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


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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Figure 1: Sld3 is a target of Rad53 in vivo.
Figure 2: At least two essential functions of Sld3 are inhibited by Rad53.
Figure 3: Sld3 and Dbf4 are the minimal substrates of Rad53 for the block to origin firing.
Figure 4: Separation of function analysis of the intra-S checkpoint.

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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.

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

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Correspondence to John F. X. Diffley.

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The authors declare no competing financial interests.

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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. (PDF 1276 kb)

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Zegerman, P., Diffley, J. Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation. Nature 467, 474–478 (2010).

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