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The Dbf4–Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4

Abstract

Eukaryotic DNA replication uses kinase regulatory pathways to facilitate coordination with other processes during cell division cycles and response to environmental cues. At least two cell cycle-regulated protein kinase systems, the S-phase-specific cyclin-dependent protein kinases (S-CDKs) and the Dbf4–Cdc7 kinase (DDK, Dbf4-dependent protein kinase) are essential activators for initiation of DNA replication1,2,3,4,5. Although the essential mechanism of CDK activation of DNA replication in Saccharomyces cerevisiae has been established6,7, exactly how DDK acts has been unclear. Here we show that the amino terminal serine/threonine-rich domain (NSD) of Mcm4 has both inhibitory and facilitating roles in DNA replication control and that the sole essential function of DDK is to relieve an inhibitory activity residing within the NSD. By combining an mcm4 mutant lacking the inhibitory activity with mutations that bypass the requirement for CDKs for initiation of DNA replication, we show that DNA synthesis can occur in G1 phase when CDKs and DDK are limited. However, DDK is still required for efficient S phase progression. In the absence of DDK, CDK phosphorylation at the distal part of the Mcm4 NSD becomes crucial. Moreover, DDK-null cells fail to activate the intra-S-phase checkpoint in the presence of hydroxyurea-induced DNA damage and are unable to survive this challenge. Our studies establish that the eukaryote-specific NSD of Mcm4 has evolved to integrate several protein kinase regulatory signals for progression through S phase.

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Figure 1: An inhibitory activity within the Mcm4 NSD is responsible for the dependency of cells on DDK for viability.
Figure 2: The status of the Mcm4 N terminus determines the efficiency of DDK-independent cell proliferation.
Figure 3: Removal of the N-terminal inhibitory domain of Mcm4 allows DDK-independent initiation of DNA replication and S phase progression.
Figure 4: Deletion of the N-terminal inhibitory domain of Mcm4 does not bypass the requirement for DDK for cell survival and checkpoint activation in the presence of hydroxyurea.

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Acknowledgements

We thank A. Stenlund and H.-G. Wendel for critical reading of the manuscript, J. Li, H. Araki and J. Diffley for plasmids and yeast strains. This work was supported by a grant from the US National Institute of General Medical Sciences.

Author Contributions Y.-J.S. and B.S. designed this study, analysed the data and wrote the paper. Y.-J.S. performed the experiments.

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Correspondence to Bruce Stillman.

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Sheu, YJ., Stillman, B. The Dbf4–Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4. Nature 463, 113–117 (2010). https://doi.org/10.1038/nature08647

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