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Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast

Nature volume 445, pages 281–285 (2007)Cite this article

Abstract

Cyclin-dependent kinases (CDKs) drive major cell cycle events including the initiation of chromosomal DNA replication. We identified two S phase CDK (S-CDK) phosphorylation sites in the budding yeast Sld3 protein that, together, are essential for DNA replication. Here we show that, when phosphorylated, these sites bind to the amino-terminal BRCT repeats of Dpb11. An Sld3–Dpb11 fusion construct bypasses the requirement for both Sld3 phosphorylation and the N-terminal BRCT repeats of Dpb11. Co-expression of this fusion with a phospho-mimicking mutant in a second essential CDK substrate, Sld2, promotes DNA replication in the absence of S-CDK. Therefore, Sld2 and Sld3 are the minimal set of S-CDK targets required for DNA replication. DNA replication in cells lacking G1 phase CDK (G1-CDK) required expression of the Cdc7 kinase regulatory subunit, Dbf4, as well as Sld2 and Sld3 bypass. Our results help to explain how G1- and S-CDKs promote DNA replication in yeast.

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Figure 1: Sld3 is an essential CDK substrate.
Figure 2: CDK-phosphorylated Sld3 binds to Dpb11.
Figure 3: Dpb11–Sld3 interaction is the essential function of Sld3 CDK phosphorylation sites and the N terminus of Dpb11.
Figure 4: DNA replication in the absence of S-CDK activity.
Figure 5: CDK bypass and Dbf4 overexpression allows replication in G1 cells.

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Acknowledgements

We thank H. Araki and colleagues for sharing unpublished information. We are grateful to J. A. Tercero for making strain y1187 and S. Sweet for making strain y2003 and providing extracts. We are grateful to ARIAD pharmaceuticals for providing the ARGENT Regulated Heterodimerization Kit. We thank J. Gannon for assistance with the BIACORE 3000. We are grateful to N. O’Reilly and the Peptide Synthesis Facility at the London Research Institute. We thank members of our laboratory for helpful discussion and criticism of the manuscript. P.Z. is supported by a Cancer Research UK fellowship.

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

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  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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Correspondence to John F. X. Diffley.

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This file contains detailed Supplementary Methods, Supplementary Figures 1-7 providing data supporting the paper’s main conclusions (described in the main text) and a Supplementary Table of yeast strain geneotypes used in the manuscript. (PDF 767 kb)

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Zegerman, P., Diffley, J. Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. Nature 445, 281–285 (2007). https://doi.org/10.1038/nature05432

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