Effect of heparin or removal of lysine-rich histone fraction on the poly (ADP-ribose) polymerase, DNA polymerase and template activity of isolated swine aortic nuclei.
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Aorta↗
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Results for “DNA Polymerase I”
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Purification and properties of spleen necrosis virus DNA polymerase.
DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.
Animals↗
The role of DNA polymerases in DNA repair in Escherichia coli: DNA polymerase I-dependent repair following chemical alkylation of permeabilized bacteria.
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Alkanesulfonates↗
Distinctive properties of mammalian DNA polymerases.
DNA polymerase-alpha and -beta can be distinguished from one another by the differential effects of N-ethylmaleimide, KCl, ara-CTP and temperature, as well as on the basis of sedimentation. The sensitivity of DNA polymerase-beta to elevated temperatures as compared to DNA polymerase-alpha provides a new means of distinguishing between these two enzymes even in crude extracts and a possible probe for determining their function. DNA polymerase-alpha and -beta share several properties in common, including the ability to readily incorporate dUTP in place of dTTP. The Km for dUTP varies from 10 to 30 micron with different preparations of DNA polymerase-alpha and -beta. Thus, in mammalian cells, dUMP could be incorporated into DNA, and if excised by an endonuclease, would lead to discontinuities. Initial analyses of fidelity in direct comparative studies indicate that beta-class DNA polymerases are highly accurate in base selection when copying poly[d(A-T)]. Less than one molecule of dGMP is incorporated for every 12 000-45 000 molecules of dAMP and dTMP polymerized. DNA polymerase-alpha is somewhat less accurate, making one mistake for every 4000-10 000 correct nucleotides incorporated. Since both polymerases lack an exonucleolytic activity, this accuracy must be the result of selectivity for the complementary nucleotide by the polymerase.
Animals↗
Two temperature-sensitive polA mutants: an approach to the role in vivo of DNA polymerase I.
DNA polymerase I (Pol I) purified from a strain carrying polA12 demonstrates a defect primarily in the polymerase portion of the molecule. The mutant enzyme is altered in its structure and template specificity. Pol I isolated from strains carrying polAexl are primarily defective in 5' in equilibrium 3' exonuclease. The polAexl lesion renders strains conditonally lethal.
DNA Nucleotidyltransferases↗
Utilization of mismatched initiator termini by avian myeloblastosis virus DNA polymerase.
DNA synthesis by avian myeloblastosis virus was studied using poly(C) as template and modified oligo(dG) as primer. The addition of one noncomplementary base to the 3'-end of the primer has no important effect on synthesis. The mispaired base is incorporated into the product and the apparent Km (for primer) and the V of the reaction remain unchanged. This confirms the absence of a 3' leads to 5'-exodeoxynuclease activity using a template that is transcribed faithfully rather than one that can undergo a slippage reaction.
Avian Leukosis Virus↗
Serological analysis of cellular and viral DNA polymerases by an antiserum to DNA polymerase gamma of human lymphoblasts.
An antiserum has been prepared against a highly purified DNA polymerase gamma from NC37 cells, a normal human lymphoblast cell line. The antiserum does not possess enzyme neutralizing activity, but does bind specifically to DNA polymerase gamma. When tested in a double antibody immunoprecipitation assay, the antibody does not cross-react with DNA polymerases alpha or beta, purified from NC37 cells, or with reverse transcriptases of avian, murine, or primate RNA tumor viruses. Antisera prepared against purified reverse transcriptases similarly do not recognize DNA polymerase gamma, either in an enzyme neutralization assay or in the more sensitive double antibody immunoprecipitation assay. The availability of an antiserum to DNA polymerase gamma will allow the further characterization of enzyme activities isolated from cellular material and suspected of being related to viral reverse ttranscriptases. In those cases where such activities do not immunologically resemble known viral DNA polymerases, the anti-DNA polymerase gamma will help determine the viral or cellular nature of the unknown activity.
Antigen-Antibody Reactions↗
Interaction of Cibacron Blue F3GA with Escherichia coli DNA polymerase I and with T4 DNA polymerase.
Individual rapid procedures for the enrichment of Escherichia coli DNA polymerase I and of bacteriophage T4 DNA polymerase free of endonuclease activity are described using Blue dextran-Sepharose chromatography. The blue dye of Blue dextran-Sepharose selectively binds to the deoxynucleoside triphosphate substrate site of the E. coli but not the T4 enzyme indicating that the catalytic sites of these two enzymes which catalyze the same polymerization reaction in vitro are quite distinct.
Anthracenes↗
Pheasant virus DNA polymerase is related to avian leukosis virus DNA polymerase at the active site.
The DNA polymerase from Amherst pheasant virus (APV), a member of the pheasant virus species of retroviruses, was compared to the DNA polymerases of avian leukosis viruses (ALV) and a reticuloendotheliosis virus (spleen necrosis virus (SNV)). Immunoglobulin inhibition tests and competition immunoassays showed that APV and ALV DNA polymerases are closely related at their active sites. The determinants common to their active sites are not shared by SNV DNA polymerase. Bu using a species-specific radioimmunoassay, it was shown that both APV and SNV DNA polymerases are grossly different from ALV DNA polymerase. The specificity of the relationship of the active sites of APV and ALV DNA polymerases was confirmed by a heterologous radioimmunoassay. Our data indicate that pheasant viruses are evolutionarily linked to ALV.
Avian Leukosis Virus↗
Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate.
The effect of the nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine) on herpes simplex virus type 1 DNA synthesis was examined. Acycloguanosine inhibited herpesvirus DNA synthesis in virus-infected cells. The synthesis of host cell DNA was only partially inhibited in actively growing cells at acycloguanosine concentrations several hundred-fold greater than the 50% effective dose for herpes simplex virus type 1. Studies using partially purified enzymes revealed that the triphosphate of this compound inhibited the virus-induced DNA polymerases (DNA nucleotidyltransferases) to a greater degree than the DNA polymerase of the host cell, that the inhibition was dependent upon the base composition of the template, and that the triphosphate was a better substrate for the virus-induced polymerases than for the alpha cellular DNA polymerases.
Animals↗
Further characterization of a cell-free system for measuring replicative and repair DNA synthesis with cultured human fibroblasts and evidence for the involvement of DNA polymerase alpha in DNA repair.
DNA repair synthesis can be specifically measured in osmotically opened, confluent cultured human fibroblasts after exposure to DNA damaging agents such that both induction and mediation of DNA repair synthesis can take place in this cell-free system. Alternatively, by utilizing osmotically shocked, log phase cells and altering the DNA precursors, pH and ionic strength, replicative DNA synthesis can be specifically monitored. Autoradiographic studies show that virtually all of the nuclei from the lysates of the confluent, UV-iradiated cells are lightly labeled in the fashion characteristic of DNA repair. By contrast, only a fraction of nuclei is labeled in a population of unperturbed, opened log phase cells and the labeling is heavy and characteristic of replicative synthesis. Furthermore, equilibrium density gradient sedimentation shows that DNA synthesis in lysates of log-phase cells is semiconservative, whereas that with UV-irradiated cells is repair synthesis. This open cell system has been used to study the enzymology of DNA repair. Thus, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerases beta and gamma, does not inhibit either replicative or repair synthesis. By contrast, aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits DNA repair and replicative synthesis in both intact and permeabilized cells. Finally, phage T4 UV-exonuclease stimulates repair synthesis, but only when phage T4 UV-endonuclease is also added to the UV-irradiated nuclei.
4-Nitroquinoline-1-oxide↗
DNA polymerases of anucleated cells. Isolation and characterization of two DNA polymerases from human platelets.
Two different DNA polymerases have been purified and characterized from human platelets. In the mitochondrial fraction a unique activity of the polymerase gamma type has been found. The same enzyme is found in the extramitochondrial supernatant. A second DNA polymerase, called 'cytoplasmic' DNA polymerase has been found in the 10000 x g supernatant of human platelets. The following properties of the latter DNA polymerase from human platelets are identical to those of DNA polymerase alpha from normal cells: DEAE-cellulose and phosphocellulose chromatography, size, thermal stability, phosphonoacetic acid and ethidium bromide inhibition. However, some of its properties, like high resistance to N-ethylmaleimide and the lack of DNA polymerization using synthetic RNA primers, are those of DNA polymerase beta.
Blood Platelets↗
Relationship of Bacillus subtilis DNA polymerase III to bacteriophage PBS2-induced DNA polymerase and to the replication of uracil-containing DNA.
In vivo studies of PBS2 phage replication in a temperature-sensitive Bacillus subtilis DNA polymerase III (Pol III) mutant and a temperature-resistant revertant of this mutant have suggested the possible involvement of Pol III in PBS2 DNA synthesis. Previous results with 6-(p-hydroxyphenylazo)-uracil (HPUra), a specific inhibitor of Pol III and DNA replication in uninfected cells, suggest that Pol III is not involved in phage DNA replication, due to its resistance to this drug. Experiments were designed to examine possible explanations for this apparent contradiction. First, assays of the host Pol III and the phage-induced DNA polymerase activities in extracts indicated that a labile Pol III did not result in a labile phage-induced enzyme, suggesting that this new polymerase is not a modified HPUra-resistant form of Pol III. Indeed the purified phage-induced enzyme was resistant to the active, reduced form of HPUra under all assay conditions tested. Since in vitro Pol III was capable of replicating the uracil-containing DNA found in this phage, the sensitivity of the purified enzyme to reduced HPUra was examined using phage DNA as template-primer and dUTP as substrate; these new substrates did not affect the sensitivity of the host enzyme to the drug.
Bacillus cereus↗
Nuclear localisation of DNA polymerase alpha and DNA synthesis in polyoma virus infected mouse cells.
Studies using inhibitors of DNA synthesis have shown that DNA polymerase alpha is located in nuclei of polyoma virus infected mouse cells to the same degree as these nuclei are engaged in DNA replication. These results indicate that either the enzyme is actively transported into nuclei concomitant with the onset of DNA synthesis, or that it is bound much more strongly in nuclei during DNA replication. In any case, these observations support the hypothesis that DNA polymerase alpha is involved in the replication of cellular and viral DNA.
Animals↗
[DNA polymerases of animal cells: III. DNA replication and DNA polymerase alpha (author's transl)].
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Animals↗
On the fidelity of DNA replication. Lack of exodeoxyribonuclease activity and error-correcting function in avian myeloblastosis virus DNA polymerase.
Homogeneous DNA polymerase ("reverse transcriptase") from avian myeoblastosis virus was assayed for exodeoxyribonuclease activity. The substrates were defined template-initiator complexes in which different radioactive nucleotides were present at the 3'-OH termini of the initiator. Even when the number of molecules of enzyme was equal to the number of initiator termini there was no significant release of radioactivity with any of the template-initiator combinations tested. Under similar conditions, the nuclease activity associated with either Escherichia coli or T4DNA polymerases rendered more than 90% of the initiator termini acid-soluble. The ratio of exodeoxyribonuclease activity to protein with avian myeoblastosis DNA polymerase is less than 0.003% of that obtained with E. coli DNA polymerase I. Furthermore, avian myeloblastosis virus DNA polymerase failed to excise mispaired terminal nucleotides in both the presence and absence of polymerization.
Avian Leukosis Virus↗
Role of silicon on diatom metabolism. IX. Differential synthesis of DNA polymerases and DNA-binding proteins during silicate starvation and recovery in Cylindrotheca fusiformis.
During recovery from silicate-starvation, a period of active DNA synthesis, synchronized cells of Cylindrotheca fusiformis incorporated 3 times more L-[U-14C]aspartate than did starved cells. Of the diatoms's four DNA polymerases, A and D are synthesized during silicate recovery, indicating that they are involved in silicate-dependent DNA replication. Polymerase B, and the chloroplast enzyme, polymerase C, are synthesized during silicate-starvation and their levels are unaffected by the addition of silicate. DEAE-Sephadex analysis of the DNA-binding proteins, labeled with [14C]- and [3H]asparate, shows that only three proteins are synthesized in cells recovering from silicate-starvation. Two of these proteins correspond to polymerases A and D, while the function of the third protein is not known. At least 15 other proteins are present in silicate-starved cells and their synthesis is repressed upon the addition of silicate. Models are proposed which describe the modes by which silicate might regulate DNA synthesis in the diatom.
Amino Acids↗
In vitro transcription of theavian oncornavirus genome by the RNA-directed DNA polymerase: analysis of DNA transcripts synthesized in reconstructed enzymatic reactions.
We have analyzed the DNA products synthesized in vitro in reconstructed reactions containing purified avian oncornavirus genome RNA and RNA-directed DNA polymerase. The results of these studies indicate that: (i) the initial DNA product synthesized on either 70S RNA or reconstituted 35S RNA-tRNAtrp template - primer complexes in the presence of low concentrations of deoxynucleoside triphosphates consists of several discrete size classes, none of which exceed 200 nucleotides in length; (ii) large DNA transcripts (about 2,000 nucleotides) can be synthesized on both 70S RNA and the 35S RNA - tRNAtrp complex by increasing the deoxynucleoside triphosphate concentration; and (iii) DNA synthesized by detergent-disrupted virus is considerably longer than DNA synthesized in reconstructed reactions.
Avian Leukosis Virus↗