aphidicolin and Carcinoma--Ehrlich-Tumor

Two species of DNA polymerase alpha free of primase activity were identified in extracts of Ehrlich mouse cells that had been infected with minute virus of mice. Primase-free forms of DNA polymerase alpha eluted with 150 and 180 mM NaCl during ion-exchange chromatography on DEAE-cellulose columns, exhibited sedimentation coefficients of 11 S and 8.2 S, respectively, and were inhibited by aphidicolin, N2-(p-n-butylphenyl)-9-(2-deoxy-beta-D-ribofuranosyl)guanine 5'-triphosphate, and 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)adenine 5'-triphosphate. The ratio of primase-free DNA polymerase alpha to the DNA polymerase alpha-primase complex increased from 1.5 to greater than 100 during the course of infection, and free primase was produced during the MVM replicative cycle.

Topics: Animals; Aphidicolin; Carcinoma, Ehrlich Tumor; Cell Line; Chromatography, Ion Exchange; Diterpenes; DNA Polymerase II; DNA Primase; DNA, Viral; Enzyme Induction; Mice; Minute Virus of Mice; Mutation; Parvoviridae Infections; RNA Nucleotidyltransferases; Virus Replication

To asses the possible roles of the two active forms of mouse DNA polymerase alpha: primase--DNA-polymerase alpha complex (DNA replicase) and DNA polymerase alpha free from primase activity (7.3S polymerase), in nuclear DNA replication the correlation of their activity levels with the rate of nuclear DNA replication was determined and a comparison made of their catalytic properties. The experiments using either C3H2K cells, synchronized by serum starvation, or Ehrlich culture cells, arrested at the S phase by aphidicolin, showed DNA replicase to increase in cells in the S phase to at least six times that of the G0-phase cells but 7.3S polymerase to increase but slightly in this phase. This increase in DNA replicase activity most likely resulted from synthesis of a new enzyme, as shown by experiments using a specific monoclonal antibody, aphidicolin and cycloheximide. Not only with respect to the presence or absence of primase activity, but in other points as well the catalytic properties of these two forms were found to differ; DNA replicase preferred the activated calf thymus DNA with wide gaps of about 100 nucleotides long as a template-primer, while the optimal gap size for 7.3S polymerase was 40-50 nucleotides long. Size analysis of the products synthesized on M13 single-stranded circular DNA with a single 17-nucleotide primer by DNA replicase and 7.3S polymerase suggested the ability of DNA replicase to overcome a secondary structure formed in single-stranded DNA to be greater than that of 7.3S polymerase.

Topics: Animals; Aphidicolin; Base Sequence; Carcinoma, Ehrlich Tumor; Cell Cycle; Cell Line; Cycloheximide; Diterpenes; DNA Polymerase II; DNA-Directed DNA Polymerase; DNA, Circular; Mice; Mice, Inbred C3H; Nucleic Acid Conformation; Templates, Genetic

Topics: Animals; Aphidicolin; Carcinoma, Ehrlich Tumor; Cell Survival; Cells, Cultured; Diterpenes; DNA; DNA Polymerase II; DNA Repair; Interphase; Mice; Ultraviolet Rays

A partially purified preparation of DNA polymerase alpha, obtained from the cytosol of Ehrlich ascites tumour cells, has been found to catalyze the conversion of MVM parvovirus, SS DNA (5 kilobases) to RF in vitro. The reaction initiates at a natural 55 base pair hairpin which exists at the 3' terminus of MVM SS DNA. The SS leads to RF conversion is sensitive to aphidicolin, resistant to ddTTP and is promoted by purine ribonucleoside 5' triphosphates, a phenomenon which could not be explained simply by stabilization effects on the in vitro deoxynucleotide precursor pool. In the absence of rNTPs, nascent complementary strands frequently terminate prematurely at a preferred location, between 1300 and 1700 nucleotides from the initiating 3' hairpin terminus. This in vitro system, involving self-primed parvovirus DNA synthesis, provides a convenient assay for those components of the mammalian replicative DNA polymerase complex which are required for the elongation of nascent DNA chains.

Topics: Animals; Aphidicolin; Carcinoma, Ehrlich Tumor; Cytosol; Diterpenes; DNA Polymerase II; DNA Replication; DNA-Directed DNA Polymerase; DNA, Single-Stranded; DNA, Viral; Kinetics; Mice; Parvoviridae; Virus Replication

The effects of aphidicolin, a specific inhibitor of DNA alpha-polymerase, have been studied on various cellular end-points and on DNA strand break repair. In the concentration range 0.02-2 micrograms/ml DNA synthesis was strongly inhibited resulting in a concomittant loss of cell proliferation ability; RNA and protein synthesis were unaffected in this range. At these concentrations PLD repair in X-irradiated plateau-phase cells was unaffected even after 7 hours treatment with aphidicolin; however, at higher concentrations (greater than 2 micrograms/ml) PLD repair was inhibited. We show that in the low concentration range (less than 2 micrograms/ml) PLD repair can be seen in exponentially growing cells and from experiments with synchronized cells we establish that the PLD repair observed can be attributed to the S-phase population, the survival of G1-cells not being affected by aphidicolin. The 'promotion' of PLD repair in exponentially growing cells was in excess of that observed for the same cells in balanced salt solution in which PLD repair is usually observed. At high concentrations (greater than 2 micrograms/ml) of aphidicolin, both X-irradiated and control S-cells were killed increasingly as concentration increased. The repair of DNA strand breaks (single and double) was unaffected in the low concentration range, but a strong inhibition was observed at high concentration. It is concluded from these results that alpha-polymerase, which is strongly inhibited at low concentrations of aphidicolin as evidenced by the inhibition of DNA synthesis, plays no major part in the repair of DNA strand breaks or in the repair of PLD.

Topics: Animals; Antibiotics, Antineoplastic; Aphidicolin; Carcinoma, Ehrlich Tumor; Cell Cycle; Cell Survival; Diterpenes; DNA Polymerase II; DNA Repair; DNA, Neoplasm; Kinetics; Mice; Nucleic Acid Synthesis Inhibitors