E R Stout

DNA polymerase from maize seedlings.

Abstract 1. A protein fraction which promotes the incorporation of deoxyribonucleotides into acid-insoluble product has been extracted and partially purified from young maize seedlings ( Zea mays L.). The characteristics of this enzyme are analogous to those of DNA polymerases (deoxyribonucleosidetriphosphate: DNA deoxyribonucleotidyltransferase, EC 2.7.7.7) isolated from other organisms. The bulk of the activity was recovered in the supernatant fraction of a seedling homogenate after centrifugation at 105 000 × g and was precipitated from (NH 4 ) 2 SO 4 solution between 40 and 50% saturation. Chromatography of the solubilized precipitate on DEAE-cellulose resulted in a preparation which incorporated 100 pmoles dAMP/min per mg of protein into acid-insoluble product. 2. DNA polymerase activity depended upon the simultaneous presence of dATP, dCTP, dGTP, dTTP, DNA and Mg 2+ . The rate of formation of the acid-insoluble product was 2–4-fold greater with heat-denatured DNA than native DNA. All species of DNA tested effectively promoted product formation. Incorporation of deoxyribonucleotides was linear with time for at least 1 h of incubation at 30° and was first order with respect to enzyme concentration. The optimal pH was 8.4 in Tris-HCl and 8.8 in glycine-NaOH. 3. The product of the reaction was insoluble in acid, ethanol and ether. It was hydrolyzed by pancreatic deoxyribonuclease and was resistant to the action of pancreatic ribonuclease. The incorporation of each of the four deoxyribonucleotides into the product was a function of the base composition of native DNA. With heat-denatured DNA, the incorporation of dAMP was increased relative to the other deoxyribonucleotides.

Reversible Inhibition of Bovine Parvovirus DNA Replication by Aphidicolin and L-Canavanine

The replication of the autonomous parvovirus, bovine parvovirus (BPV), has been studied in virus-infected cells. Gel electrophoresis was used to determine the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, and L-canavanine, an inhibitor of protein synthesis, on viral DNA replication. Synchronized cell cultures were infected with 32P-labelled or unlabelled BPV in the presence or absence of aphidicolin and L-canavanine. Cells were harvested at various times post-infection, and DNA was electrophoresed and blotted. When aphidicolin was added to cells at the time of infection, then removed 8 h later, BPV replicative form DNA (RF) synthesis began within 2 h after its removal. This preceded the peak of cellular DNA synthesis by 2 h, unlike an uninhibited infection, when viral RF synthesis follows the peak of S phase by 2 to 4 h. Furthermore, if aphidicolin was added at any point during the replication cycle, BPV DNA synthesis stopped. This effect was shown to be completely reversible and indicated that aphidicolin did not disrupt the replication apparatus required for viral DNA synthesis. L-Canavanine inhibited synthesis of the virus-specific proteins NP-1 and VP3 and synthesis of BPV DNA. Upon removal of L-canavanine, viral protein synthesis was detected by 30 min followed by viral DNA synthesis. These results indicate that a specific S phase function other than cellular DNA synthesis is required for initiation of BPV DNA synthesis, that DNA polymerase alpha plays a major role in BPV DNA replication in vivo, and that these inhibitors can be used to inhibit reversibly various stages of BPV DNA replication.

Purification and comparative mechanistic studies of an endogenous inhibitor of maize RNA polymerase

Abstract 1. A low molecular weight inhibitor of maize DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) has been purified to chromatographic homogeneity from a high-speed supernatant fraction which also contains the soluble enzyme. 2. Chemical characterization of the inhibitor has revealed the presence of an aromatic nucleus which apparently contains an attached —COOH group and one or more sulfur-containing side chains. 3. Studies using known inhibitors of RNA polymerase for comparative purposes demonstrated that the maize inhibitor acts much the same as α-amanitin in its ability to interrupt polymerization. Initiation by maize RNA polymerase was greatly enhanced in the presence of the maize inhibitor. 4. On the basis of resistance to rifamycins and inhibition by α-aminitin, the soluble maize RNA polymerase was shown to be equivalent to the nucleoplasmic RNA polymerase II of animals.

Possible sequences for nuclear accumulation of parvoviral proteins

Parvoviral genomes have been searched for sequences which may code for the nuclear transport of viral proteins. Sequences similar to those which regulate the nuclear transport of T antigen and yeast mating type protein were detected within the sequences coding for capsid and non-capsid proteins.

RNA polymerase from nucleoli of maize seedlings

Abstract RNA polymerase I (nucleosidetriphosphate: RNA nucleotidytransferase, EC 2.7.7.6) was purified 1600-fold from nucleoli of maize seedlings. The enzyme was eluted from anion exchange resin at low ionic strength ahead of RNA polymerase II. On DEAE—Sephadex, polymerase I was chromatographed into forms Ia and Ib. The molecular weight was 500 000 daltons by glycerol gradient centrifugation. The activity was partially inhibited by α-amanitin although it was much less sensitive to this toxin than polymerase II from the same source.

Purified DNA polymerase γ replicates bovine parvovirus DNA to a unit-length product

Abstract DNA polymerase gamma, purified from fetal bovine liver, replicated virion single-stranded DNA from bovine parvovirus to a unit-length double-stranded DNA molecule. This product was not nicked and was covalently linked to the 3′ hairpin primer. The reaction was inhibited by dideoxythymidine 5′-triphosphate, but was unaffected by ATP or aphidicolin. Double-stranded viral DNA was not a functional template for purified DNA polymerase gamma.

IN VITRO SYNTHESIS OF PARVOVIRAL DNA

ABSTRACT We have developed a nuclear lysate system capable of synthesizing unit length parvoviral DNA in vitro . In this system RF, RI, and single stranded DNA was synthesized. It was necessary to supplement the nuclear lysates with polyamines to prevent degradation of template and product DNA. The results show that viral RF DNA pulse-labeled in vivo and in vitro was chased into single stranded viral DNA in nuclear lysates. The single stranded DNA was synthesized by a continuous mechanism through replicative intermediates. The results of density labeling experiments suggest that the single stranded DNA was of one polarity. The RF DNA synthesized in nuclear lysates had identical restriction enzyme patterns to virion DNA replicated in vitro with E. Coli DNA polymerase 1.