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)

The nature of transcription of the avian retrovirus RNA genome by the alpha form of the viral RNA-directed DNA polymerase has been investigated. Transcription was most efficient when Mn2+ was provided as the divalent metal ion. The patterns of DNA transcription using 70S RNA, 35S RNA-tRNAtrp, or 35S RNA-oligo(dT)12-18 template-primer complexes by the alpha DNA polymerase were essentially identical to those obtained using the alphabeta form. The alpha DNA polymerase appears to be deficient in the synthesis of true duplex DNA but is able to synthesize hairpin-structured DNA initiated at the 5' terminus of the viral genome on the tRNAtrp primer molecule.
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PMID:In vitro transcription of the avian retrovirus genome by the alpha form of the viral RNA-directed DNA polymerase. 7 62

The secondary structure of the hydrogen bonded hybrids polycytidylate-oligodeoxguanylate (poly(rC)-(dG)12-18 and poly (2'-oMe) cytidylate-oligodeoxyguanylate (poly (rCm)-(dG)12-18 was studied at several magnesium and manganese ion concentrations. These hybrids are effective template-primer complexes for the synthesis of poly(dG) by avian myeloblastosis virus (AMV) DNA polymerase under disparate ionic conditions. Circular dichroism spectra and thermal melting data were obtained as a function of ion concentration, including conditions that allow optimum rates of poly (dG) synthesis by each complex. These studies demonstrate that both hybrids can change conformation and stability depending on their ionic environment. Comparison of enzyme activity and physical data suggest that the polymerase recognizes particular secondary structure features. Changes in the activity of the AMV polymerase can be induced by varying the Mg++ and Mn++ concentrations alone and in combination. These variations in enzyme activity are correlated with observed changes in the base-stacking alignment of the synthetic template primers. The ions, therefore, seem to affect enzyme activity by altering the conformation of the polnucleotide complexes.
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PMID:The effect of magnesium and manganese ions on the structure and template activity for reverse transcriptase of polyribocytidylate and its 2'-0-methyl derivative. 7 65

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

The 3' terminus of tRNA was enzymatically elongated by an oligo(A) tail. A fragment of DNA polymerase I (E. coli) was used in the presence of manganese to phase and synthesize a cleavable primer at the oligo(A)-tRNA template. When the threedimensional structure of oligo(A)-tRNA is being unfolded under conditions where the primer is still hybridized at the oligo(A) tail, the DNA polymerase I fragment transcribes oligo(A)-tRNA into DNA. Reverse transcription is slowed down and its fidelity suspended by the 1-methyladenine in oligo(A)-tRNAPhe(yeast). The reaction is stopped by the highly modified Y-base present in this template. Approximately full length transcripts can be obtained from oligo(A)-tRNA3Gly(E.coli). The transcription products were characterized by sequence analysis.
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PMID:Reverse transcription of tRNA. 7 22

The biochemical properties of DNA polymerase purified from Mason-Pfizer monkey virus were studied, with respect to synthetic and natural template-primer utilization. Thes studies revealed the following new information about the Mason-Pfizer monkey virus enzyme: (a) Mason-Pfizer monkey virus polymerase was found to prefer template: primer molar nucleotide ratios of 2.5-5: 1 for optimal rates of synthesis with poly(C) .(dG)12-18 as template-primer. (b) Poly(A)-directed synthesis was stimulated by the addition of low concentrations of inorganic phosphate to the reaction mixture. (c) Poly(2' -O-methyl-cytidylate), poly(rCm), was the only template studied for which Mn2+ proved the preferred divalent cation. Combinations of divalent cations stimulated rather than inhibited poly(rCm)-directed poly(dG) synthesis by the Mason-Pfizer monkey virus enzyme. (d) Heteropolymeric regions of rabbit globin mRNA and avian myeloblastosis virus 70 S RNA could be copied by the Mason-Pfizer monkey virus polymerase with oligo(dT), oligo(U) or in the case of avian myeloblastosis virus RNA, endogenous primers. In all such studies, Mg2+ was the preferred divalent cation and a distinct preference for the DNA primer in the reverse transcription of natural RNAs was observed. These new findings necessitated comparative studies with the DNA polymerases from Rauscher murine leukemia virus and murine mammary tumor virus, as representative type C and type B retroviruses. Although the Mason-Pfizer monkey virus enzyme was found to share some properties in common with both type C and type B mammalian viral enzymes, certain of the above properties rendered it unique among the polymerases examined.
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PMID:Template-specific requirements for DNA synthesis by the Mason-Pfizer monkey virus DNA polymerase: unique aspects. 7 24

A virus, similar to the murine mammary tumor viruses (MuMTV) of the laboratory mouse Mus musculus, was identified in the milk of M. cervicolor popaeus mice. The virus was morphologically indistinguishable from the type-B MuMTV and was thus termed MC-MTV. Radioimmunoassays for the 52,000-dalton major envelope glycoprotein and the 28,000-dalton major internal protein of MuMTV demonstrated that MC-MTV shared some antigenic determinants with both of these MuMTV proteins. This reactivity was clearly different, however, from that observed with all MuMTV tested from M. musculus. MC-MTV had a density of 1.16 g/ml in sucrose and a virion-associated DNA polymerase with a divalent cation preference for Mg2+ over Mn2+. Radioimmunoassays clearly differentiated MC-MTV from the other viruses previously identified from M. cervicolor, i.e., M432, CERV-CI, and CERV-CII. These studies thus identified the first virus from another species that is immunologically related to the MuMTV of M. musculus. Particles similar to MC-MTV were also observed in a spontaneous M. cervicolor popaeus mammary tumor.
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PMID:Characterization of a new virus from Mus cervicolor immunologically related to the mouse mammary tumor virus. 8 34

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


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