Yeast DNA Replication

  • Judith L. Campbell
  • Martin Budd
  • Colin Gordon
  • Ambrose Jong
  • Kevin Sweder
  • Alexander Oehm
  • Mary Gilbert
Part of the Basic Life Sciences book series (BLSC, volume 40)


We have explored various strategies for exploiting the yeast genetic and biochemical system for understanding DNA replication. Because of the long time that has intervened between the isolation of random replication mutants of yeast and the identification of the gene products affected, an alternative approach to elucidating the molecular basis of replication has been needed. One such alternative involves purifying replication proteins, isolating the genes encoding them, and constructing the appropriate mutant from the cloned gene. We have applied this “reverse genetics” strategy to three different replication activities: DNA polymerases, single-stranded DNA binding proteins, and proteins that bind to autonomously replicating sequence (ARS) elements.


Orotic Acid Autonomously Replicate Sequence High Copy Number Plasmid hnRNP Protein Thymidylate Kinase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Badaracco, G., L. Capucci, P. Plevani, and L.M.S. Chang (1983) Polypeptide structure of DNA polymerase I from Saccharomyces cerevisiae. J. Biol. Chem. 258:10720–10726.PubMedGoogle Scholar
  2. 2.
    Beyer, A.J., M.E. Christensen, B.W. Walker, and W.M. LeStourgeon (1977) Identification and characterization of the packaging proteins of core 40S hnRNP particles thymidylate synthetase. Cell 11:127–138.PubMedCrossRefGoogle Scholar
  3. 3.
    Bisson, I., and J. Thorner (1977) Thymidine 5’-monophosphate-requiring mutants of Saccharomyces cerevisiae are deficient in thymidylate synthetase. J. Bacteriol. 132:44–50.PubMedGoogle Scholar
  4. 4.
    Bram, R.J., and R.D. Rornberg (1985) Specific protein binding for upstream activating sequences in polymerase II promoters. Proc. Natl. Acad. Sci., USA 82:43–47.PubMedCrossRefGoogle Scholar
  5. 5.
    Campbell, J.L. (1983) Yeast DNA replication. In Genetic Engineering: Principles and Methods, Vol. 5, J.K. Setlow and A. Hollaender, eds. Plenum Press, New York, pp. 109–156.Google Scholar
  6. 6.
    Campbell, J.L. (1986) Eukaryotic DNA replication. Ann. Rev. Biochem. Vol. 55 (in press).Google Scholar
  7. 7.
    Celniker, S.E., K. Sweder, F. Srienc, J.E. Bailey, and J.L. Campbell (1984) Deletion mutations affecting autonomously replicating sequence ARS1 of Saccharomyces cerevisiae. Mol. Cell. Biol. 4:2455–2466.PubMedGoogle Scholar
  8. 8.
    Chang, L.M.S. (1977) DNA polymerases from bakers’ yeast. J. Biol. Chem. 252:1873–1880.PubMedGoogle Scholar
  9. 9.
    Chelm, B.D., and E.P. Geidushek (1979) Gel electrophoretic separation of transcription complexes: An assay for RNA polymerase selectivity and a method for promoting mapping. Nucl. Acids Res. 7:1851–1867.PubMedCrossRefGoogle Scholar
  10. 10.
    Cobianchi, F., D.N. Sen Gupta, B.Z. Zmudzka, and S.H. Wilson (1986) Structure of rodent helix-destabilizing proteins revealed by cDNA cloning. J. Biol. Chem. 261:3536–3543.PubMedGoogle Scholar
  11. 11.
    Dodson, M., J. Roberts, R. McMacken, and H. Echols (1985) Specialized nucleoprotein structures at the origin of replication of bacteriophage λ: Complexes with λO protein and with λO, λP, and Escherichia coli Dnaβ proteins. Proc. Natl. Acad. Sci., USA 82:4678–4682.PubMedCrossRefGoogle Scholar
  12. 12.
    Dumas, L.B., J.P. Lussky, E.F. McFarland, and J. Shampay (1982) New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication. Mol. Gen. Genet. 187:42–46.PubMedCrossRefGoogle Scholar
  13. 13.
    Formosa, T., R. Burke, and B. Alberts (1983) Affinity purification of bacteriophage T4 proteins essential for DNA replication and genetic recombination. Proc. Natl. Acad. Sci., USA 80:2442.PubMedCrossRefGoogle Scholar
  14. 14.
    Fried, M., and D. Crothers (1981) Equilibria and kinetics of Lac repressor-operator interactions by Polyacrylamide gel electrophoresis. Nucl. Acids Res. 9:6505–6525.PubMedCrossRefGoogle Scholar
  15. 15.
    Fuller, R.S., B.E. Funnell, and A. Romberg (1984) The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites. Cell 38:889–900.PubMedCrossRefGoogle Scholar
  16. 16.
    Game, J.C. (1976) Yeast cell-cycle mutant cde21 is a temperature-sensitive thymidylate auxotroph. Mol. Gen. Genet. 146:313–315.PubMedCrossRefGoogle Scholar
  17. 17.
    Garner, M.M, and A. Revzin (1981) A gel electrophoresis method for quantifying the bending of proteins to specific DNA regions: Application to components of the Escherichia coli lactose Operon regulating system. Nucl. Acids Res. 9:3047–3060.PubMedCrossRefGoogle Scholar
  18. 18.
    Hartwell, L. (1974) Genetic control of the cell division cycle in yeast. J. Mol. Biol. 59:183–194.CrossRefGoogle Scholar
  19. 19.
    Herrick, G., and B. Alberts (1976) Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus. J. Biol. Chem. 251:2124–2132.PubMedGoogle Scholar
  20. 20.
    Herrick, G., and B. Alberts (1976) Nucleic acid helix-coil transition mediated by helix-unwinding proteins from calf thymus. J. Biol. Chem. 251:2133–2141.PubMedGoogle Scholar
  21. 21.
    Herrick, G., and B. Alberts (1976) Single-stranded DNA structure and DNA polymerase activity in the presence of nucleic acid helix-unwinding proteins from calf thymus. J. Biol. Chem. 251:2142–2146.PubMedGoogle Scholar
  22. 22.
    Hieter, P., C. Mann, M. Snyder, and R.W. Davis (1985) Mitotic stability of yeast chromosomes: A colony color assay that measures nondisjunction and chromosome loss. Cell 40:381–392.PubMedCrossRefGoogle Scholar
  23. 22a.
    Johnson, L.M., M. Snyder, L.M.S. Chang, R.W. Davis, and J.L. Campbell (1985) Isolation of the gene encoding yeast DNA polymerase I. Cell 43:369–377.PubMedCrossRefGoogle Scholar
  24. 23.
    Johnston, L.H., and J.C. Game (1978) Mutants of yeast with depressed DNA synthesis. Mol. Gen. Genet. 161:205–214.PubMedGoogle Scholar
  25. 24.
    Johnston, L.H., and K.A. Nasmyth (1978) Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase. Nature 274:891–894.PubMedCrossRefGoogle Scholar
  26. 25.
    Jong, A.Y.S., and J.L. Campbell (1984) Characterization of Saccharomyces cerevisiae thymidylate kinase, the CDC8 gene product. J. Biol. Chem. 259:14394–14398.PubMedGoogle Scholar
  27. 26.
    Jong, A.Y.S., and J.L. Campbell (1986) Isolation of the gene encoding yeast single-stranded nucleic acid binding protein I (SSB-1). Proc. Natl. Acad. Sci., USA 83:877–881.PubMedCrossRefGoogle Scholar
  28. 27.
    Jong, A.Y.S., R. Aebersold, and J.L. Campbell (1985) Multiple species of single-stranded DNA binding proteins in Saccharomyces cerevisiae. J. Biol. Chem. 260:16367–16374.PubMedGoogle Scholar
  29. 28.
    Jong, A.Y.S., C.L. Kuo, and J.L. Campbell (1984) The CDC8 gene of yeast encodes thymidylate kinase. J. Biol. Chem. 259:11052–11058.PubMedGoogle Scholar
  30. 29.
    Kearsey, S. (1984) Structural requirements for the function of a yeast chromosomal replicator. Cell 37:299–307.PubMedCrossRefGoogle Scholar
  31. 30.
    Koshland, D., J.C. Kent, and L.H. Hartwell (1985) Genetic analysis of the mitotic transmission of minichromosomes. Cell 40:393–403.PubMedCrossRefGoogle Scholar
  32. 31.
    Kuo, C.L., and J.L. Campbell (1983) Isolation of yeast DNA replication mutants in permeabilized cells. Proc. Natl. Acad. Sci., USA 80:6465–6469.PubMedCrossRefGoogle Scholar
  33. 32.
    LeStourgeon, W.M., A.L. Beyer, M.E. Christensen, B.W. Walker, S.M. Poupore et al. (1977) The packaging proteins of core hnRNP particles and the maintenance of proliferative cell states. Cold Spring Harbor Symp. Quant. Biol. 42:885–898.CrossRefGoogle Scholar
  34. 33.
    Merrill, B.M., M.B. LoPresti, K.L. Stone, and K.R. Williams (1986) High pressure liquid chromatography purification of UP1 and UP2, two related single-stranded nucleic acid-binding proteins from calf thymus. J. Biol. Chem. 261:878–883.PubMedGoogle Scholar
  35. 34.
    Pandolfo, M., O. Valentini, G. Biamonti, C. Morandi, and S. Riva (1985) Single stranded DNA binding proteins derived from hnRNP proteins by proteolysis in mammalian cells. Nucl. Acids Res. 13:6577–6590.PubMedCrossRefGoogle Scholar
  36. 35.
    Parker, C., and J. Topol (1984) A Drosophila RNA polymerase II transcription factor contains a promoter-region-specific DNA binding activity. Cell 36:357–369.PubMedCrossRefGoogle Scholar
  37. 36.
    Plevani, P., G. Badaracco, C. Augh, and L.M.S. Chang (1984) DNA polymerase I and DNA primase complex in yeast. J. Biol. Chem. 259:7532–7539.PubMedGoogle Scholar
  38. 37.
    Plevani, P., M. Foiari, P. Valsasnini, and G. Badaracco (1985) Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex. J. Biol. Chem. 260:7102–7107.PubMedGoogle Scholar
  39. 38.
    Sapp, M., H. Konig, H.D. Riedl, A. Richter, and R. Knippers (1985) A newly detected class of mammalian single strand-specific DNA-binding proteins. J. Biol. Chem. 260:1550–1556.PubMedGoogle Scholar
  40. 39.
    Sclafani, R.A., and W.L. Fangman (1984) Yeast gene CDC8 encodes thymidylate kinase and is complemented by herpes thymidine kinase gene TK. Proc. Natl. Acad. Sci., USA 81:5821–5825.PubMedCrossRefGoogle Scholar
  41. 40.
    Shortle, D., P. Novick, and D. Dotstein (1984) Construction and genetic characterization of temperature-sensitive mutant alleles of the yeast actin gene. Proc. Natl. Acad. Sci., USA 81:4889–4893.PubMedCrossRefGoogle Scholar
  42. 41.
    Singh, H., and L.B. Dumas (1984) A DNA primase that copurifies with the major DNA polymerase from the yeast Saccharomyces cerevisiae. J. Biol. Chem. 259:7936–7940.PubMedGoogle Scholar
  43. 42.
    Srienc, F., J.E. Bailey, and J.L. Campbell (1985) Effect of ARS1 mutations on chromosome stability in Saccharomyces cerevisiae. Mol. Cell. Biol. 5:1676–1684.PubMedGoogle Scholar
  44. 43.
    Sugino, A., H. Kojo, B.D. Greenberg, P.O. Brown, and K.C. Kim (1981) In vitro replication of yeast 2-um plasmid DNA. In The Initiation ofDNA Replication (ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. 11), Academic Press, Inc., New York, pp. 529–533.Google Scholar
  45. 44.
    Takanami, M., S. Tabata, A. Oka, K. Sugimoto, H. Sasaki, S. Yasuda, and Y. Hirota (1983) The Escherichia coli origin of replication: Essential structure for bidirectional replication. In Mechanisms of DNA Replication (ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. 10), Alan R. Liss, Inc., New York, pp. 257–273.Google Scholar
  46. 45.
    Valentini, O., G. Biamonti, M. Pandolfo, C. Morandi, and S. Riva (1984) Mammalian single-stranded DNA binding proteins and heterogeneous nuclear RNA proteins have common antigenic determinants. Nucl. Acids Res. 13:337–346.CrossRefGoogle Scholar
  47. 46.
    Walker, J.M., K. Gooderham, J.B.B. Hastings, E. Mayes, and E.W. Johns (1980) The primary structures of non-histone chromosomal proteins HMG 1 and 2. FEBS Lett. 122:264–270.PubMedCrossRefGoogle Scholar
  48. 47.
    Williams, K.R., E.K. Spicer, M.B. LoPresti, R.A. Guggenheimer, and J.W. Chase (1983) Limited proteolysis studies on the Escherichia coli single-stranded DNA binding protein. J. Biol. Chem. 258:3346–3355.PubMedGoogle Scholar
  49. 48.
    Williams, K.R., K.L. Stone, M.B. LoPresti, B.M. Merrill, and S.R. Planck (1985) Amino acid sequence of the UP1 calf thymus helix-destabilizing protein and its homology to an analogous protein from mouse myeloma. Proc. Natl. Acad. Sci., USA 82:5666–5670.PubMedCrossRefGoogle Scholar
  50. 49.
    Wintersberger, E. (1974) Absence of a low-molecular-weight DNA polymerase from nuclei of the yeast, Saccharomyces cerevisiae. Eur. J. Biochem. 50:197–202.PubMedCrossRefGoogle Scholar
  51. 50.
    Wintersberger, E. (1978) Yeast DNA polmerases: Antigenic relationship, use of RNA primer and associated exonuclease activity. Eur. J. Biochem. 84:167–172.PubMedCrossRefGoogle Scholar
  52. 51.
    Wintersberger, U., and E. Wintersberger (1970) Studies on deoxyribonucleic acid polymerases from yeast. 1. Partial purification and properties of two DNA polymerases from mitochondria-free cell extracts. Eur. J. Biochem. 13:11–19.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1986

Authors and Affiliations

  • Judith L. Campbell
    • 1
  • Martin Budd
    • 1
  • Colin Gordon
    • 1
  • Ambrose Jong
    • 1
  • Kevin Sweder
    • 1
  • Alexander Oehm
    • 1
  • Mary Gilbert
    • 1
  1. 1.Divisions of Biology and ChemistryCalifornia Institute of TechnologyPasadenaUSA

Personalised recommendations