Mochihiko Ohashi

Aphidicolin: A specific inhibitor of DNA polymerases in the cytosol of rat liver

Abstract Aphidicolin, a tetracyclic diterpenoid, is known to be antiviral and to inhibit the incorporation of thymidine into DNA of cultured human embryonic lung cells. We examined effects of the compound on the activity of several DNA polymerases obtained from subcellular fractions of rat liver. Aphidicolin at a concentration of 15 μg/ml caused a 85% reduction in level of the activity of crude and partially purified DNA polymerases from the cytosol. However, aphidicolin even at a concentration of 75 μg/ml failed to affect the activity of crude and partially purified DNA polymerases from nuclear and mitochondrial fractions.

Aphidicolin does inhibit repair replication in HeLa cells.

Abstract Aphidicolin was shown to be a specific inhibitor of eukaryotic DNA polymerase α. We have examined the effect of aphidicolin on repair synthesis as well as replication of HeLa cell DNA, and found that it inhibits not only DNA replication but also UV-induced DNA repair in hydroxyurea-arabinosyl cytosine treated cells.

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS

Aphidicolin at 2 μg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I‐cell stage. However, at 40 μg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 μg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 μg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase β or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.

Two-dimensional electrophoresis on the layers of cellulose acetate membrane☆

Abstract A new type of two-dimensional electrophoresis for analysis of protein using cellulose acetate membrane has been developed. Prior to the separation, proteins in a sample are concentrated to a narrow zone on a strip of cellulose acetate according to “steady-state stacking” of isotachophoresis. Electroendosmotic counterflow on cellulose acetate membranes is advantageous for the isotachophoretic concentration of large sample volumes. The concentrated protein zone is then subjected to electrophoretic separation on the same strip. This first-dimensional separation including the concentrating process is named “concentrating electrophoresis.” Iso-electric focusing on several layers of cellulose acetate membrane is performed in the second-dimensional step. Many kinds of detection methods can be applied to the layers among which proteins are distributed. The novel two-dimensional electrophoresis takes only 5 h to perform.

Aphidicolin resistant mutant of which DNA polymerase α is induced by this drug

Abstract Mutants resistant to aphidicolin, a specific inhibitor of DNA polymerase α of eukaryotic cells, were selected from cultured FM3A cells, derived from mouse mammary carcinoma. One of them, designated as Aph 212, grew in the presence of 1 μg/ml of the drug, which did not permit wild type cells to grow. The resistance of Aph 212 cells to aphidicolin seems to be due to the increment of the activity of DNA polymerase α when Aph 212 cells were cultivated in the presence of the drug.

Possible involvement of DNA polymerases α and β in bleomycin-induced unscheduled DNA synthesis in permeable Hela cells

Abstract DNA polymerases involved in bleomycin-induced unscheduled DNA synthesis in some permeable human cells and rodent cells were studied by using selective inhibitors (aphidicolin, 2′,3′-dideoxythymidine-5′-triphosphate and N-ethylmaleimide) for DNA polymerases. The results suggest that both DNA polymerases α and β are involved in bleomycin-induced unscheduled DNA synthesis in permeable HeLa-S3 cells and probably in some other permeable human cells (HEp-2, KB and WI-38 VA-13 cells). Bleomycin-induced unscheduled DNA synthesis in some permeable rodent cells ( SR- C3H He , Balb c 3T3, 3Y1 and XC cells) is mostly attributed to DNA polymerase β.

Changes in fidelity levels of DNA polymerases α-1, α-2, and β during ageing in rats

Abstract DNA polymerases (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase EC 2.7.7.7.) were extracted from the regenerating livers of rats of various ages. The extracts were separated into three DNA polymerase fractions ( α -1, α -2, and β ) by phosphocellulose column chromatography, and their fidelity levels were then monitored with the synthetic template-primer, poly (dA-dT), poly dA-dT 10 , or poly dC-poly dG. The fidelity levels of the three DNA polymerases from regenerating liver of rats younger than 20 months were high, while those of DNA polymerases from rats older than 20 months were significantly lower with similar profiles on all three template-primers. On the other hand, the fidelity levels of enzymes from 23- and 26-month-old rats were similar. These results indicate that the levels of error-prone DNA polymerases increase rapidly in the regenerating liver of rats from ages 20 to 23 months. This may due to the amplification of DNA polymerase gene mutations by an error-prone enzyme itself. However, the cells in which mutations in the functional gene occur may undergo cell death because the fidelity levels of the DNA polymerases in the older animals did not increase.

Age-associated changes in the template-reading fidelity of DNA polymerase α from regenerating rat liver

Abstract DNA polymerases (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase EC 2.7.7.7.) were extracted from regenerating livers from young and aged rats. DNA polymerase α was separated and partially purified by DEAE-cellulose column chromatography, polyethyleneglycol precipitation, and phosphocellulose column chromatography, and fidelity levels were then monitored with the synthetic template-primer poly (dG-dC). The fidelity level of the DNA polymerase from regenerating liver of a 4-month-old rat was very high, while that of the DNA polymerase from a 24-month-old rat was significantly decreased. To confirm this result, DNA was synthesized on poly (dG-dC) in a reaction mixture containing [ 32 P]dTTP, and the synthetic polynucleotide was purified and digested with Hha I restriction endonuclease. After hydrolysis, the oligonucleotides were developed by two dimensional thin layer chromatography on PEI cellulose plates. Spots containing [ 32 P]dTMP were observed when DNA polymerase from a 24 month-old rat was used, but none was found in polynucleotides synthesized using DNA polymerase from a 4 month-old rat. Nearest neighbor analysis suggested that dG-dT and dC-dT pairs were constructed by mis-incorporation due to DNA polymerase α .

Differences in DNA synthesis in vitro using isolated nuclei from regenerating livers of young and aged rats.

To detect changes in DNA synthesis during ageing, we compare DNA synthesis in the livers of young and aged rats. As an intermediate between an in vivo system using intact cells and an in vitro system using purified DNA polymerases, isolated nuclei were prepared and used as the machinery for DNA synthesis. The DNA synthesizing capacity of nuclei from regenerating liver was higher than that of nuclei from normal liver and these capacities from liver and regenerating liver were lower in nuclear preparations from aged rats. DNA synthesis using isolated nuclei was stimulated by ATP and the cytoplasmic preparation. The cytoplasmic preparation from regenerating rat liver was found to stimulate DNA synthesis more than the preparation from normal liver. The activity in regenerating liver from young rats was also greater than in that from aged rats. It is well known that DNA replication is inhibited by aphidicolin and DNA repair by ddTTP. We examined the effects of aphidicolin and ddTTP on DNA synthesis using the nuclear system. Surprisingly, the inhibition by aphidicolin was 30% of total DNA synthesis using the nuclear system from young rats. On the other hand, the inhibition by ddTTP was approximately 80%. We measured the sizes of the DNA synthesized in the presence of both inhibitors. DNA synthesis was allowed to proceed for 10 min using isolated nuclei from regenerating liver of young rats and the size of the DNA was determined by sucrose density gradient centrifugation analysis. DNA products appeared in two fractions. Following a chase of 50 min in the presence or absence of aphidicolin, the short DNA product grew larger in both cases, although the amount of DNA in the presence of aphidicolin was approximately 90% that in its absence. In the same experiment using nuclei from aged rats, the amount in the presence of aphidicolin was approximately 60% that in its absence. These results suggest that DNA polymerase beta is closely related to abnormal replication when DNA polymerases alpha and delta are inhibited and that the effect of cytosol on DNA synthesis, as well as the DNA synthetic capacity of isolated nuclei, becomes lower in regenerating rat liver during ageing.

Enzyme-linked immunodetection of proteins on Coomassie blue-stained two-dimensional cellulose acetate membranes

Cellulose acetate membrane on which proteins are separated by two-dimensional electrophoresis and visualized by Coomassie staining is directly subjected to an enzyme-linked immunodetection method using a horseradish peroxidase-conjugated second antibody. The Coomassie-stained and immunochemically stained spots are distinguishable by their colors, such as blue with Coomassie blue staining and purple with horseradish peroxidase staining. Two kinds of lighting make the distinction more apparent. Coomassie-stained protein patterns are clear in the transmitted light. However, the immunochemically stained protein spots are obvious in the reflected light, in which Coomassie-stained patterns are obscure. The procedures do not require proteins to transfer onto nitrocellulose paper in contrast to proteins in polyacrylamide gel. The simple procedure excludes the nonspecific binding of the first and second antibodies to blocking proteins such as bovine serum albumin or gelatin because blocking protein is not used. With this method, matching of the Coomassie blue-stained spot with the immunochemically stained spot is done accurately and easily.