLV DNA polymerase, upon analysis by polyacrylamide gel electrophoresis, showed one major polypeptide of mol wt 80,000. Estimation of molecular weight from the sedimentation rate of the purifed enzyme in a glycerol gradient was consistent with a structure containing one polypeptide. M-MuLV DNA polymerase could transcribe ribopolymers, deoxyribopolymers, and heteropolymers as efficiently as did purified DNA polymerase from avian myeloblastosis virus (AMV). M-MuLV DNA polymerase, however, transcribed native 70S viral RNA less efficiently than did AMV DNA polymerase. Addition of oligo(dT) enhanced five to tenfold the transcription of 70S viral RNA by M-MuLV DNA polymerase. Purified enzyme also exhibited nuclease activity (RNase H) that selectively degraded the RNA moiety of the RNA-DNA hybrid. It did not degrade single-stranded RNA, single-stranded DNA, double-stranded RNA, and double-stranded DNA. M-MuLV DNA polymerase-associated RNase H acted as a random exonuclease. When [3-H]poly(A)-poly(dT) was used as a substrate, the size of the M-MuLV DNA polymerase-associated RHase H digested product was larger than the size of the digestion products by AMV DNA polymerase. The oligonucleotide digestion products could be further digested to 5'-AMP by snake venom phosphodiesterase, indicating that the products were terminated by 3'-OH groups. Alkaline hydrolysis of the oligonucleotide digestion products generated pAp, suggesting that M-MuLV DNA polymerase-associated RNase H cleaves at the 3' side of the 3',5'-phosphodiester bond. The ratios of the rates of DNA polymerase activity and RNase H activity were not significantly different in the murine and avian enzymes.
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PMID:Studies on reverse transcriptase of RNA tumor viruses III. Properties of purified Moloney murine leukemia virus DNA polymerase and associated RNase H. 4 25

Storage of the dimeric (alphabeta) form of avian myeloblastosis virus (AMV) DNA polymerase in glycerol resulted in the release of the smaller alpha subunit, as detected by glycerol gradient sedimentation. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme stored in glycerol showed the concomitant appearance of several polypeptides and a lowering in the level of both beta and alpha components. This reduction appears to be the result of cleavages introduced by traces of hydrolytic activity present in glycerol samples. An enhancement of alpha subunit released, as detected by activity profile, was also achieved upon direct but limited exposure of purified avian myeloblastosis virus DNA polymerase to carboxymethyl-cellulose-bound trypsin matrix. Electrophoretic analysis of digested enzyme revealed a progressive fragmentation, with simultaneous increase in the alpha subunit and decrease in the beta subunit.
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PMID:Mechanism of release of active alpha subunit from dimeric alpha beta avian myeloblastosis virus DNA polymerase. 5 80

Highly purified preparations of RNA-directed DNA polymerase from avian myeloblastosis virus (AMV) contain a Mn2+-activated endonuclease activity capable of nicking supercoiled DNA. This endonuclease activity co-sediments in glycerol gradients with the alphabeta form of AMV DNA polymerase, and co-chromatographs with DNA polymerase activity on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. It is also present in AMV alphabeta-DNA polymerase purified by electrophoresis through nondenaturing polyacrylamide gels and subsequently chromatographed on poly(C)-agarose. alphabeta-associated endonuclease is co-immunoprecipitated with DNA polymerase activity by antiserum directed against alphabeta holoenzyme. The alpha form of AMV DNA polymerase lacks this activity. In its enzymatic properties, alphabeta-associated endonuclease resembles the endodeoxyribonuclease activity associated with the AMV p32 protein, which has been shown to be structurally related to the beta (but not the alpha) subunit of AMV DNA polymerase.
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PMID:Endonuclease activity of purified RNA-directed DNA polymerase from avian myeloblastosis virus. 8 98

RNA-directed DNA polymerase was purified from spleens of Balb/c and NMRI mice infected with Rauscher murine leukemia virus. The method includes cell fractionation and lysis of microsomal fraction, chromtography on Sephadex G-200 and phosphocellulose. Estimation of molecular weight from the sedimentation rate of the purified enzyme in a glycerol gradient was consistent with a structure containing one polypeptide with a molecular weight of 70,000. Purified RLV DNA polymerase from spleen could transcribe purified DNA polymerase from purified virions. This simple preparation method offers a procedure for large scale preparation of the RNA-directed DNA polymerase which can be used for synthesis of DNA complementary to mRNA.
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PMID:Purification of RNA-directed DNA polymerase from mouse spleen infected with Rauscher leukemia virus. 8 71

RNA-directed DNA polymerase was isolated from the liver, spleen, and myeloblasts of chickens that had been infected with virus of avian myeloblastosis. The enzyme was chromatographically purified from the myeloblasts and brought to about 1,000-fold concentration. The method consisted in cell fractionation, lysis of the microsomal fraction, chromatography on Sephadex G-200 and phosphocellulose, as well as ultracentrifugation in the glycerol gradient. The cellular DNA polymerases alpha and beta were clearly separated from the DNA polymerase of avian myeloblastosis virus and could be distinguished from one another by template-specific reactions. The viral DNA polymerase activities in the microsomal fractions of liver, spleen, and myeloblasts were compared with one another. The amount of avian myeloblastosis virus and related DNA polymerase recorded from the myeloblasts was about six times that in the liver and four times higher than that in the spleen. The procedure described, together with the use of cell fractionation and gel filtration, is an appropriate method for biochemical detection of avian oncorna viruses in cells.
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PMID:[Separation of cellular and viral DNA polymerase from oncornavirus infected chicken cells]. 9 56

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.
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PMID:Three forms of DNA polymerase from Drosophila melanogaster embryos. Purification and properties. 9 4

A new DNA polymerase was partially purified from cell-free extracts of a continuous rat cell-line (XC). The XC cells had been transformed by the Prague strain of Rous sarcoma virus but did not produce infectious virus. The molecular weight of the DNA polymerase is 70,000, as estimated by glycerol gradient centrifugation and by Sephadex gel filtration. This enzyme can be distinguished from the other cellular DNA polymerases by its elution pattern on DNA-cellulose column chromatography, its molecular weight, and its primer-template specificity. The enzyme has some characteristics of the murine leukemia virus reverse transcriptase. It is partially inhibited by immunoglobulin G purified from rabbit antiserum prepared against Rauscher leukemia virus reverse transcriptase, but is not inhibited by IgG from rat antiserum prepared against avian myeloblastosis virus reverse transcriptase. However, the XC cell enzyme can be distinguished from the murine leukemia virus reverse transcriptase by its inefficiency in copying an oligo(dG)12-poly(rC)primer-template.
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PMID:Partial purification and characterization of DNA polymerases from a Rous sarcoma virus-transformed rat cell line. 17 Sep 87

We have partially purified and characterized two separate DNA polymerase activities associated with Epstein-Barr virus (EB virus). One activity is present in EB virus producer cell lines but not in nonproducer or negative cell lines. It adheres more strongly to DEAE-cellulose than any host cell enzymes, eluting at 210 to 270 mM potassium phosphate buffer. Further elution from phosphocellulose and sedimentation in glycerol gradients yields an enzyme purified 900-fold with an S value of 8.3. The second DNA polymerase activity co-purifies with EB viral particles, elutes at low salt from DEAE-cellulose (40 to 60 mM potassium phosphate buffer) and phosphocellulose (100 mM), and has an S value of 9.5 on glycerol gradient sedimentation. These two enzymes are referred to for convenience as the EB virus-induced DNA polymerase and the EB virion-associated DNA polymerase. The EB virus-induced polymerase can be distinguished from host alpha, beta, and the virion-associated polymerase in 1) being resistant to salt inhibition, 2) having a more basic pH optima in Tris buffer (pH 9.5), and 3) having a 10-fold lower saturating concentration for the activated DNA template. The EB virion-associated polymerase is distinguished from host alpha, beta, and the EB virus-induced polymerase, because it cannot utilize synthetic deoxy- and ribohomopolymer primer-templates in place of the activated calf thymus DNA template in DNA polymerase assays. Neither of the EB virus-associated polymerases can copy the ribohomopolymers dT10poly(rA) or dG12-18(poly(rC) efficiently and therefore can be distinguished from host gamma polymerase and reverse transcriptase. The activity of the EB virus-induced and virion-associated polymerases are unaffected both by antibody to alpha polymerase, and by antiserum with high antibody titers to EB early antigen and viral capsid antigen.
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PMID:Two Epstein-Barr virus-associated DNA polymerase activities. 21 39


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