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Measurement of Geminin Activity in Xenopus Egg Extracts

  • Thomas J. McGarry
Part of the Methods in Molecular Biology™ book series (MIMB, volume 296)

Abstract

Geminin is an unstable protein that inhibits DNA replication by interfering with the assembly of prereplication complex at replication origins. Geminin is thought to prevent a second round of replication during late S- or G2-phase. The protein is destroyed by ubiquitin-dependent proteolysis during mitosis, allowing a new round of replication in the next cell cycle. This chapter describes protocols for measuring the stability of geminin and two of its activities, the inhibition of replication and the inhibition of pre-RC loading.

Key Words

Geminin DNA Replication ubiquitin-dependent proteolysis prereplication complex rereplication cell cycle S-phase mitosis anaphase-promoting complex 

References

  1. 1.
    McGarry, T. J. and Kirschner, M. W. (1998) Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93, 1043–1053.PubMedCrossRefGoogle Scholar
  2. 2.
    Wohlschlegel, J. A., Dwyer, B. T., Dhar, S. K., Cvetic, C., Walter, J. C., and Dutta, A. (2000) Inhibition of eukaryotic DNA replication by geminin binding to cdt1. Science 290,2309–2312.PubMedCrossRefGoogle Scholar
  3. 3.
    Tada, S., Li, A., Maiorano, D., Mechali, M., and Blow, J. J. (2001) Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nat. Cell Biol 3, 107–113.PubMedCrossRefGoogle Scholar
  4. 4.
    Murray, A. W. (1991) Cell cycle extracts. Methods Cell Biol. 36, 581–605.PubMedCrossRefGoogle Scholar
  5. 5.
    Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991) Cyclin is degraded by the ubiquitin pathway [see comments]. Nature 349, 132–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Wu, M. and Gerhart, J. (1991). Raising Xenopus in the laboratory, in Methods in Cell Biology, Vol. 36 (Kay, B. K. and Peng, H. B., eds.), Academic, San Diego, pp. 3–18.Google Scholar
  7. 7.
    Lustig, K. D., Stukenberg, P. T., McGarry, T. J., et al. (1997) Small pool expression screening: identification of genes involved in cell cycle control, apoptosis, and early development. Methods Enzymol. 283, 83–99.PubMedCrossRefGoogle Scholar
  8. 8.
    Blow, J. J. and Laskey, R. A. (1986) Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell 47, 577–587.PubMedCrossRefGoogle Scholar
  9. 9.
    Kubota, Y., Mimura, S., Nishimoto, S., Takisawa, H., and Nojima, H. (1995) Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell 81, 601–609.PubMedCrossRefGoogle Scholar
  10. 10.
    Lygerou, Z. and Nurse, P. (2000) License withheld-geminin blocks DNA replication. Science 290, 2271–2273.PubMedGoogle Scholar
  11. 11.
    Quinn, L. M., Herr, A., McGarry, T. J., and Richardson, H. (2001) The Drosophila geminin homolog: roles for geminin in limiting DNA replication, in anaphase and in neurogenesis. Genes Dev. 15, 2741–2754.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Thomas J. McGarry

There are no affiliations available

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