The mode of action by aphidicolin on DNA polymerase a from the nuclear fraction of seaurchin blastulae was studied. The inhibition of DNA polymerase a by aphidicolin was uncompetitive with activated DNA and competitive with the four deoxynucleoside triphosphates using activated DNA as a template-primer. For truncated (residual or limited) DNA synthesis with only three deoxynucleoside triphosphates, aphidicolin inhibited the residual synthesis more strongly in the absence of dCTP than in the absence of each of the other three deoxynucleoside triphosphates. The inhibition was reversed with excess dCTP but not with the other three deoxynucleoside triphosphates. That is, aphidicolin inhibited DNA polymerase a by competing with dCTP with a K , value of 0.5 pg/ml and by not competing with the other three deoxynucleoside triphosphates. dTMP incorporation with the activated DNA was more sensitive to aphidicolin than dGMP or dTMP incorporation with poly(dC) . (dG)12-~8 or poly(dA) . (dT)12-18. Similar resuIts were obtained for DNA polymerase a (B form) from mouse myeloma MOPC 104E.We have previously reported that aphidicolin, a tetracyclic diterpene-tetraol [1,2], inhibits mitotic division of sea-urchin embryos but not meiotic maturational divisions of starfish oocytes [3,4]. Among the macromolecular syntheses that we have examined in vivo, only DNA synthesis is sensitive to aphidicolin [3]. Of the three DNA polymerase species in seaurchin embryos [6-81, only DNA polymerase x is sensitive to aphidicolin at a dose similar to that which inhibits mitosis. DNA polymerases fl and y are resistant to extremely high doses of aphidicolin [3]. It has been reported that rat liver DNA polymerase a is sensitive to aphidicolin but not DNA polymerases fl and y [5]. From these results, we have concluded that DNA polymerase a is the replicative enzyme [3]. In this paper we report that inhibition of DNA polymerase x activity by aphidicolin is due to competition with dCTP.

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Approximately 2,500-fold purifications of DNA polymerase-beta from the nuclear fraction of blastulae of the sea urchin, Hemicentrotus pulcherrimus, was performed. The enzyme preparation, which was devoid of DNase and terminal deoxynucleotidyl transferase as contaminants, showed a sedimentation constant of 3.0 S in a sucrose density gradient, a molecular weight of 50,000 by gel filtration, and an isoelectric point of pH 8.1. The enzyme activity was resistant to sulfhydryl group inhibitors. Its optimal pH was 9.0-9.5 in Tris-maleate buffer and 10.0 in glycine buffer. The optimal NaCl concentration for the activity was 30-60 mM and about half of the activity remained at 0.4 M NaCl. As a template-primer, the enzyme preferred synthetic homopolymers to activated DNA. The order of this preference was as follows; poly (dA)-oligo (dT)12-18 greater than poly (rA)-oligo (dT)12-18 greater than activated DNA. The above results indicate that the enzyme corresponds to DNA polymerase-beta from vertebrate cells.

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Cyclic guanosine 3',5'-monophosphate (cGMP) was injected into fertilized starfish eggs in order to study the effects of this cyclic nucleotide on early embryogenesis. cGMP at approximate intracellular concentrations higher than 0.05 mM causes achromosomal cleavage in the presence of 1 mM caffeine, that is, each blastomere is devoid of a nucleus but not spindle and asters. The treated embryos cleave slower than control, never form blastulae, and disintegrate just before blastulation. CAMP, dibutyryl cGMP and dibutyryl CAMP failed to cause achromosomal cleavage. These results suggest that unphysiologically high levels of intracellular cGMP inhibit in vivo DNA synthesis of fertilized starfish eggs.Aphidicolin is an inhibitor of eukaryotic DNA polymerase CY and inhibits replicative DNA synthesis in eukaryotes (lkegami et al., '78; Oguro et al., '79; Wist and Prydz, '79; Huberman, '81). Fertilized echinoderm eggs whose replication is inhibited by this drug cleave achromosomally (Nagano et al., '81; Yamada et al., '85; Hirai et al., '84), i.e., each blastomere is devoid of a nucleus or chromosomes but not spindle and asters. AphidiColin-treated embryos never form blastulae and disintegrate before blastulation.It has been reported that cGMP inhibits the DNA polymerase CY activity of rat liver and yeast (Eckstein, '81). So, we investigated the effects of cGMP on early embryogenesis of the starfish by the microinjection method. The results show that cGMP also caused achromosomal cleavage in fertilized starfish eggs. MATERIALS AND METHODSThe starfish, Asterina pectinifera, were collected during their breeding season at Hashirimizu (Kanagawa) and Asamushi (Aomori) and were kept in laboratory aquaria supplied with running sea water. Maturing oocytes were obtained from ovarian fragments by treatment with M 1-methyladenine (1-MeAde). 1-MeAde (Sigma, USA) was dissolved in distilled water at a concentration of 5 mM and diluted with sea water before use. Spermatozoa were obtained from isolated testis and diluted with sea water. Histidine was added to the sperm sus ension to give a final concentration of lo-' M in order to increase their motility. The oocytes were inseminated in normal sea water 40 min after 1-MeAde treatment and allowed to develop at 20°C. Fertilized eggs were transferred to sea water containing 1 mM caffeine when the first polar body was expelled (65 min after the 1-MeAde treatment). In experiments involving microinjection of reagents into fertilized eggs, injection was carried out in the period between the appearance of the first and the second polar bodies (Fig. 1). The injected oocytes were allowed to develop in the presence of 1 mM caffeine at 20°C. Microinjection was carried out according to the method of Hiramoto ('74). The amount of reagent solutions injected into an oocyte was 100 pl. This volume is approximately 1/40Address reprint requests to