Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo

Nature volume 423, pages 720–725 (2003)Cite this article

Abstract

Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function. Some are essential for cell viability1,2, emphasising their importance for fundamental processes of cell biology but complicating their analysis. We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined3. Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a ‘heat-inducible-degron’ cassette that targets the protein for proteolysis at 37 °C (ref. 4). By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells. We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks. The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Direct inactivation of essential proteins by fusion to a heat-inducible degron.
Figure 2: Three new DNA replication proteins identified by functional proteomics.
Figure 3: Cdc102 and Cdc105 are essential for an early step of origin replication.
Figure 4: Cdc102 and Cdc105 are important for the normal progression of DNA replication forks away from origins.

Similar content being viewed by others

References

  1. Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003)

    Article  ADS  CAS  Google Scholar 

  2. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002)

    Article  ADS  CAS  Google Scholar 

  3. Labib, K., Tercero, J. A. & Diffley, J. F. X. Uninterrupted MCM2-7 function required for DNA replication fork progression. Science 288, 1643–1647 (2000)

    Article  ADS  CAS  Google Scholar 

  4. Dohmen, R. J., Wu, P. & Varshavsky, A. Heat-inducible degron: a method for constructing temperature-sensitive mutants. Science 263, 1273–1276 (1994)

    Article  ADS  CAS  Google Scholar 

  5. Belli, G., Gari, E., Aldea, M. & Herrero, E. Functional analysis of yeast essential genes using a promoter-substitution cassette and the tetracycline-regulatable dual expression system. Yeast 14, 1127–1138 (1998)

    Article  CAS  Google Scholar 

  6. Dragon, F. et al. A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 417, 967–970 (2002)

    Article  ADS  CAS  Google Scholar 

  7. Wu, L. F. et al. Large-scale prediction of Saccharomyces cerevisiae gene function using overlapping transcriptional clusters. Nature Genet. 31, 255–265 (2002)

    Article  CAS  Google Scholar 

  8. Uetz, P. et al. A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000)

    Article  ADS  CAS  Google Scholar 

  9. Ito, T. et al. A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc. Natl Acad. Sci. USA 98, 4569–4574 (2001)

    Article  ADS  CAS  Google Scholar 

  10. Takayama, Y. et al. GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast. Genes Dev. 17, 1153–1165 (2003)

    Article  CAS  Google Scholar 

  11. Kubota, Y. et al. A novel ring-like complex of Xenopus proteins essential for the initiation of DNA replication. Genes Dev. 17, 1141–1152 (2003)

    Article  CAS  Google Scholar 

  12. Santocanale, C. & Diffley, J. F. X. A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication. Nature 395, 615–618 (1998)

    Article  ADS  CAS  Google Scholar 

  13. Yabuki, N., Terashima, H. & Kitada, K. Mapping of early firing origins on a replication profile of budding yeast. Genes Cells 7, 781–789 (2002)

    Article  CAS  Google Scholar 

  14. Bousset, K. & Diffley, J. F. X. The Cdc7 protein kinase is required for origin firing during S phase. Genes Dev. 12, 480–490 (1998)

    Article  CAS  Google Scholar 

  15. Donaldson, A. D., Fangman, W. L. & Brewer, B. J. Cdc7 is required throughout the yeast S phase to activate replication origins. Genes Dev. 12, 491–501 (1998)

    Article  CAS  Google Scholar 

  16. Donaldson, A. D. et al. CLB5-dependent activation of late replication origins in S. cerevisiae. Mol. Cell 2, 173–183 (1998)

    Article  CAS  Google Scholar 

  17. McCarroll, R. M. & Fangman, W. L. Time of replication of yeast centromeres and telomeres. Cell 54, 505–513 (1988)

    Article  CAS  Google Scholar 

  18. Gavin, A. C. et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, 141–147 (2002)

    Article  ADS  CAS  Google Scholar 

  19. Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002)

    Article  ADS  CAS  Google Scholar 

  20. Kumar, A. et al. Subcellular localization of the yeast proteome. Genes Dev. 16, 707–719 (2002)

    Article  CAS  Google Scholar 

  21. Lindner, K., Gregan, J., Montgomery, S. & Kearsey, S. E. Essential role of MCM proteins in premeiotic DNA replication. Mol. Biol. Cell 13, 435–444 (2002)

    Article  CAS  Google Scholar 

  22. Levy, F., Johnston, J. A. & Varshavsky, A. Analysis of a conditional degradation signal in yeast and mammalian cells. Eur. J. Biochem. 259, 244–252 (1999)

    Article  CAS  Google Scholar 

  23. Tercero, J. A., Labib, K. & Diffley, J. F. X. DNA synthesis at individual replication forks requires the essential initiation factor, Cdc45p. EMBO J. 19, 2082–2093 (2000)

    Article  CAS  Google Scholar 

  24. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnol. 17, 1030–1032 (1999)

    Article  CAS  Google Scholar 

  25. Tasto, J. J., Carnahan, R. H., McDonald, W. H. & Gould, K. L. Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe. Yeast 18, 657–662 (2001)

    Article  CAS  Google Scholar 

  26. Labib, K., Diffley, J. F. X. & Kearsey, S. E. G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus. Nature Cell Biol. 1, 415–422 (1999)

    Article  CAS  Google Scholar 

  27. Kamimura, Y., Tak, Y.-S., Sugino, A. & Araki, H. Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae. EMBO J. 20, 2097–2107 (2001)

    Article  CAS  Google Scholar 

  28. Drury, L. S., Perkins, G. & Diffley, J. F. X. The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast. EMBO J. 16, 5966–5976 (1997)

    Article  CAS  Google Scholar 

  29. Desdouets, C. et al. Evidence for a Cdc6p-independent mitotic resetting event involving DNA polymerase α. EMBO J. 17, 4139–4146 (1998)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Diffley for support and encouragement in the early stages of this project and for comments on the manuscript, together with N. Jones. We are grateful to members of our laboratory for helpful discussions, S. Pepper for helping to analyse mass spectrometry data, and to M. Segurado and E. Rawson for their assistance. We thank E. Schiebel and K. Gould for plasmids; E. Schiebel for help in growing fermentor cultures; and H. Araki and Y. Kamimura for assistance with ChIP and for communicating unpublished data, together with H. Takisawa. A.S.-D. thanks F. Perez-Campo for her support. This work was funded by Cancer Research UK, from whom K.L. receives a Senior Cancer Research Fellowship, and by the European Community, from whom A.S.-D. receives a Marie Curie Fellowship.

Author information

Author notes

  1. Masato Kanemaki and Alberto Sanchez-Diaz: These authors contributed equally to this work

  2. Karim Labib: Correspondence and requests for materials should be addressed to K.L.

Authors and Affiliations

  1. Cancer Research UK, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, M20 4BX, Manchester, UK

    Masato Kanemaki, Alberto Sanchez-Diaz, Agnieszka Gambus & Karim Labib

Authors
  1. Masato Kanemaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alberto Sanchez-Diaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Agnieszka Gambus
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karim Labib
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karim Labib.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Additional information

This paper was published online as an Article on 25 May 2003, but appeared as a Letter for a short period between 30 May and 5 June 2003, due to a production error. It has now been restored in its Article format.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kanemaki, M., Sanchez-Diaz, A., Gambus, A. et al. Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo. Nature 423, 720–725 (2003). https://doi.org/10.1038/nature01692

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature01692

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing