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1.
Dissociation of alpha beta DNA polymerase of avian myeloblastosis virus by dimethyl sulfoxide. 总被引:5,自引:5,他引:0
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D P Grandgenett 《Journal of virology》1976,17(3):950-961
The alpha beta DNA polymerase of avian myeloblastosis virus was treated with dimethyl sulfoxide to dissociate the enzyme subunits. The dimethyl sulfoxide treated enzymes were passed over phosphocellulose to purify and characterize the dissociated subunits as well as to remove the dimethyl sulfoxide. RNA-directed DNA polymerase, RNase H, and nucleic acid-binding activity were monitored, as well as the subunit structure (on sodium dodecyl sulfate-polyacrylamide gels) of the various enzyme species obtained. With 30% dimethyl sulfoxide, the majority of DNA polymerase and RNase H activities as well as the alpha subunit were displaced from the alpha beta DNA polymerase position on phosphocellulose (0.23 M potassium phosphate) to the alpha DNA polymerase position (0.1 M). The association of DNA polymerase and RNase H activities with the alpha subunit suggests that alpha is the enzymatically active subunit in alpha beta. In addition to alpha DNA polymerase, a minor polymerase species eluted from phosphocellulose at 0.4 M potassium phosphate. The dissociated beta subunit eluted from phosphocellulose at a wide range of salt concentrations (0.28 to 0.5 M potassium phosphate). The dissociated beta subunit bound 3H-labeled murine leukemia virus RNA and [3H]poly(dT)-poly(dA) approximately 20-fold more avidly than alpha DNA polymerase alone. In contrast to the results with the alpha subunit, there was no correlation between DNA polymerase and RNase H activity profiles and the elution profile of the beta subunit from phosphocellulose. These observations suggest the beta subunit is either enzymatically inactive or possesses limited DNA polymerase and RNase H activity when compared with the alpha subunit. 相似文献
2.
Sequence relatedness between the subunits of avian myeloblastosis virus reverse transcriptase. 总被引:9,自引:0,他引:9
One- and two-diminsional tryptic and chymotryptic peptide maps of 125-I-labeled alpha and alphabeta avian myeloblastosis virus DNA polymerase demonstrate that the alpha polypeptide of the one and two subunit enzymes are structurally similar, if not identical. Furthermore, the beta subunit contains the same major 125I-labeled peptides as alpha, plus several additional peptides. These relationships and the fact that aging of purified alphabeta avian myeloblastosis virus DNA polymerase increases the proportion of alpha DNA polymerase that can be isolated from the alphabeta enzyme by phosphocellulose chromatography, suggests that alpha is derived from beta by proteolytic cleavage. 相似文献
3.
Activation of an Mg2+-dependent DNA endonuclease of avian myeloblastosis virus alpha beta DNA polymerase by in vitro proteolytic cleavage. 总被引:14,自引:12,他引:2
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Partial chymotryptic digestion of purified avian myeloblastosis virus alpha beta DNA polymerase resulted in the activation of a Mg2+-dependent DNA endonuclease activity. Incubation of the polymerase-protease mixture in the presence of super-coiled DNA and Mg2+ permitted detection of the cleaved polymerase fragment possessing DNA nicking activity. Protease digestion conditions were established permitting selective cleavage of beta to alpha, which contained DNA polymerase and RNase H activity and to a family of polypeptides ranging in size from 30,000 to 34,000 daltons. These latter beta-unique fragments were purified by polyuridylate-Sepharose 4B chromatography and were shown to contain both DNA binding and DNA endonuclease activities. We have demonstrated that this group of polymerase fragments derived by chymotryptic digestion of alpha beta DNA polymerase is similar to the in vivo-isolated avian myeloblastosis virus p32pol in size, sequence, and DNA endonuclease activity. 相似文献
4.
Endonuclease activity of purified RNA-directed DNA polymerase from avian myeloblastosis virus. 总被引:17,自引:0,他引:17
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. 相似文献
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7.
DNA-processing activities associated with the purified alpha, beta 2, and alpha beta molecular forms of avian sarcoma virus RNA-dependent DNA polymerase. 总被引:2,自引:2,他引:0
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The RNA-dependent DNA polymerase purified from B77 avian sarcoma virus exhibited two distinct DNA-processing activities. The alpha and beta 2 isoenzymes possessed an endodeoxyribonuclease activity capable of nicking simian virus 40 superhelical DNA, whereas the alpha beta isoenzyme performed as an untwisting topoisomerase. Both activities associated with the three molecular forms of the retroviral DNA polymerase were dependent on the presence of either Mn2+ or Mg2+ ions. From analysis of the denaturated DNA products, it is apparent that the alpha and beta 2 isoenzymes introduced two nicks, one per each strand in the superhelical simian virus 40 DNA molecules, whereas the alpha beta polymerase converted these supercoiled molecules to the relaxed covalently closed circular form. The notion that the DNA-processing activities are located on the DNA polymerase molecules was supported by the following: (i) the three isoenzymes were of a high purity; (ii) the activities cosedimented in glycerol gradients with the DNA polymerase activities of the alpha, beta 2, and alpha beta molecular forms; and (iii) immunoglobulin directed against the purified polymerase immunoprecipitated the DNA-processing activities. Chemical treatments of the DNA polymerase molecules (with pyridoxalphosphate, iodoacetamide, and sulfhydryl reagents), which inhibited the polymerase activity, also suppressed the endonucleolytic and topoisomerase activities, suggesting that cystein and amino groups play an important role in the active sites of the DNA-processing activities as well. 相似文献
8.
Binding properties of avian myeloblastosis virus DNA polymerases to nucleic acid affinity columns. 总被引:4,自引:4,他引:0
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A new method for the analysis and purification of the RNA-directed DNA polymerase of RNA tumor viruses has been developed. This nucleic acid affinity chromatography system utilizes an immobilized oligo (dT) moiety annealed with poly (A). The alpha and alphabeta DNA polymerases of avain myeloblastosis virus bound effectively to poly (A) oligo (dT)-cellulose. Alpha DNA polymerase did not bind effectively to poly (A) oligo (dT)-cellulose, poly (A)-cellulose, or to cellulose. Alphabeta bound to oligo (dT)-cellulose and cellulose at the same extent (approximately 30%), indicating that this enzyme did not bind specifically to the oligo (DT) moiety only. However, alphabeta bound to poly (A)-cellulose two to three times better than to cellulose itself, showing that alphabeta could bind to poly (A) without a primer. Alphabeta DNA polymerase also bound to poly (C)-cellulose, whereas alpha did not. These data show that the alpha DNA polymerase is defective in binding to nucleic acids if the beta subunit is not present. Data is presented which demonstrates that the alphabeta DNA polymerase bound tighter to poly (A). oligo (DT)-cellulose and to calf thymus DNA-cellulose than the alpha DNA polymerase, suggesting that the beta subunit or, at least part of it is responsible for this tighter binding. In addition, alphabeta DNA polymerase is able to reversibly transcribe avian myeloblastosis virus 70S RNA approximately fivefold faster than alpha DNA polymerase in the presence of Mg2+ and equally efficient in the presence of Mn2+. alpha DNA polymerase transcribed 9S globin m RNA slightly better than alphabeta with either metal ion. 相似文献
9.
A study was made of the DNA polymerase of reptilian type C virus isolated from Russell's viper spleen cells. Simultaneous detection experiments demonstrated the presence of 70S RNA and RNA-dependent DNA polymerase activity in reptilian type C virions. The endogenous activity was dependent on the addition of all four deoxynucleotide triphosphates and demonstrated an absolute requirement for a divalent cation. The reptilian viral DNA polymerase elutes from phosphocellulose at 0.22 M salt. In this respect, it is similar to the avian (avian myeloblastosis virus; AMV) viral enzyme but is different from the mammalian (Rauscher leukemia virus; RLV) viral enzyme which elutes at 0.4 M salt. The molecular weight of the viper DNA polymerase as estimated from glycerol gradient centrifugation is 109,000. It is a smaller enzyme than the AMV DNA polymerase (180,000 daltons) and somewhat larger than the RLV enzyme (70,000 daltons). A comparison of other properties of the type C reptilian DNA polymerase with the enzyme found in other type C oncogenic viruses is made. 相似文献
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Serological Analysis of the Deoxyribonucleic Acid Polymerase of Avian Oncornaviruses II. Comparison of Avian Deoxyribonucleic Acid Polymerases 总被引:15,自引:7,他引:8
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Monospecific antiserum prepared against the isolated deoxyribonucleic acid (DNA) polymerase of avian myeloblastosis virus (AMV) neutralized the endogenous ribonucleic acid-instructed DNA polymerase activity of detergent-disrupted virus. The viral polymerase was serologically unrelated to the seven major structural polypeptides of AMV. Furthermore, the viral enzyme was distinguished from normal cellular DNA polymerases by serological criteria; thus, antiserum against the viral enzyme neutralized its homologous antigen but not normal cellular DNA polymerases. Neutralization by antibody of viral DNA polymerase activity was observed with all avian leukemia-sarcoma viruses tested, irrespective of viral antigenic subtype. The DNA polymerase activity of avian reticuloendotheliosis virus, and of a variety of mammalian oncornaviruses, was not neutralized by antisera against the AMV polymerase. Immunological analysis of the RSValpha(O) mutant, which is deficient in DNA polymerase activity, shows this mutant to lack demonstrable polymerase antigen. Viral polymerase was identified by immunofluorescence as a cytoplasmic constituent in virus-producing chicken cells; polymerase antigen was not detected in uninfected (gs(-)) chicken cells. 相似文献
12.
Association of DNA primase with the beta/gamma subunits of DNA polymerase alpha from Drosophila melanogaster embryos 总被引:10,自引:0,他引:10
L S Kaguni J M Rossignol R C Conaway G R Banks I R Lehman 《The Journal of biological chemistry》1983,258(15):9037-9039
The DNA polymerase and primase activities of the intact DNA polymerase alpha from early embryos of Drosophila melanogaster co-sediment in native glycerol gradients. However, the activities are separated in glycerol gradients containing 2.8 M urea after treatment of the enzyme with 3.4 M urea. The 182,000-dalton alpha subunit which is required for DNA polymerase activity (Kaguni, L.S., Rossignol, J.-M., Conaway, R. C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S.A. 80, 2221-2225) is not required for DNA primase activity. Instead, primase activity resides in the 60,000-dalton (beta) and/or the 50,000-dalton (gamma) subunit. Neither polymerase nor primase has been found in association with the 73,000-dalton polypeptide which co-purifies with the intact enzyme. 相似文献
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14.
Purification of the α Subunit of Avian Myeloblastosis Virus DNA Polymerase by Polyuridylic Acid-Sepharose
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Duane P. Grandgenett 《Journal of virology》1976,20(1):348-350
The alpha subunit of the avian myeloblastosis virus DNA polymerase could be readily purified to near homogeneity using a polyuridylic acid-Sepharose column chromatography step. 相似文献
15.
RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses II. Directing Influence of RNA in the Reaction 总被引:30,自引:16,他引:14
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The role of ribonucleic acid (RNA) in deoxyribonucleic acid (DNA) synthesis with the purified DNA polymerase from the avian myeloblastosis virus has been studied. The polymerase catalyzes the synthesis of DNA in the presence of four deoxynucleoside triphosphates, Mg(2+), and a variety of RNA templates including those isolated from avian myeloblastosis, Rous sarcoma, and Rauscher leukemia viruses; phages f2, MS2, and Qbeta; and synthetic homopolymers such as polyadenylate.polyuridylic acid. The enzyme does not initiate the synthesis of new chains but incorporates deoxynucleotides at 3' hydroxyl ends of primer strands. The product is an RNA.DNA hybrid in which the two polynucleotide components are covalently linked. Free DNA has not been detected among the products formed with the purified enzyme in vitro. The DNA synthesized with avian myeloblastosis virus RNA after alkaline hydrolysis has a sedimentation coefficient of 6 to 7S. 相似文献
16.
Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex. 总被引:14,自引:14,他引:0
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K Moelling 《Journal of virology》1976,18(2):418-425
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RNA-dependent DNA polymerase of avian sarcoma virus B77. I. Isolation and partial characterization of the alpha, beta2, and alphabeta forms of the enzyme. 总被引:10,自引:0,他引:10
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. 相似文献
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