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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Bell, S. P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002)
Diffley, J. F. Regulation of early events in chromosome replication. Curr. Biol. 14, R778–R786 (2004)
Masai, H. & Arai, K. Cdc7 kinase complex: a key regulator in the initiation of DNA replication. J. Cell. Physiol. 190, 287–296 (2002)
Sclafani, R. A. Cdc7p-Dbf4p becomes famous in the cell cycle. J. Cell Sci. 113, 2111–2117 (2000)
Stillman, B. Cell cycle control of DNA replication. Science 274, 1659–1663 (1996)
Tanaka, S. et al. CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast. Nature 445, 328–332 (2007)
Zegerman, P. & Diffley, J. F. Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. Nature 445, 281–285 (2007)
Rao, P. N. & Johnson, R. T. Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis. Nature 225, 159–164 (1970)
Forsburg, S. L. Eukaryotic MCM proteins: beyond replication initiation. Microbiol. Mol. Biol. Rev. 68, 109–131 (2004)
Laskey, R. A., Fairman, M. P. & Blow, J. J. S phase of the cell cycle. Science 246, 609–614 (1989)
Gambus, A. et al. GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nature Cell Biol. 8, 358–366 (2006)
Moyer, S. E., Lewis, P. W. & Botchan, M. R. Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc. Natl Acad. Sci. USA 103, 10236–10241 (2006)
Pacek, M., Tutter, A. V., Kubota, Y., Takisawa, H. & Walter, J. C. Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Mol. Cell 21, 581–587 (2006)
Masumoto, H., Muramatsu, S., Kamimura, Y. & Araki, H. S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast. Nature 415, 651–655 (2002)
Francis, L. I., Randell, J. C., Takara, T. J., Uchima, L. & Bell, S. P. Incorporation into the prereplicative complex activates the Mcm2–7 helicase for Cdc7–Dbf4 phosphorylation. Genes Dev. 23, 643–654 (2009)
Hardy, C. F., Dryga, O., Seematter, S., Pahl, P. M. & Sclafani, R. A. mcm5/cdc46-bob1 bypasses the requirement for the S phase activator Cdc7p. Proc. Natl Acad. Sci. USA 94, 3151–3155 (1997)
Lei, M. et al. Mcm2 is a target of regulation by Cdc7–Dbf4 during the initiation of DNA synthesis. Genes Dev. 11, 3365–3374 (1997)
Sheu, Y. J. & Stillman, B. Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression. Mol. Cell 24, 101–113 (2006)
Weinreich, M. & Stillman, B. Cdc7p–Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway. EMBO J. 18, 5334–5346 (1999)
Devault, A., Gueydon, E. & Schwob, E. Interplay between S-cyclin-dependent kinase and Dbf4-dependent kinase in controlling DNA replication through phosphorylation of yeast Mcm4 N-terminal domain. Mol. Biol. Cell 19, 2267–2277 (2008)
Cho, W. H., Lee, Y. J., Kong, S. I., Hurwitz, J. & Lee, J. K. CDC7 kinase phosphorylates serine residues adjacent to acidic amino acids in the minichromosome maintenance 2 protein. Proc. Natl Acad. Sci. USA 103, 11521–11526 (2006)
Montagnoli, A. et al. Identification of Mcm2 phosphorylation sites by S-phase-regulating kinases. J. Biol. Chem. 281, 10281–10290 (2006)
Aparicio, O. M., Weinstein, D. M. & Bell, S. P. Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91, 59–69 (1997)
Masai, H. et al. Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin. J. Biol. Chem. 281, 39249–39261 (2006)
Zou, L. & Stillman, B. Formation of a preinitiation complex by S-phase cyclin CDK-dependent loading of Cdc45p onto chromatin. Science 280, 593–596 (1998)
Jiang, W., McDonald, D., Hope, T. J. & Hunter, T. Mammalian Cdc7–Dbf4 protein kinase complex is essential for initiation of DNA replication. EMBO J. 18, 5703–5713 (1999)
Ogi, H., Wang, C. Z., Nakai, W., Kawasaki, Y. & Masumoto, H. The role of the Saccharomyces cerevisiae Cdc7–Dbf4 complex in the replication checkpoint. Gene 414, 32–40 (2008)
Shimada, K., Pasero, P. & Gasser, S. M. ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase. Genes Dev. 16, 3236–3252 (2002)
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.
Author information
Authors and Affiliations
Corresponding author
Supplementary information
Supplementary Information
This file contains a Supplementary Table, Supplementary Figures S1-S12 with Legends, Supplementary Notes and Supplementary References. (PDF 4366 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/nature08647
This article is cited by
-
CDC7-independent G1/S transition revealed by targeted protein degradation
Nature (2022)
-
Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase
Nature Structural & Molecular Biology (2022)
-
Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions
Nature Communications (2022)
-
A rethink about enzymes that drive DNA replication
Nature (2022)
-
Yeast Stn1 promotes MCM to circumvent Rad53 control of the S phase checkpoint
Current Genetics (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.