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)

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV) produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a sedimentation value of approximately 80 to 100S and a density of 1.23 g/cm3. This RNP complex is not derived from the mature virion. An endogenous RNA-directed DNA polymerase activity is associated with the RNP complex. The enzyme activity was completely neutralized by anti-REV DNA polymerase antibody but not by anti-avian myeloblastosis virus DNA polymerase antibody. The DNA product from the endogenous RNA-directed DNA polymerase reaction of the RNP complex hybridized to REV RNA but not to avian leukosis virus RNA. The RNA extracted from the RNP hybridized only to REV-specific complementary DNA synthesized from an endogenous DNA polymerase reaction of purified REV. The size of the RNA in the RNP is 30 to 35S, which represents the subunit size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activity in the viral RNP in infected cells and the maturation process of 60S virion RNA of REV are discussed.
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PMID:Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells. 8 19

We report on the discovery and isolation of DNA- and RNA-containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of approximately 90 and approximately 25 kbp. The deoxyribonucleoprotein-ribonucleoprotein complexes contain RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large-cell lymphoma cells by restriction digestion with Msp-I, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two-dimensional (isoelectric focusing/M(r)) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of approximately 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific activities. Using the DNP/RNP complexes a discrete DNA polymerase alpha product of approximately 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase alpha inhibitor aphidicolin. RNA polymerase assays in the presence of excess alpha-amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex-associated enzyme activities to be cell-cycle dependent. The DNA and RNA polymerase-related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities. 142 73

The Holy Grail of gerontologists investigating cellular senescence is the mechanism responsible for the finite proliferative capacity of somatic cells. In 1973, Olovnikov proposed that cells lose a small amount of DNA following each round of replication due to the inability of DNA polymerase to fully replicate chromosome ends (telomeres) and that eventually a critical deletion causes cell death. Recent observations showing that telomeres of human somatic cells act as a mitotic clock, shortening with age both in vitro and in vivo in a replication dependent manner, support this theory's premise. In addition, since telomeres stabilize chromosome ends against recombination, their loss could explain the increased frequency of dicentric chromosomes observed in late passage (senescent) fibroblasts and provide a checkpoint for regulated cell cycle exit. Sperm telomeres are longer than somatic telomeres and are maintained with age, suggesting that germ line cells may express telomerase, the ribonucleoprotein enzyme known to maintain telomere length in immortal unicellular eukaryotes. As predicted, telomerase activity has been found in immortal, transformed human cells and tumour cell lines, but not in normal somatic cells. Telomerase activation may be a late, obligate event in immortalization since many transformed cells and tumour tissues have critically short telomeres. Thus, telomere length and telomerase activity appear to be markers of the replicative history and proliferative potential of cells; the intriguing possibility remains that telomere loss is a genetic time bomb and hence causally involved in cell senescence and immortalization.
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PMID:Telomere loss: mitotic clock or genetic time bomb? 172 17

Two different systems of dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in separate laboratories detected analogous patterns of dye bands in virions of avian myeloblastosis virus (AMV). At least 11 of the dye bands co-migrated with the major polypeptides reported in Rous sarcoma virus. Particles with the morphology of the AMV core component, obtained after exposure of AMV to the nonionic surfactant Sterox SL, contained major polypeptides p12, p27, p60, p64, p91, and p98. The polypeptide p12 has been previously shown to be the major constituent of the inner ribonucleoprotein (RNP) of the AMV core, and has been designated p12(N). Two RNP polypeptides, p64 and p91, co-electrophoresed with purified AMV DNA polymerase and have now been designated p64(P) and p91(P). The polypeptide p27 has been identified as a probable constituent of the core shell, and has accordingly now been designated p27(C). In comparison to virions of AMV, the AMV core component contained a greatly reduced amount of polypeptide p15 and appeared to lack a major polypeptide, p19. Consequently, these polypeptides may be associated either with the exterior of the core shell or the interior of the viral envelope. Glycopeptides were not detected in AMV cores, in agreement with earlier reports that they reside in external projections from the viral envelope.
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PMID:Structural studies of avian myeloblastosis virus: comparison of polypeptides in virion and core component by dodecyl sulfate-polyacrylamide gel electrophoresis. 412 94

The protein of Visna virus, disrupted by 8 M guanidine hydrochloride and heating, was resolved into 10 polypeptides by agarose gel column chromatography in 6 M guanidine hydrochloride. Two of the peaks contained glycopolypeptides. Nonidet-disrupted virions were resolved into two fractions by potassium tartrate gradient centrifugation, with densities of 1.08 and 1.24 g/ml, respectively. About 70% of the viral DNA polymerase directed by added template was released into the light fraction, in which very little endogenous enzyme activity was detected. Also released into the light fraction were all of the glycopolypeptides, 50% of the viral RNA, and a part of each of the other viral protein components. The data indicate that extensive degradation of subviral structures occurred, even under mild conditions for virion disruption. The 1.24-g/ml fraction was composed of 50% of the viral RNA, most of the endogenous DNA polymerase activity (80%), and a major internal polypeptide (GuHCl6) with an estimated mol wt of 28,000. Two other polypeptides were also consistently detected in the heavy fraction, but they constituted less than 25% of the ribonucleoprotein complex, compared with 75% for GuHCl6.
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PMID:Substructures and polypeptides of Visna virus. 413 79

A native ribonucleoprotein (RNP) complex of avian myeloblastosis virus was prepared under conditions that gave optimal cDNA synthesis. The complex was an autonomous transcriptional unit capable of synthesizing DNA complementary to the RNA virus genome in the absence of exogenous reverse transcriptase (RNA-dependent DNA nucleotidyltransferase), genomic RNA, and primer. The RNA of the RNP complex cannot be translated in an in vitro cell-free translational system. The RNP contains intact viral RNA, the two subunits of the reverse transcriptase (beta and alpha), the p32 polypeptide resulting from the cleavage of the beta subunit into the alpha subunit, and p12. The principal polypeptide constituent of the RNP complex is the highly basic protein p12, which occurs at a molar ratio of 40:1 in relation to the beta subunit of the polymerase. When examined by the electron microscope, the RNP complex appears similar to the beaded structure of chromatin fiber. A significant portion of these molecules are circular, with headlike structures attached. The circular nature of the proviral DNA and the ability of the RNP complex to generate large intact cDNA copies from the natural primer end suggest that the 5' and 3' ends of the viral RNA are in proximity when in the RNP complex.
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PMID:Native ribonucleoprotein is an efficient transcriptional complex of avian myeloblastosis virus. 615 28

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.
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PMID:Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase. 748 31

Macronuclear telomeres in Oxytricha exist as DNA-protein complexes in which the termini of the G-rich strands are bound by a 97-kDa telomere protein. During telomeric DNA replication, the replication machinery must have access to the G-rich strand. However, given the stability of telomere protein binding, it has been unclear how this is accomplished. In this study we investigated the ability of several different DNA polymerases to access telomeric DNA in Oxytricha telomere protein-DNA complexes. Although DNA bound by the telomere protein is not degraded by micrococcal nuclease or labeled by terminal deoxynucleotidyltransferase, this DNA serves as an efficient primer for the addition of telomeric repeats by telomerase, a specialized RNA-dependent DNA polymerase (ribonucleoprotein reverse transcriptase), EC 2.7.7.49. Moreover, in the presence of a suitable complementary C-rich DNA template, AMV reverse transcriptase and the E. coli Klenow fragment will also elongate DNA bound by the telomere protein. These findings indicate that the 3' terminus and the Watson-Crick base pairing positions are exposed in the protein complex. We propose that the telomere protein can serve a dual role at the telomere by protecting the DNA phosphate backbone from degradation while simultaneously exposing the DNA bases for replication.
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PMID:DNA bound by the Oxytricha telomere protein is accessible to telomerase and other DNA polymerases. 750 21

Telomerase is a ribonucleoprotein DNA polymerase that catalyzes the de novo synthesis of telomeric simple sequence repeats. We describe the purification of telomerase and the cloning of cDNAs encoding two protein subunits from the ciliate Tetrahymena. Two proteins of 80 and 95 kDa copurified and coimmunoprecipitated with telomerase activity and the previously identified Tetrahymena telomerase RNA. The p95 subunit specifically cross-linked to a radiolabeled telomeric DNA primer, while the p80 subunit specifically bound to radiolabeled telomerase RNA. At the primary sequence level, the two telomerase proteins share only limited homologies with other polymerases and polymerase accessory factors.
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PMID:Purification of Tetrahymena telomerase and cloning of genes encoding the two protein components of the enzyme. 777 9

The nucleoside analog 2'-deoxy-2'-fluoroguanosine (2'-fluorodGuo) is phosphorylated by cellular enzymes and reversibly inhibits influenza virus replication in chick embryo cells within the first 4 h of infection. RNA hybridization studies revealed that primary and secondary transcription of influenza virus RNA were blocked at a compound concentration of 10 microM, but no inhibition of cell protein synthesis was seen even at high compound concentrations (200 microM). In vitro, the triphosphate of 2'-fluorodGuo is a competitive inhibitor of influenza virus transcriptase activity from disrupted virus, with a Ki of 1.0 microM. The cellular polymerases DNA polymerase alpha and RNA polymerase II were only weakly inhibited or were insusceptible to 2'-fluorodGTP. In kinetic studies with the influenza virus transcriptase, 2'-fluorodGTP, in the absence of GTP, blocked elongation of the virus RNA chain. Similarly, by using purified ribonucleoprotein complexes it was found that the addition of a single nucleotide of 2'-fluorodGTP to the virus RNA caused chain termination, which resulted in the blockage of further virus transcription. Furthermore, the specificity for influenza virus transcriptase was confirmed when the transcriptase from partially resistant virus was found to be 10-fold less susceptible to 2'-fluorodGTP (Ki = 13.1 microM).
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PMID:Inhibition of influenza virus transcription by 2'-deoxy-2'-fluoroguanosine. 858 25


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