Masaki Shioda

The mode of action of aphidicolin on DNA synthesis in isolated nuclei

Abstract Aphidicolin (a specific inhibitor of DNA polymerase-α) inhibited DNA synthesis in isolated nuclei from sea urchin embryos but ddTTP (an inhibitor of DNA polymerases-β and -γ) did not, indicating that DNA polymerase-α was responsible for DNA synthesis in isolated nuclei. DNA synthesis in isolated nuclei was inhibited by aphidicolin noncompetitively with respect to each of dNTPs indicating that properties of in situ DNA polymerase activity in isolated nuclei are different from those of the purified DNA polymerase-α which was inhibited by aphidicolin competitively with respect to dCTP and noncompetitively with respect to the other 3 dNTPs. Similar results were obtained using HeLa cell nuclei.

Selective inhibition of DNA polymerase α by a polysaccharide purified from slime of Physarumpolycephalum

Abstract A polysaccharide was purified from the slime of a myxomycete, Physarum polycephalum , and its inhibitory effect on eukaryotic DNA polymerases was examined. Almost all the calf thymus DNA polymerase α activity was inhibited with higher than 0.2 mg/ml of the polysaccharide, when the assay was carried out with activated DNA as a template. The inhibitory effect occurred regardless of the amounts of the enzyme and deoxyribonucleotides, however, kinetic analysis revealed that the inhibition occurs competitively with the template DNA, the K i value being 4 μg/ml. Inhibition was observed for DNA polymerase α, but not for DNA polymerases β and γ from various eukaryote species.

Cytoplasmic location of DNA polymerase-α and -β of sea urchin eggs

Abstract Unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus , were quantitatively separated into nucleate and anucleate halves by isopycnic centrifugation. Approximately 32 % of DNA polymerase-α and 34 % of DNA poly-merase-β activities were found in the anucleate halves. From the diameters, the volume of the anucleate half was calculated to be 24.5 % that of the egg. It is concluded that nearly all DNA polymerase-α and -β are localized in the cytoplasm and it is suggested that DNA polymerases are bound to a cytoplasmic granule.

Transition of DNA polymerase-α and endoplasmic reticulum during gastrulation of the sea urchin

Abstract Changes in the activity of DNA polymerase-α and in subcellular distribution were studied during gastrulation of the sea urchin, Hemicentrotus pulcherrimus . Although the activity of DNA polymerase-α for each embryo was constant up to the blastula stage as reported previously, the enzyme activity increased during gastrulation by about twofold prior to an increase in its DNA content. Thereafter the enzyme activity remained constant at a high level until the early pluteus stage. During gastrulation, an increase in the fraction of DNA polymerase-α was associated with the rough endoplasmic reticulum. During the period between the gastrula and pluteus stages, the cytoplasmic DNA polymerase-α activity decreased gradually with a concomitant increase of activity in the nucleus fraction. The timing of this increase in the nucleus coincided with the increase of DNA content per embryo. These results suggest that DNA polymerase-α accumulates on the rough endoplasmic reticulum during gastrulation and then translocates to the nucleus for DNA synthesis as seen before the blastula stage. DNA polymerase-α obtained from gastrula nuclei did not associate with the endoplasmic reticulum from gastrulae. DNA polymerase-α obtained from the gastrula endoplasmic reticulum membranes became bound to the salt-washed membranes from gastrulae but not to those from unfertilized eggs. Likewise, DNA polymerase-α from the rough endoplasmic reticulum of unfertilized eggs became attached to salt-washed membranes from unfertilized eggs, but not to those from gastrulae. This suggests that DNA polymerase-α is synthesized anew, and a transition of both DNA polymerase-α and endoplasmic reticulum occurs at the gastrula stage.

Differential inhibition of eukaryotic DNA polymerases by halenaquinol sulfate, a p-hydroquinone sulfate obtained from a marine sponge

Halenaquinol sulfate, a p‐hydroquinone sulfate obtained from a marine sponge, inhibited the activity of eukaryotic DNA polymerases in varying degrees; the K i values for DNA polymerases α, β, δ and ε were 1.3, 80, 17.5 and 2.0 νM, respectively, whereas it was less effective against E. coli DNA polymerase I. The inhibition occurred competitively with each of dATP and dTTP, but non‐competitively with dCTP, dGTP and the template DNA. Thus, halenaquinol sulfate is demonstrated to be a potential inhibitor of DNA polymerases α and ε, and be a useful tool for analysing the DNTP binding sites of DNA polymerases.

Microtubule-associated protein-2 stimulates DNA synthesis catalyzed by the nuclear matrix

Microtubule-associated protein-2 (MAP-2) isolated from porcine brains stimulated DNA synthesis catalyzed by the nuclear matrix isolated from Physarum polycephalum in the presence of activated DNA as exogenous templates. The degree of the stimulation depended on the amount of the nuclear matrix, but not on that of the template. MAP-2 also stimulated DNA polymerase alpha activity solubilized from nuclei, but not DNA polymerase beta activity. These results suggest that MAP-2 stimulates DNA synthesis by interacting with the putative DNA replication machinery including DNA polymerase alpha bound to the matrix. Similar stimulation occurred in the nuclear matrix isolated from HeLa and rat ascites hepatoma cells, which strongly suggests that MAP-2 is involved in the control of DNA replication in eukaryotic cells.

Localization of DNA polymerase α on the nuclear membrane in sea urchin embryos

Subnuclear localization of DNA polymerase alpha was studied in sea urchin embryos. Blastula nuclei treated with EDTA and potassium phosphate released subnuclear components bearing most of the nuclear DNA polymerase alpha. These components were suggested to be a part of nuclear membrane based on their buoyant densities (1.177 and 1.136 g/cm3) in isopyknic centrifugation and the nuclear pore-like structure. Contamination with DNA and endoplasmic reticulum membrane to the subnuclear components was shown to be negligible. These results suggested that DNA polymerase alpha associates with nuclear membrane of sea urchin embryos. Nuclear membrane deprived of DNA polymerase alpha was able to associate with nuclear DNA polymerase alpha from blastulae and the cytoplasmic enzyme of unfertilized eggs efficiently, but not with the cytoplasmic enzyme of gastrulae. This result suggests that the nuclear membrane is originates from the endoplasmic reticulum with which DNA polymerase alpha associates in unfertilized eggs.

Initiation of DNA synthesis in a high molecular weight fraction of Xenopus egg extract.

Xenopus egg extract was fractionated by gel‐filtration column chromatography and DNA synthetic activity was examined using double‐stranded DNA as a template. The major activity eluted had an apparent molecular mass of about 300 kDa. Product analyses showed that de novo DNA synthesis occurs with formation of replication bubbles, thereby suggesting that this fraction catalyzes the initiation of DNA replication. Activities of DNA polymerase α‐primase and DNA helicase overlapped with the DNA synthetic activity, but the elution profiles of the enzymes differed from that of the DNA synthetic activity. Therefore, this 300‐kDa fraction may contain a component which differs from the above enzymes yet is essential for initiation of DNA replication.

Effects of Okadaic Acid on Embryonic Development of the Starfish Asterina pectinifera

The effects of okadaic acid, an inhibitor of protein phosphatases types 1 and 2A, from the sponge Halichondria okadai Kadota, on the embryonic development of the starfish Asterina pectinifera, were investigated. When cultured in okadaic acid from fertilization, the embryo divided synchronously without any abnormalities such as lysis, swelling or morphological changes different from control embryos up to the 32-cell stage. However, okadaic acid prevented development before the onset of blastulation. Cytological examination showed that chromosomes condensed but did not align in a plane in the mitotic apparatus in each of the blastomeres of the treated embryo at the sixth cleavage, suggesting that this was the root cause of the arrest of further development.

Subcellular localization of DNA polymerase γ and changes in its activity in sea urchin embryos

Abstract 1. 1. Subcellular localization and changes in the activity of DNA polymerase γ were examined in sea urchin eggs and embryos. 2. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopyenic centrifugation. 3. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. 5. The γ-activity per mitochondrial DNA remained constant during embryogenesis. 6. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase γ) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.