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. 1983 Nov;80(21):6465–6469. doi: 10.1073/pnas.80.21.6465

Isolation of yeast DNA replication mutants in permeabilized cells.

C Kuo, H Nuang, J L Campbell
PMCID: PMC390134  PMID: 6356128

Abstract

A random population of temperature-sensitive mutants was screened by assaying for defects in DNA synthesis in a permeabilized yeast DNA replication system. Twenty mutants defective in in vitro DNA synthesis have been isolated. In this paper we describe eight of these mutants. Seven of them fall into three complementation groups--cdc2, cdc8, and cdc16--involved in the control of the cell-division cycle. Because synthesis in vitro represents propagation of replication forks active in vivo at the time of permeabilization, our finding that cdc2 and cdc16 mutants can incorporate dTMP into DNA in such permeabilized cells at 23 degrees C but not at 37 degrees C supports the conclusion that these two mutations directly affect DNA synthesis at replication forks. Such an involvement was previously suggested by in vivo analysis for CDC2 but was less clear for CDC16. Finally, the usefulness of our screening procedure is demonstrated by the isolation of replication mutants in previously undescribed complementation groups. One strain shows a serious defect in in vivo DNA synthesis but normal RNA synthesis.

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Selected References

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

  1. Arendes J., Kim K. C., Sugino A. Yeast 2-microns plasmid DNA replication in vitro: purification of the CDC8 gene product by complementation assay. Proc Natl Acad Sci U S A. 1983 Feb;80(3):673–677. doi: 10.1073/pnas.80.3.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bisson L., Thorner J. Thymidine 5'-monophosphate-requiring mutants of Saccharomyces cerevisiae are deficient in thymidylate synthetase. J Bacteriol. 1977 Oct;132(1):44–50. doi: 10.1128/jb.132.1.44-50.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Celniker S. E., Campbell J. L. Yeast DNA replication in vitro: initiation and elongation events mimic in vivo processes. Cell. 1982 Nov;31(1):201–213. doi: 10.1016/0092-8674(82)90420-2. [DOI] [PubMed] [Google Scholar]
  5. Conrad M. N., Newlon C. S. Saccharomyces cerevisiae cdc2 mutants fail to replicate approximately one-third of their nuclear genome. Mol Cell Biol. 1983 Jun;3(6):1000–1012. doi: 10.1128/mcb.3.6.1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Culotti J., Hartwell L. H. Genetic control of the cell division cycle in yeast. 3. Seven genes controlling nuclear division. Exp Cell Res. 1971 Aug;67(2):389–401. doi: 10.1016/0014-4827(71)90424-1. [DOI] [PubMed] [Google Scholar]
  7. Dumas L. B., Lussky J. P., McFarland E. J., Shampay J. New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication. Mol Gen Genet. 1982;187(1):42–46. doi: 10.1007/BF00384381. [DOI] [PubMed] [Google Scholar]
  8. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hartwell L. H., Culotti J., Reid B. Genetic control of the cell-division cycle in yeast. I. Detection of mutants. Proc Natl Acad Sci U S A. 1970 Jun;66(2):352–359. doi: 10.1073/pnas.66.2.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Hartwell L. H. Macromolecule synthesis in temperature-sensitive mutants of yeast. J Bacteriol. 1967 May;93(5):1662–1670. doi: 10.1128/jb.93.5.1662-1670.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hartwell L. H., Mortimer R. K., Culotti J., Culotti M. Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics. 1973 Jun;74(2):267–286. doi: 10.1093/genetics/74.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hartwell L. H. Sequential function of gene products relative to DNA synthesis in the yeast cell cycle. J Mol Biol. 1976 Jul 15;104(4):803–817. doi: 10.1016/0022-2836(76)90183-2. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Hereford L. M., Hartwell L. H. Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis. J Mol Biol. 1974 Apr 15;84(3):445–461. doi: 10.1016/0022-2836(74)90451-3. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Johnston L. H., Nasmyth K. A. Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase. Nature. 1978 Aug 31;274(5674):891–893. doi: 10.1038/274891a0. [DOI] [PubMed] [Google Scholar]
  19. Johnston L. H., Thomas A. P. A further two mutants defective in initiation of the S phase in the yeast Saccharomyces cerevisiae. Mol Gen Genet. 1982;186(3):445–448. doi: 10.1007/BF00729467. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kuo C. L., Campbell J. L. Purification of the cdc8 protein of Saccharomyces cerevisiae by complementation in an aphidicolin-sensitive in vitro DNA replication system. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4243–4247. doi: 10.1073/pnas.79.14.4243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mark D. F., Chase J. W., Richardson C. C. Genetic mapping of trxA, a gene affecting thioredoxin in Escherichia coli K12. Mol Gen Genet. 1977 Oct 20;155(2):145–152. doi: 10.1007/BF00393153. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Zeman L. J., Lusena C. V. In vivo and in vitro synthesis of yeast mitochondrial DNA. Methods Cell Biol. 1975;12:273–283. doi: 10.1016/s0091-679x(08)60960-7. [DOI] [PubMed] [Google Scholar]

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