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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1980 Dec;77(12):7049–7053. doi: 10.1073/pnas.77.12.7049

DNA polymerase alpha mutants from a Drosophila melanogaster cell line.

A Sugino, K Nakayama
PMCID: PMC350438  PMID: 6784118

Abstract

Aphidicolin, a tetracyclic diterpenoid antibiotic, is a specific inhibitor of DNA synthesis in vivo and DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) alpha of eukaryotic organisms. After ethyl methanesulfonate mutagenesis, we have recovered mutants of Drosophila melanogaster Schneider cell line no. 2 that grow at concentrations of aphidicolin that completely inhibit wild-type cells. The DNA polymerase alpha from one of these mutants, aph-10, is much more resistant to inhibition by the drug; the apparent Ki of the wild-type enzyme is 12 nM aphidicolin, whereas the apparent Ki of the aph-10 polymerase is more than 100 nM. (The apparent Km for dCTP is the same for both enzymes.) Another mutant, aph-13, overproduces DNA polymerase alpha at least 8-fold. The DNA polymerase of this mutant has the same apparent Km and Ki for dNTPs and aphidicolin as does wild-type polymerase.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arens M., Yamashita T., Padmanabhan R., Tsuruo T., Green M. Adenovirus deoxyribonucleic acid replication. Characterization of the enzyme activities of a soluble replication system. J Biol Chem. 1977 Nov 25;252(22):7947–7954. [PubMed] [Google Scholar]
  2. Ayusawa D., Iwata K., Kozu T., Ikegami S., Seno T. Increase in dATP pool in aphidicolin-resistant mutants of mouse FM3A cells. Biochem Biophys Res Commun. 1979 Dec 14;91(3):946–954. doi: 10.1016/0006-291x(79)91971-5. [DOI] [PubMed] [Google Scholar]
  3. Banks G. R., Boezi J. A., Lehman I. R. A high molecular weight DNA polymerase from Drosophila melanogaster embryos. Purification, structure, and partial characterization. J Biol Chem. 1979 Oct 10;254(19):9886–9892. [PubMed] [Google Scholar]
  4. Brakel C. L., Blumenthal A. B. Multiple forms of Drosophila embryo DNA polymerase: evidence for proteolytic conversion. Biochemistry. 1977 Jul 12;16(14):3137–3143. doi: 10.1021/bi00633a016. [DOI] [PubMed] [Google Scholar]
  5. Bucknall R. A., Moores H., Simms R., Hesp B. Antiviral effects of aphidicolin, a new antibiotic produced by Cephalosporium aphidicola. Antimicrob Agents Chemother. 1973 Sep;4(3):294–298. doi: 10.1128/aac.4.3.294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cozzarelli N. R. The mechanism of action of inhibitors of DNA synthesis. Annu Rev Biochem. 1977;46:641–668. doi: 10.1146/annurev.bi.46.070177.003233. [DOI] [PubMed] [Google Scholar]
  7. Hanaoka F., Kato H., Ikegami S., Oashi M., Yamada M. Aphidicolin does inhibit repair replication in HeLa cells. Biochem Biophys Res Commun. 1979 Mar 30;87(2):575–580. doi: 10.1016/0006-291x(79)91833-3. [DOI] [PubMed] [Google Scholar]
  8. Ikegami S., Taguchi T., Ohashi M., Oguro M., Nagano H., Mano Y. Aphidicolin prevents mitotic cell division by interfering with the activity of DNA polymerase-alpha. Nature. 1978 Oct 5;275(5679):458–460. doi: 10.1038/275458a0. [DOI] [PubMed] [Google Scholar]
  9. Knopf K. W., Yamada M., Weissbach A. HeLa cell DNA polymerase gamma: further purification and properties of the enzyme. Biochemistry. 1976 Oct 5;15(20):4540–4548. doi: 10.1021/bi00665a032. [DOI] [PubMed] [Google Scholar]
  10. Krokan H., Schaffer P., DePamphilis M. L. Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid. Biochemistry. 1979 Oct 2;18(20):4431–4443. doi: 10.1021/bi00587a025. [DOI] [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Nishimura M., Yasuda H., Ikegami S., Ohashi M., Yamada M. Aphidicolin resistant mutant of which DNA polymerase alpha is induced by this drug. Biochem Biophys Res Commun. 1979 Dec 14;91(3):939–945. doi: 10.1016/0006-291x(79)91970-3. [DOI] [PubMed] [Google Scholar]
  14. Oguro M., Suzuki-Hori C., Nagano H., Mano Y., Ikegami S. The mode of inhibitory action by aphidicolin on eukaryotic DNA polymerase alpha. Eur J Biochem. 1979 Jul;97(2):603–607. doi: 10.1111/j.1432-1033.1979.tb13149.x. [DOI] [PubMed] [Google Scholar]
  15. Ohashi M., Taguchi T., Ikegami S. Aphidicolin: a specific inhibitor of DNA polymerases in the cytosol of rat liver. Biochem Biophys Res Commun. 1978 Jun 29;82(4):1084–1090. doi: 10.1016/0006-291x(78)90298-x. [DOI] [PubMed] [Google Scholar]
  16. Pedrali-Noy G., Mazza G., Focher F., Spadari S. Lack of mutagenicity and metabolic inactivation of aphidicolin by rat liver microsomes. Biochem Biophys Res Commun. 1980 Apr 29;93(4):1094–1103. doi: 10.1016/0006-291x(80)90601-4. [DOI] [PubMed] [Google Scholar]
  17. Schaller H., Nüsslein C., Bonhoeffer F. J., Kurz C., Nietzschmann I. Affinity chromatography of DNA-binding enzymes on single-stranded DNA-agarose columns. Eur J Biochem. 1972 Apr 24;26(4):474–481. doi: 10.1111/j.1432-1033.1972.tb01789.x. [DOI] [PubMed] [Google Scholar]
  18. Schimke R. T., Kaufman R. J., Alt F. W., Kellems R. F. Gene amplification and drug resistance in cultured murine cells. Science. 1978 Dec 8;202(4372):1051–1055. doi: 10.1126/science.715457. [DOI] [PubMed] [Google Scholar]
  19. Schneider I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 1972 Apr;27(2):353–365. [PubMed] [Google Scholar]
  20. Sheinin R., Humbert J. Some aspects of eukaryotic DNA replication. Annu Rev Biochem. 1978;47:277–316. doi: 10.1146/annurev.bi.47.070178.001425. [DOI] [PubMed] [Google Scholar]
  21. Wahl G. M., Padgett R. A., Stark G. R. Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells. J Biol Chem. 1979 Sep 10;254(17):8679–8689. [PubMed] [Google Scholar]
  22. Weissbach A. Eukaryotic DNA polymerases. Annu Rev Biochem. 1977;46:25–47. doi: 10.1146/annurev.bi.46.070177.000325. [DOI] [PubMed] [Google Scholar]
  23. Wist E., Prydz H. The effect of aphidicolin on DNA synthesis in isolated HeLa cell nuclei. Nucleic Acids Res. 1979 Apr;6(4):1583–1590. doi: 10.1093/nar/6.4.1583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yoshida S., Yamada M., Masaki S. Inhibition of DNA polymerase-alpha and -beta of calf thymus by 1-beta-D-arabinofuranosylcytosine-5'-triphosphate. Biochim Biophys Acta. 1977 Jul 15;477(2):144–150. doi: 10.1016/0005-2787(77)90230-1. [DOI] [PubMed] [Google Scholar]

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