In eukaryotic cells, cyclin-dependent kinases (CDKs) have an important involvement at various points in the cell cycle. At the onset of S phase, active CDK is essential for chromosomal DNA replication1, although its precise role is unknown. In budding yeast (Saccharomyces cerevisiae), the replication protein Sld2 (ref. 2) is an essential CDK substrate3, but its phospho-mimetic form (Sld2-11D) alone neither affects cell growth4 nor promotes DNA replication in the absence of CDK activity4, suggesting that other essential CDK substrates promote DNA replication. Here we show that both an allele of CDC45 (JET1) and high-copy DPB11, in combination with Sld2-11D, separately confer CDK-independent DNA replication. Although Cdc45 is not an essential CDK substrate, CDK-dependent phosphorylation of Sld3, which associates with Cdc45 (ref. 5), is essential and generates a binding site for Dpb11. Both the JET1 mutation and high-copy DPB11 by-pass the requirement for Sld3 phosphorylation in DNA replication. Because phosphorylated Sld2 binds to the carboxy-terminal pair of BRCT domains in Dpb11 (ref. 4), we propose that Dpb11 connects phosphorylated Sld2 and Sld3 to facilitate interactions between replication proteins, such as Cdc45 and GINS. Our results demonstrate that CDKs regulate interactions between BRCT-domain-containing replication proteins and other phosphorylated proteins for the initiation of chromosomal DNA replication; similar regulation may take place in higher eukaryotes.
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We thank J. Diffley and P. Zegerman for sharing unpublished information; B. Stillman for anti-Orc6 antibody; A. Calzada, M. Foiani and M. Lopes for protocol of two-dimensional gel assay; H. Masukata for protocols of density labelling and detection of DNA in a CsCl density gradient; S. Endo, E. Nakashima, Y. Suzuki and S. Yamaguchi for technical assistance; Y. Tanaka for discussion; and J. Diffley, K. Furuya, H. Masukata and K. Sugimoto for critical reading of the manuscript. This study was partly supported by grants to H.A. and S.T. from Ministry of Education, Culture, Sports, Science and Technology, Japan.
Author Contributions S.M. isolated and characterized novel mutations of DPB11; Y.K. analysed the interaction of Sld3 with other proteins; K.H. analysed in vitro interaction between Dpb11 and Sld3; T.U. analysed the interaction between Sld3 and Dpb11, alanine-substituted sld3 mutants and phosphorylation of Sld3; S.T. isolated and characterized the JET1 mutation, and analysed the relationship between Cdc45/Jet1 and Sld3; H.A. organized this study and wrote the paper. All authors discussed the results and commented on the manuscript.
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
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Tanaka, S., Umemori, T., Hirai, K. et al. CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast. Nature 445, 328–332 (2007) doi:10.1038/nature05465
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