Letter | Published:

Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation

Nature volume 467, pages 474478 (23 September 2010) | Download Citation

Abstract

The initiation of eukaryotic DNA replication is regulated by three protein kinase classes: cyclin-dependent kinases (CDK), Dbf4-dependent kinase (DDK) and the DNA damage checkpoint kinases1. CDK phosphorylation of two key initiation factors, Sld2 and Sld3, promotes essential interactions with Dpb11 (refs 2–4), whereas DDK acts by phosphorylating subunits of the Mcm2-7 helicase5. CDK has an additional role in replication by preventing the re-loading of Mcm2-7 during the S, G2 and M phases6, thus preventing origin re-firing and re-replication. During the G1 phase, both CDK and DDK are downregulated, which allows origin licensing and prevents premature replication initiation3. Origin firing is also inhibited during the S phase when DNA damage or replication fork stalling activates the checkpoint kinases7,8,9,10. Here we show that, analogous to the situation in the G1 phase, the Saccharomyces cerevisiae checkpoint kinase Rad53 inhibits both CDK- and DDK-dependent pathways, which acts redundantly to block further origin firing. Rad53 acts on DDK directly by phosphorylating Dbf4, whereas the CDK pathway is blocked by Rad53-mediated phosphorylation of the downstream CDK substrate, Sld3. This allows CDK to remain active during the S phase in the presence of DNA damage, which is crucial to prevent re-loading of Mcm2-7 onto origins that have already fired6. Our results explain how checkpoints regulate origin firing and demonstrate that the slowing of S phase by the ‘intra-S checkpoint’ is primarily due to the inhibition of origin firing.

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Acknowledgements

We are grateful to K. Bousset, M. Godinho Ferreira, D. Quintana and C. Santocanale for early contributions to this project. We thank J. Gannon and T. Hunt for providing recombinant cyclin A/Cdk2, N. O’Reilly and the Peptide Synthesis Facility at the London Research Institute for the peptide arrays, and D. Toczyski for sharing information before publication. Work was supported by Cancer Research UK.

Author information

Author notes

    • Philip Zegerman

    Present address: Wellcome Trust/Cancer Research UK Gurdon Institute, Department of Zoology, University of Cambridge, Cambridge CB2 1QN, UK.

Affiliations

  1. Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK

    • Philip Zegerman
    •  & John F. X. Diffley

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Contributions

P.Z. and J.F.X.D. conceived and designed experiments and wrote the paper. P.Z. performed all experiments.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to John F. X. Diffley.

Supplementary information

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    Supplementary Information

    This file contains Supplementary Figures 1-7 with legends, Supplementary Tables 1-3 and a list of Genotypes. Supplementary Figure 7b and legend were amended on 05 May 2011.

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DOI

https://doi.org/10.1038/nature09373

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