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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.

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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.

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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for help in growing fermentor cultures; and H. Araki and Y. Kamimura for assistance with ChIP and for communicating unpublished data, together with H. Takisawa.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.

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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
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Correspondence to Karim Labib.

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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.

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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

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