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-directed RNA polymerase is responsible for gene expression. Despite its importance, many details of its function and higher-order structure still remain unknown. We report here a local sequence similarity between the second largest subunit of RNA polymerase II and bacterial RNases Ba (barnase), Bi, and St. The most remarkable similarity is that the catalytic sites of the RNases are shared with the eukaryotic RNA polymerase II subunits of Drosophila melanogaster and Saccharomyces cerevisiae. Several amino acids conserved among the RNases and the RNase-like domains of the RNA polymerase subunits are located in the neighborhood of the catalytic sites of barnase, whose three-dimensional structure has been resolved. This observation suggests the functional importance of the RNase-like domain of the RNA polymerase subunits and indicates that the RNase-like domain may have RNase activity. The location of the RNase-like domain relative to the region necessary for RNA polymerization is similar to the relative proximity of 5'----3' or 3'----5' exonuclease and the region of polymerase activity of DNA polymerase I. The RNase-like domain might work in proofreading, as in RNA-directed RNA polymerase of influenza virus, or it may contribute to RNA binding through an unknown function.
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PMID:RNase-like domain in DNA-directed RNA polymerase II. 192 68

The exonucleolytic activities associated with herpes simplex virus type-1 (HSV-1) DNA polymerase and DNase were compared. The unique properties of these nucleases were assessed by applying biochemical and immunological methods as well as by genetics. In contrast to the viral DNA polymerase, HSV DNase is equipped with a 5'-3'-exonuclease activity. Under reaction conditions optimal for HSV DNA polymerase, i.e. at high ionic strength, HSV DNase exhibited only limited endonucleolytic activity and degraded double-stranded DNA in a very processive manner and exclusively in the 5'-3' direction, producing predominantly mononucleotides. Both viral enzymes displayed significant RNase activity which could be correlated with the endogenous endonucleolytic and 5'-3'-exonucleolytic activities of the DNase and the polymerase-associated 3'-5' exonuclease. The tight linkage of polymerizing and exonucleolytic functions of the viral DNA polymerase was demonstrated by their identical response to (a) thermal inactivation, (b) drug inhibition and (c) neutralization by polyclonal antibodies reacting specifically with the N-terminal, central and C-terminal polypeptide domains of HSV-1 DNA polymerase. From the data presented it can be concluded that the cryptic 3'-5' exonuclease is the only exonucleolytic activity associated with the viral DNA polymerase.
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PMID:Comparison of exonucleolytic activities of herpes simplex virus type-1 DNA polymerase and DNase. 216 60

A cell-free system that catalyzes DNA replication was prepared from cytoplasmic extracts of Vero cells infected with African swine fever virus (ASFV). The cells were permeabilized with lysolecithin and disrupted by mild mechanical action and the nuclei were removed by low-speed centrifugation. Extracts prepared from infected cells at the time of maximal DNA replication incorporated [alpha-32P]dTTP into acid-insoluble material that was sensitive to DNase and resistant to RNase. The reaction was inhibited by phosphonoacetic acid, an inhibitor of ASFV-specific DNA polymerase. Extracts from mock-infected cells had a negligible activity. Micrococcal nuclease-treated extracts were able to replicate added virion DNA or viral replicative DNA. An increase in the mass of DNA detected by ethidium bromide staining and by dot blot hybridization with ASFV DNA showed that the incorporation was due to true replication. Plasmid DNA was also replicated, which indicates that ASFV-specific DNA polymerase does not require a virus-specific origin of replication. The pattern of fragments generated by EcoRI digestion of the in vitro product was characteristic of viral replicative DNA. Hybridization with a recombinant plasmid containing a terminal fragment of ASFV DNA confirmed the presence of dimer terminal ASFV fragments presumably generated from concatemeric replicative intermediates.
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PMID:In vitro DNA replication by cytoplasmic extracts from cells infected with African swine fever virus. 221 42

Biochemical and morphological evidence indicates that a type-C retrovirus is present in the blood of swine (both leukemic and nonleukemic) exposed to strontium-90 radiation. Nonexposed swine that were leukemic also had virus present. The virus was shown to contain an RNase-sensitive DNA polymerase activity with cation, detergent and template requirements similar to those of known viral reverse transcriptases. The buoyant density of the virus was 1.14 to 1.16 g/ml, which can be converted, by treatment with ether, to a virion core having a density of 1.20 to 1.23 g/ml. Linear regression analysis indicated a correlation between virus-associated DNA polymerase activity and the number of blast cells in the peripheral blood.
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PMID:Evidence for retrovirus in miniature swine with radiation-induced leukemia or metaplasia. 257 82

A highly selective affinity labeling procedure has been applied to map the active center of DNA primase from the yeast Saccharomyces cerevisiae. Enzyme molecules that have been modified by covalent attachment of benzaldehyde derivatives of adenine nucleotides are autocatalytically labeled by incubation with a radioactive ribonucleoside triphosphate. The affinity labeling of primase requires a template DNA, is not affected by DNase and RNase treatments, but is sensitive to proteinase K. Both the p58 and p48 subunits of yeast DNA primase appear to participate in the formation of the catalytic site of the enzyme, although UV-photocross-linking with [alpha-32P]ATP locates the ribonucleoside triphosphate binding site exclusively on the p48 polypeptide. The fixation of the radioactive product has been carried out also after the enzymatic reaction. Under this condition the RNA primers synthesized by the DNA polymerase-primase complex under uncoupled DNA synthesis conditions are linked to both DNA primase and DNA polymerase. When DNA synthesis is allowed to proceed first, the labeled RNA chains are fixed exclusively to the DNA polymerase polypeptide. These results, in accord with previous data, have been used to propose a model illustrating the interactions and the putative roles of the polypeptides of the DNA polymerase-primase complex.
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PMID:Affinity labeling of the active center and ribonucleoside triphosphate binding site of yeast DNA primase. 264 56

A tumor-derived factor that inhibits cellular DNA synthesis was identified. The factor was extractable from a small-cell lung carcinoma cell line grown in either chemically defined medium or nu/nu mice and inhibited tritiated thymidine ([3H]dThd) incorporation by tumor cell lines of autologous, allogeneic, and xenogeneic origins. The viability of nonproliferating cells from normal tissue was not affected. Tumor extract inhibitory activity was trypsin labile but was resistant to other proteases, neuraminidase, lipase, DNase, RNase, glucosidase, extremes of pH-temperature, and reducing conditions. Inhibitory activity was reversibly bound to helix pomatia lectin but not to lentil, wheat germ, or concanavalin A lectins. Purification by size-exclusion high-performance liquid chromatography yielded a bioactive unimodal 12-kilodalton (kd) peak. The bioactive 12-kd moiety could be eluted from sodium dodecyl sulfate-polyacrylamide gels. Redosing of populations of the T-lymphoblastoid cell line CEM achieved an early (24 hr) sustained depression of pulse [3H]dThd incorporation and ultimately led to decreased population density of factor-treated populations. DNA histogram analysis demonstrated no change in cell cycle phase distribution after factor treatment. 5-Bromo-2'-deoxyuridine (BrdUrd) vs. propidium iodide with the two-parameter Fluorescence-Activated Cell Sorter analysis showed relative inhibition of non-S-phase BrdUrd uptake at 24 hours. A cell-free DNA polymerase assay demonstrated significant inhibition of non-alpha-polymerase-associated DNA synthesis in factor-treated cells. These studies suggest that this tumor-derived inhibitor of DNA synthesis represents a class of cellular products involved in the autoregulation of growth by regulation of DNA synthetic activity.
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PMID:Inhibition of DNA synthesis by a small-cell lung carcinoma-derived protein. 302 Mar 1

A highly active and stable DNA polymerase was found in purified preparations of two murine sarcoma viruses. Enzyme activity is not detected in most virus preparations unless they are treated with low concentrations of a nonionic detergent such as Nonidet P-40. The incorporation of labeled thymidine triphosphate requires all four deoxyribonucleoside triphosphates and either Mg(2+) or Mn(2+). Enzyme activity is proportional to virus concentration and is linear with time up to 90 min. That the template is RNA is suggested by the reduction in polymerase activity upon treatment of murine sarcoma virus with RNase, and by the absence of detectable amounts of DNA in the virus. That the product is DNA is shown by the incorporation of all four deoxyribo-nucleoside triphosphates into an acid-insoluble product which is stable in alkali, is destroyed by DNase, sediments in alkaline sucrose gradients with a sedimentation coefficient of 7 S, and bands in isopycnic CsCl gradients with a mean buoyant density of 1.700.
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PMID:Mechanism of carcinogenesis by RNA tumor viruses. I. An RNA-dependent DNA polymerase in murine sarcoma viruses. 431 86

DNA polymerase activity can be unmasked in avian myeloblastosis virus (AMV) by treatment with the nonionic detergent Nonidet P-40. Two products are formed: (1) RNA.DNA hybrid molecules and (2) duplex DNA molecules. The kinetics of dTTP incorporation into DNA are biphasic: an initial rapid reaction for 4 min at 37 degrees C with a minimal polymerization rate of 10-20 nucleotides per see, and a second reaction at about half the initial rate. Viral RNA.DNA complexes are detected as early as 30 sec after the initiation of DNA synthesis; DNA free of template is formed subsequently. Most of the free AMV DNA forms an RNA.DNA hybrid when annealed with viral RNA. Over half of the free AMV DNA product is inferred to be double-stranded, since it is retained on hydroxyapatite columns after elution with 0.12 M phosphate buffer, and is resistant to Escherichia coli exonuclease I. Adenovirus or calfthymus DNA added to unmasked AMV stimulates DNA synthesis 4-16 times if there is no treatment with RNase, and 40-130 fold if RNase treatment precedes the enzyme assay. It is possible that two polymerases are present, or that a single enzyme forms both the RNA.DNA hybrid and the double-stranded product.
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PMID:Mechanism of carcinogenesis by RNA tumor viruses. 3. Formation of RNA, DNA complex and duplex DNA molecules by the DNA polymerase (s) of avian myeloblastosis virus. 432 24

A complex of ribosomal DNA with RNA has been isolated from ovaries of metamorphosing tadpoles of Xenopus laevis. The complex is disrupted by treatments that destroy hydrogen bonds, and the sedimentation of the DNA within the complex is sensitive to RNase. We suggest that the RNA-ribosomal DNA complex is an intermediate in the synthesis of amplified ribosomal DNA and that the RNA is a template. In addition, in a preliminary attempt to mimic the amplification process in vitro, we have demonstrated the use of this RNA from the complex as a template for DNA synthesis by a DNA polymerase isolated from ovaries of Xenopus.
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PMID:[On the role of RNA in gene amplification]. 450 50

RNase-sensitive DNA polymerase activity (RSDP) was tested in different cell fractions of Neurospora crassa cell types and its morphological mutants. This RSDP was found localized in the microsomal pellet fraction and absent in the purified nuclear pellets isolated from different N. crassa cell types: conidia, germinated conidia, and mycelia. This enzyme is capable of synthesizing a DNA product only in the presence of all four deoxyribonucleoside-5' - triphosphates and Mg2+. Removal of RNA from the pellet fraction by RNase strongly inhibited the DNA synthesis. The endogenous synthesis of DNA in the microsomal pellet fraction was associated with the formation of an RNA:DNA hybrid as analyzed by Cs2SO4 equilibrium density gradient centrifugation. The DNA product after alkali hydrolysis hybridizes with the RNA isolated from the same pellet fraction, as analyzed by elution from hydroxylapatite column at 60C. This DNa product did not hybridize with poly (A). A few mutants tested showed this RNase-sensitive DNA polymerase activity.
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PMID:RNase-sensitive DNA polymerase activity in cell fractions and mutants of Neurospora crassa. 616 50


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