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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
. 1982 Jul;79(14):4243–4247. doi: 10.1073/pnas.79.14.4243

Purification of the cdc8 protein of Saccharomyces cerevisiae by complementation in an aphidicolin-sensitive in vitro DNA replication system.

C L Kuo, J L Campbell
PMCID: PMC346646  PMID: 6812044

Abstract

DNA synthesis in vitro in Brij-treated Saccharomyces cerevisiae requires the product of the CDC8 gene (Hereford, L. M. & Hartwell, L. H. (1971) Nature (London) New Biol. 234, 171-172). Extracts of wild-type A364a yeast restore DNA synthesis in Brij-treated cdc8, a mutant containing a thermolabile cdc8 gene product. This constitutes a complementation assay by which the cdc8 gene product can be monitored during purification. A heat-stable protein responsible for this complementation has been partially purified from both wild-type A364a cells and from a cdc8 temperature-sensitive mutant. The complementation activity from the mutant is thermolabile when compared to the wild-type activity, indicating that CDC8 is the structural gene for the protein.

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Selected References

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

  1. Banks G. R. Mitochondrial DNA synthesis in permeable cells. Nat New Biol. 1973 Oct 17;245(146):196–199. doi: 10.1038/newbio245196a0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Hartwell L. H. Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. J Mol Biol. 1971 Jul 14;59(1):183–194. doi: 10.1016/0022-2836(71)90420-7. [DOI] [PubMed] [Google Scholar]
  4. Hartwell L. H. Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J Bacteriol. 1973 Sep;115(3):966–974. doi: 10.1128/jb.115.3.966-974.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hereford L. M., Hartwell L. H. Defective DNA synthesis in permeabilized yeast mutants. Nat New Biol. 1971 Dec 8;234(49):171–172. doi: 10.1038/newbio234171a0. [DOI] [PubMed] [Google Scholar]
  6. Hinkle D. C., Richardson C. C. Bacteriophage T7 deoxyribonucleic acid replication in vitro. Requirements for deoxyribonucleic acid synthesis and characterization of the product. J Biol Chem. 1974 May 10;249(9):2974–2980. [PubMed] [Google Scholar]
  7. Klein H. L., Byers B. Stable denaturation of chromosomal DNA from Saccharomyces cerevisiae during meiosis. J Bacteriol. 1978 May;134(2):629–635. doi: 10.1128/jb.134.2.629-635.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Meyer R. R., Glassberg J., Kornberg A. An Escherichia coli mutant defective in single-strand binding protein is defective in DNA replication. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1702–1705. doi: 10.1073/pnas.76.4.1702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Modrich P., Richardson C. C. Bacteriophage T7 Deoxyribonucleic acid replication in vitro. A protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity. J Biol Chem. 1975 Jul 25;250(14):5508–5514. [PubMed] [Google Scholar]
  12. Moses R. E. Replicative deoixyribonucleic acid synthesis in a system diffusible for macromolecules. J Biol Chem. 1972 Oct 10;247(19):6031–6038. [PubMed] [Google Scholar]
  13. Moses R. E., Richardson C. C. Replication and repair of DNA in cells of Escherichia coli treated with toluene. Proc Natl Acad Sci U S A. 1970 Oct;67(2):674–681. doi: 10.1073/pnas.67.2.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Newlon C. S., Fangman W. L. Mitochondrial DNA synthesis in cell cycle mutants of Saccharomyces cerevisiae. Cell. 1975 Aug;5(4):423–428. doi: 10.1016/0092-8674(75)90061-6. [DOI] [PubMed] [Google Scholar]
  15. Petes T. D., Newlon C. S. Structure of DNA in DNA replication mutants of yeast. Nature. 1974 Oct 18;251(5476):637–639. doi: 10.1038/251637a0. [DOI] [PubMed] [Google Scholar]
  16. Plevani P., Badaracco G., Ginelli E., Sora S. Effect and mechanism of action of aphidicolin on yeast deoxyribonucleic acid polymerases. Antimicrob Agents Chemother. 1980 Jul;18(1):50–57. doi: 10.1128/aac.18.1.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Prakash L., Hinkle D., Prakash S. Decreased UV mutagenesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae. Mol Gen Genet. 1979;172(3):249–258. doi: 10.1007/BF00271724. [DOI] [PubMed] [Google Scholar]
  18. Weiner J. H., Bertsch L. L., Kornberg A. The deoxyribonucleic acid unwinding protein of Escherichia coli. Properties and functions in replication. J Biol Chem. 1975 Mar 25;250(6):1972–1980. [PubMed] [Google Scholar]
  19. Wintersberger U., Hirsch J., Fink A. M. Studies on nuclear and mitochondrial DNA-replication in a temperature-sensitive mutant of Saccharomyces cerevisiae. Mol Gen Genet. 1974;131(4):291–299. doi: 10.1007/BF00264860. [DOI] [PubMed] [Google Scholar]

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