Skip to main content
NCBI home page
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 Jun;79(11):3428–3432. doi: 10.1073/pnas.79.11.3428

Protein complexes from active replicative fractions associate in vitro with the replication origins of yeast 2-micrometers DNA plasmid.

S M Jazwinski, G M Edelman
PMCID: PMC346433  PMID: 7048304

Abstract

In a search for a replication complex, the activity that replicates the 2-micrometers yeast DNA plasmid in vitro was isolated in a high molecular weight form (Mr approximately 2 X 10(6) by gel filtration and rate-zonal sedimentation from extracts prepared from cells of the budding yeast Saccharomyces. When obtained from cells in late logarithmic cultures this material or "complex" was labile compared to that from early logarithmic cultures, and it did not survive as a complex after ammonium sulfate precipitation. This suggests that, as cultures approach stationary phase and cells cease growth, the association of its protein constituents may be altered. A chimera of 2-micrometers DNA inserted into the plasmid pBR322 was used to test for binding of components of the complex. After a brief incubation of the chimera in vitro with the high molecular weight material containing replicating activity, a protein "knob" was found associated with the 2-micrometers DNA as shown by electron microscopy. This association was not random but was limited to two positions on the plasmid. In the same series of experiments, the in vitro origins of 2-micrometers plasmid replication were also mapped. Two origins were found, consistent in position with those that have been identified in vivo. Molecules utilizing both origins simultaneously in vitro were not observed, and replication in vitro was bidirectional. The location of the origins corresponded to the positions at which the protein knobs associated with 2-micrometers DNA. This and the fact that no replicative intermediates with associated complexes were detected raises the possibility that a specific protein complex may be involved in initiation of DNA replication.

Full text

PDF
3428

Images in this article

3430Image
on p.3430

Selected References

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

  1. Broach J. R., Hicks J. B. Replication and recombination functions associated with the yeast plasmid, 2 mu circle. Cell. 1980 Sep;21(2):501–508. doi: 10.1016/0092-8674(80)90487-0. [DOI] [PubMed] [Google Scholar]
  2. Hartley J. L., Donelson J. E. Nucleotide sequence of the yeast plasmid. Nature. 1980 Aug 28;286(5776):860–865. doi: 10.1038/286860a0. [DOI] [PubMed] [Google Scholar]
  3. Hartwell L. H., Culotti J., Pringle J. R., Reid B. J. Genetic control of the cell division cycle in yeast. Science. 1974 Jan 11;183(4120):46–51. doi: 10.1126/science.183.4120.46. [DOI] [PubMed] [Google Scholar]
  4. Jazwinski S. M., Edelman G. M. Acitivity of yeast extracts in cell-free stimulation of DNA replication. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3933–3936. doi: 10.1073/pnas.73.11.3933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jazwinski S. M., Edelman G. M. Replication in vitro of the 2-micrometer DNA plasmid of yeast. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1223–1227. doi: 10.1073/pnas.76.3.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Johnston G. C., Pringle J. R., Hartwell L. H. Coordination of growth with cell division in the yeast Saccharomyces cerevisiae. Exp Cell Res. 1977 Mar 1;105(1):79–98. doi: 10.1016/0014-4827(77)90154-9. [DOI] [PubMed] [Google Scholar]
  7. Kojo H., Greenberg B. D., Sugino A. Yeast 2-micrometer plasmid DNA replication in vitro: origin and direction. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7261–7265. doi: 10.1073/pnas.78.12.7261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Livingston D. M., Hahne S. Isolation of a condensed, intracellular form of the 2-micrometer DNA plasmid of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3727–3731. doi: 10.1073/pnas.76.8.3727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Livingston D. M., Kupfer D. M. Control of Saccharomyces cerevisiae 2microN DNA replication by cell division cycle genes that control nuclear DNA replication. J Mol Biol. 1977 Oct 25;116(2):249–260. doi: 10.1016/0022-2836(77)90215-7. [DOI] [PubMed] [Google Scholar]
  10. Nelson R. G., Fangman W. L. Nucleosome organization of the yeast 2-micrometer DNA plasmid: a eukaryotic minichromosome. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6515–6519. doi: 10.1073/pnas.76.12.6515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Petes T. D., Williamson D. H. Replicating circular DNA molecules in yeast. Cell. 1975 Mar;4(3):249–253. doi: 10.1016/0092-8674(75)90172-5. [DOI] [PubMed] [Google Scholar]
  12. Prem veer Reddy G., Pardee A. B. Multienzyme complex for metabolic channeling in mammalian DNA replication. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3312–3316. doi: 10.1073/pnas.77.6.3312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Scott J. H., Schekman R. Lyticase: endoglucanase and protease activities that act together in yeast cell lysis. J Bacteriol. 1980 May;142(2):414–423. doi: 10.1128/jb.142.2.414-423.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Taketo M., Jazwinski S. M., Edelman G. M. Association of the 2-micron DNA plasmid with yeast folded chromosomes. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3144–3148. doi: 10.1073/pnas.77.6.3144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Zakian V. A., Brewer B. J., Fangman W. L. Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase. Cell. 1979 Aug;17(4):923–934. doi: 10.1016/0092-8674(79)90332-5. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES