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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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ku, Tokyo I I3Aphidicolin at 2 pg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I-cell stage. However, at 40 ,ug/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 ,ug/ml it caused 90% inhibition of incorpo ration of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 pg/ml it also caused 90% inhibition of the activity of DNA polymerase a, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase , B or 7. These findings suggest that DNA polymerase a is responsible for replication of DNA in sea urchin embryos.Much of our knowledge of cellular events governed by DNA replication has been obtained by experiments using inhibitors of cellular DNA synthesis, such as arabinosyl nucleosides, N-hydroxyurea and 5-fluorouracil. However, conclusions drawn from these experiments can hardly be considered as other than suggestive, or corroborative of data obtained by other means, because arabinosyl nucleosides have undesirable effects aside from blocking DNA synthesis, and N-hydroxyurea and 5-fluorouracil do not inhibit DNA synthesis directly, but prevent deoxyribonucleoside triphosphate biosynthesis, thereby depriving DNA polymerases of necessary substrates (5).In an attempt to find chemicals that directly and selectively block DNA synthesis, we have searched for chemicals that prevent mitotic cell division of sea urchin embryos, which requires DNA synthesis (32), but do not prevent meiotic maturational divisions of starfish oocytes, which are independent of DNA synthesis (37). We found that aphidicolin (3, 6), a tetracyclic diterpene-tetrol, is one such compound (1 6). Furthermore, our recent experiments have shown that aphidicolin inhibits the activity of DNA polymerase c1 obtained from regenerating rat liver without affecting the activity of DNA polymerase p or mitochondria1 DNA polymerase (23).The functional role of the nuclear DNA polymerases in the complex process of DNA replication is not known. Correlative studies of DNA synthesis and the level of DNA polymerase activity suggest that DNA polymerase c1 may be important in DNA replication (35).However, in certain cases, DNA polymerases fl and y also increase at the time of DNA synthesis, and attempts to specify which of the DNA polymerases is replicative and to assign roles to the various polymerases have not yet been successful (35). 119