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)

An RNA directed DNA polymerase was purified over 2500 fold from gibbon ape leukemia virus by successive column chromatography on Sephadex G100, DEAE cellulose, phosphocellulose and hydroxyapatite. The purified DNA polymerase has a molecular weight of 68 000, a pH optimum of 7.5, a Mn2+ optimum of 0.8 mM, and KCl optimum of 80 mM. The purified enzyme transcribes heteropolymeric regions of viral 60-70 S RNA isolated from avian myeloblastosis virus, Rauscher murine leukemia virus and simian sarcoma virus and it is inhibited by antiserum prepared against either gibbon ape leukemia virus or simian sarcoma virus DNA polymerases.
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PMID:Purification and characterization of gibbon ape leukemia virus DNA polymerase. 6 92

Procedures were established for the isolation and partial purification of DNA polymerase, RNA polymerase and poly(A) polymerase activities from the cytoplasm and nuclei of NIH-Swiss mouse embryos. Based on the elution pattern of these enzyme activities from DEAE-cellulose and phosphocellulose columns in Tris-HCl buffer, pH 8.0, the apparent basicities of the enzymes can be arranged as follows: cytoplasmic(C) poly(A) polymerase greater than (C)DNA polymerase beta greater than (C)DNA polymerase alpha and nuclear(N) poly(A) polymerase greater than (N)DNA polymerase greater than (N)RNA polymerase I greater than (N)RNA polymerase II. Twenty rifamycins, including rifamycin B, rifamycin S, rifamycin SV, and rifamycin SV derivatives, were examined for their ability to inhibit the above mentioned nucleic acid polymerizing enzymes and Simian sarcoma virus type I (SSV-1) reverse transcriptase. Rifamycin SV 3'-formyldiphenylhydrazone, rifamycin SV 3'-formyl-n-octyloxime (AF/013) and rifamycin SV 3'-formyldiphenylmethyloxime (AF/05) inhibited all the tested enzyme activities. Rifamycin SV 3'-formylpropylphenyloxime (AF/015) inhibited cellular nucleic acid polymerase activities but not SSV-1 DNA polymerase activity. Rifamycin SV 3'-formyldinitrophenylhydrazone (AF/DNFL) strongly inhibited reverse transcriptase activity but did not inhibit cellular DNA polymerase activities. AF/DNFI slightly inhibited RNA and poly(A) polymerase activities. Rifamycin SV 3'-formyldipropylhydrazone (AF/DPI) and 2,6-dimethyl-4-N-benzyldemethyl-rifampicin (AF/ABDMP) slightly inhibited reverse transcriptase activity but did not inhibit cellular nucleic acid polymerase activities. Active rifamycin derivatives inhibited enzyme reactions by interacting with the enzyme proteins. Nascent polynucleotide chain elongation continued although at a reduced rate in the presence of inhibitor. The addition of increasing concentrations of nonionic detergent (Triton X-100) to rifamycin-inhibited enzyme reactions fully restored enzyme activities. The presence of highly lipophilic 3'-side chains on active rifamycins and the reversibility of enzyme inhibition by Triton X-100 suggest that the tested nucleic acid polymerizing enzymes may have hydrophobic regions with which inhibitory rifamycins interact.
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PMID:Interaction of rifamycins with mammalian nucleic acid polymerizing enzymes. 6 93

Three forms of the RNA-dependent DNA polymerase were isolated from highly purified avian sarcoma virus B77 grown in duck embryo fibroblasts, using sequential chromatography on DEAE-cellulose, phosphocellulose, and poly(U)-cellulose. One form, which sedimented with about 5.2 S, contained only one species of polypeptide, with a molecular weight of 63,000; a second sedimented with about 7.8 S and contained only one species of polypeptide with a molecular weight of 81,000; and a third form, which sedimented with about 7.3 S, contained two species of polypeptides with molecular weights of 63,000 and 81,000. The molecular constitution of the three enzyme forms were therefore alpha, beta2, and alphabeta. All three possessed almost the same specific activity with poly(rA)-oligo(dT) as the primer-template. Forms alpha and alphabeta of avian sarcoma virus DNA polymerase have already been described in the literature; form beta2 is a new form. All three forms possessed ribonuclease H activity, the relative specific activities of the alpha, beta2, and alphabeta forms being about 1:4:5. All three enzyme forms were inhibited by antiserum to the alphabeta form, but whereas the alpha and alphabeta forms could be inhibited about 95%, the maximum degree of inhibition of the beta2 form was about 80%. The three enzyme forms also differed with respect to heat stability at 46 degrees, the monomeric alpha form of the enzyme being only about one-half as stable as the two dimeric forms.
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PMID:RNA-dependent DNA polymerase of avian sarcoma virus B77. I. Isolation and partial characterization of the alpha, beta2, and alphabeta forms of the enzyme. 6 34

Lysates of Moloney murine sarcoma-leukemia virus [M-MSV(MLV)], a virus complex grown in the rat cell line 78A-1, were found to contain three RNase H species separable by polycytidylic acid[poly(C)]-agarose chromatography. RNase H activity (RNase H I) associated with RNA-directed DNA polymerase eluted at 0.23 M KCI from poly(C)-agarose. RNase H II, which eluted from poly(C)-agarose at 0.12 M KCI and was not associated with DNA polymerase activity, was shown to be identical to an RNase H species (designated RNase H II) previously isolated from M-MSV(MLV) by a different procedure (G. F. Gerard and D. P. Grandgenett, J. Virol. 15:785-797, 1975). M-MSV(MLV) RNase H II was established to be a random exohybridase that requires free-chain termini in its hybrid substrate for activity. Lysates of Rickard feline leukemia virus also contained RNase H activity not associated with DNA polymerase activity that eluted from poly(C)-agarose at 0.12 M KCl. A third species of enzyme from M-MSV(MLV) lysates, called RNase H III, did not bind to poly(C)-agarose in 0.06 M KCl. RNase H III was purified from lysates of M-MSV(MLV) and M-MLV (grown in mouse cells) by sequential chromatography on poly(C)-agarose, DEAE-cellulose, phosphocellulose, and polyuridylic acid-Sepharose. Purified RNase H III (i) was free of any associated DNA polymerase activity, (ii) had an apparent molecular weight of 30,000 determined by Sephadex G-100 gel filtration, (iii) had an absolute requirement for Mn2+ (1 mM optimum) for the degradation of [3H](A)n.(dT)n, (iv) was inhibited by the presence of any salt in reaction mixtures, and (v) was endoribonucleolytic in its mode of action as indicated by the size distribution of limited degradation products of [3H](A)n.(dT)n. RNase H III was inhibited by antisera prepared against Rauscher MLV and simian sarcoma virus reverse transcriptase, and the quantity of RNase H III and RNase H I present in lysates of M-MLV were reduced and increased proportionately if virus was lysed in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. These results indicate that RNase H III is a proteolytic cleavage product of DNA polymerase-RNase H. Substantial RNase H activity that did not bind to poly(C)-agarose in 0.06 M KCl was also found in lysates of Harvey MSV(MLV), Rauscher MLV, and Rickard feline leukemia virus, but not in lysates of avian myeloblastosis virus.
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PMID:Multiple RNase H activities in mammalian type C retravirus lysates. 7 33

An RNA-direct DNA polymerase was purified from human melanoma tissue by successive column chromatography on DEAE-cellulose (DE-23 and DE-52) and phosphocellulose. The purified reverse transcriptase has a mol. wt. of 68,000, a pH optimum of 8.0, a Mn2+ optimum of 0.6 mM, and a KCl optimum of 60 mM. The purified enzyme transcribes (rA)n - (dT)12, (rC)n - (dG)18, (Ome-rC)n - (dG)18 and a 70s RNA from Rauscher leukemia virus (RLV), but failed to transcribe (dA)n - (dT)12. This enzyme has no terminal deoxynucleotidyl transferase activity. Serological studies have shown that the reverse transcriptase from human melanoma tissue is antigenically not related to DNA polymerases from Simian sarcoma virus (SiSV), Avian myeloblastosis virus (AMV), RLV, and human spleen of a patient with myelofibrosis. The purified enzyme showed a close antigenic resemblance to DNA polymerases from baboon endogenous virus (BEV) and rhabdomyosarcoma virus (RD-114), the endogenous virus of the cat.
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PMID:Biochemical and immunological characterization of a reverse transcriptase from human melanoma tissue. 8 88

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

An RNA-directed DNA polymerase was purified from a cell line derived from a radiation-induced lymphoma in NIH Swiss mice which produced non-infectious type C virus particles. The enzyme was isolated from a high speed particulate fraction which bands at a density of 1.16--1.19 g/ml in a sucrose gradient, and purified by successive chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite. The purified DNA polymerase has a molecular weight of 68 000, a pH optimum of 7.5, a KCl optimum of 50 mM, and a Mn2+ optimum of 0.25 mM. It prefers (dT)15 . (A)n to (dT)15 . (dA)n as the primer template and transcribes the poly(C) strand of (dG)15 .(C)n and (dG)15 . (OMeC)n. It transcribes heteropolymeric regions of avian myeloblastosis virus 70 S RNA, and is inhibited by antiserum to Rauscher murine leukemia virus DNA polymerase. Comparison of the properties of DNA polymerase purified from radiation-induced lymphoma cells with the DNA polymerase purified from non-defective murine type C RNA tumor viruses shows that the mouse lymphoma enzyme is both biochemically and immunologically related to murine leukemia virus DNA polymerases.
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PMID:Characterization of an RNA-directed DNA-polymerase from a cell line derived from a radiation-induced lymphoma in mice. 9 May 22

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

Immunoglobulin G (IgG) was isolated from ejaculates of intact and vasectomized men by precipitation with ammonium sulphate and DEAE-cellulose ionexchange chromatography. Velocity centrifugation revealed that all of the IgG from intact males was 7S protein while less than 40% of the seminal IgG of vasectomized men cosedimented with the 7S marker; the remaining, immunologically unidentifiable, protein was considerably smaller and heterogeneous in size. Only the 7S IgG from the post-vasectomy ejaculates inhibited the activity of a DNA polymerase from the seminal fluid of an intact male. These results suggest that formation of antibody reactive with the seminal fluid DNA polymerase is one manifestation of a vasectomy-associated autoimmune response in man.
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PMID:Inhibition of human seminal fluid DNA polymerase by an IgG fraction of seminal plasma from vasectomized men. 10 59

Extracts of Novikoff hepatoma cells contain factors capable of stimulating in vitro DNA synthesis several fold. The activity can be resolved into three separate protein peaks on DEAE-Sephadex. Two of these, factors II and III, have been purified and partially characterized. Both factors increase the initial rate of DNA synthesis and allow synthesis to proceed much longer. If either factor is added after synthesis by the DNA polymerase has reached a plateau, resumption of synthesis occurs. The factors appear to have different modes of action or sites of action since they show an additive effect even when a single one is used at saturating conditions. These factors are present in normal rat liver but at a concentration less than 5% of that found in the tumor cells. When tested with several highly purified DNA polymerases (DNA nucleotidyltransferase, EC 2.7.7.7), the factors show a much greater stimulation of homologous, non-mitochondrial enzymes (rat liver nuclear-, rat liver cytoplasmic-, or Novikoff-DNA polymerases) when compared with rat liver or calf liver mitochondrial-, Escherichia coli I-, or sea urchin nuclear-DNA polymerases. The mechanism of action of these factors is not known at present. No enzymatic activity has been associated with factor III. Highly purified, but not homogeneous, preparations of factor II contain low levels of endonuclease; it has not been established whether endonuclease is a contaminant or is responsible for the stimulating activity.
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PMID:Stimulation of DNA polymerase by factors isolated from Novikoff hepatoma. 16 86


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