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Analysis of replication profiles reveals key role of RFC-Ctf18 in yeast replication stress response

Nature Structural & Molecular Biology volume 17, pages 1391–1397 (2010)Cite this article

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Abstract

Maintenance of genome integrity relies on surveillance mechanisms that detect and signal arrested replication forks. Although evidence from budding yeast indicates that the DNA replication checkpoint (DRC) is primarily activated by single-stranded DNA (ssDNA), studies in higher eukaryotes have implicated primer ends in this process. To identify factors that signal primed ssDNA in Saccharomyces cerevisiae, we have screened a collection of checkpoint mutants for their ability to activate the DRC, using the repression of late origins as readout for checkpoint activity. This quantitative analysis reveals that neither RFCRad24 and the 9-1-1 clamp nor the alternative clamp loader RFCElg1 is required to signal paused forks. In contrast, we found that RFCCtf18 is essential for the Mrc1-dependent activation of Rad53 and for the maintenance of paused forks. These data identify RFCCtf18 as a key DRC mediator, potentially bridging Mrc1 and primed ssDNA to signal paused forks.

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Figure 1: RFCCtf18 is the only RFC-like clamp loader implicated in the DRC pathway.
Figure 2: Hierarchical clustering analysis of replication profiles places RFCCtf18 in the DRC pathway.
Figure 3: RFCCtf18 acts together with Mrc1 in the replication stress response.
Figure 4: Replication-fork maintenance is compromised in hydroxyurea-treated ctf18Δ cells.
Figure 5: The DDC pathway represses dormant origins in hydroxyurea-treated ctf18Δ mutants.

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Acknowledgements

We thank O. Aparicio (University of Southern California), S. Elledge (Howard Hughes Medical Institute), M.P. Longhese (Università di Milano-Bicocca), J. Boeke (The Johns Hopkins University School of Medicine), J. Campbell (California Institute of Technology), M. McAlear (Wesleyan University), D. Cortez (Vanderbilt University School of Medicine) and A. Verreault (Institute for Research in Immunology and Cancer) for plasmids, strains and antibodies; C. Alabert, A. Chabes, M. Weinreich, J. Cau, H. Tourrière and members of the Pasero laboratory for discussions and critical reading of the manuscript; E. Schwob and the DNA combing facility of Montpellier for providing silanized coverslips; and the Montpellier RIO Imaging microscopy facility. L.C. was supported by a European Molecular Biology Organization (EMBO) long-term fellowship. Work in the Pasero laboratory is supported by FRM Fondation pour la Recherche Médicale (FRM) (Equipe FRM), Agence Nationale de la Recherche (ANR), Institut National du Cancer (INCa) and the EMBO Young Investigator Programme.

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  1. Laure Crabbé

    Present address: Present address: The Salk Institute for Biological Studies, La Jolla, California, USA.,

  2. Philippe Pasero and Armelle Lengronne: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Human Genetics, Centre National de la Recherche Scientifique, Unité Propre de Recherche 1142, Montpellier, France

    Laure Crabbé, Aubin Thomas, Philippe Pasero & Armelle Lengronne

  2. Inserm U847, Centre Hospitalier Universitaire de Montpellier, Institut de Recherche en Biothérapie, Hôpital Saint Eloi, Université Montpellier 1, Montpellier, France

    Véronique Pantesco & John De Vos

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Contributions

L.C., A.L. and P.P. designed experiments, and L.C. and A.L. performed experiments. A.T. did the bioinformatic analysis. V.P. and J.D.V. performed the microarray hybridizations. A.L. and P.P. wrote the manuscript.

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Correspondence to Philippe Pasero or Armelle Lengronne.

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Supplementary Figures 1-5, Supplementary Table 2 and Supplementary Methods (PDF 605 kb)

Supplementary Table 1

Position of active replication origins in the 20 S. cerevisiae strains analyzed in this study (XLS 150 kb)

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Crabbé, L., Thomas, A., Pantesco, V. et al. Analysis of replication profiles reveals key role of RFC-Ctf18 in yeast replication stress response. Nat Struct Mol Biol 17, 1391–1397 (2010). https://doi.org/10.1038/nsmb.1932

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