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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.

We have recently found that aphidicolin, a tetracyclic diterpene-tetraol produced by several fungi, blocks DNA synthesis of sea urchin embryos by interfering with the activity of DNA polyermase alpha. These cells fail to proliferate in the presence of aphidicolin. In continuation of these studies, we determined the drug-sensitive stage in the first cell cycle of the sea urchin Clypeaster japonicus embryo. In continuous exposure to aphidicolin (2 micrograms/ml) from five minutes after fertilization, mitotic division of the embryo was completely suppressed. Embryos were exposed to the drug at progressively later intervals and their capability for cytokinesis was examined. Evidence was thereby obtained that aphidicolin acts at the S-period to inhibit DNA synthesis resulting in developmental arrest of the embryo.

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