CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast

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Abstract

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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Figure 1: CDK-catalysed essential phosphorylation of the Sld3 protein.
Figure 2: Phosphorylation-dependent interaction between Dpb11 and Sld3.
Figure 3: CDK-independent increase of cellular DNA content.
Figure 4: CDK-independent chromosome DNA replication.

References

  1. 1

    Schwob, E. & Labib, K. Regulating initiation events in yeasts. In DNA Replication and Human Disease 295–311 (Cold Spring Harbor Press, New York, 2006)

  2. 2

    Kamimura, Y., Masumoto, H., Sugino, A. & Araki, H. Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication. Mol. Cell. Biol. 18, 6102–6109 (1998)

  3. 3

    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)

  4. 4

    Tak, Y. S., Tanaka, Y., Endo, S., Kamimura, Y. & Araki, H. A CDK-catalysed regulatory phosphorylation for formation of the DNA replication complex Sld2-Dpb11. EMBO J. 25, 1987–1996 (2006)

  5. 5

    Kamimura, Y., Tak, Y. S., Sugino, A. & Araki, H. Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae. EMBO J. 20, 2097–2107 (2001)

  6. 6

    Diffley, J. F. X. Regulation of early events in chromosome replication. Curr. Biol. 14, R778–R786 (2004)

  7. 7

    Blow, J. J. & Dutta, A. Preventing re-replication of chromosomal DNA. Nature Rev. Mol. Cell Biol. 6, 476–486 (2005)

  8. 8

    Sivaprasad, U., Dutta, A. & Bell, S. P. Assembly of pre-replication complexes. In DNA Replication and Human Disease 63–88 (Cold Spring Harbor Press, New York, 2006)

  9. 9

    Bell, S. P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002)

  10. 10

    Kearsey, S. E. & Cotterill, S. Enigmatic variations: divergent modes of regulating eukaryotic DNA replication. Mol. Cell 12, 1067–1075 (2003)

  11. 11

    Mendez, J. & Stillman, B. Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins. Bioessays 25, 1158–1167 (2003)

  12. 12

    Walter, J. C. & Araki, H. Activation of pre-replication complexes. In DNA Replication and Human Disease 89–104 (Cold Spring Harbor Press, New York, 2006)

  13. 13

    Kanemaki, M. & Labib, K. Distinct roles for Sld3 and GINS during establishment and progression of eukaryotic DNA replication forks. EMBO J. 25, 1753–1763 (2006)

  14. 14

    Nguyen, V. Q., Co, C. & Li, J. J. Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature 411, 1068–1073 (2001)

  15. 15

    Mendenhall, M. D. & Hodge, A. E. Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62, 1191–1243 (1998)

  16. 16

    Araki, H., Leem, S. H., Phongdara, A. & Sugino, A. Dpb11, which interacts with DNA polymerase II (ε) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint. Proc. Natl Acad. Sci. USA 92, 11791–11795 (1995)

  17. 17

    Glover, J. N., Williams, R. S. & Lee, M. S. Interactions between BRCT repeats and phosphoproteins: tangled up in two. Trends Biochem. Sci. 29, 579–585 (2004)

  18. 18

    Zegerman, P. & Diffley, J. F. X. Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. Nature advance online publication doi:10.1038/nature05432 (13 December 2006).

  19. 19

    Labib, K., Diffley, J. F. & Kearsey, S. E. G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus. Nature Cell Biol. 1, 415–422 (1999)

  20. 20

    Bishop, A. C. et al. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395–401 (2000)

  21. 21

    Masai, H. & Arai, K. Cdc7 kinase complex: a key regulator in the initiation of DNA replication. J. Cell. Physiol. 190, 287–296 (2002)

  22. 22

    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)

  23. 23

    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)

  24. 24

    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)

  25. 25

    Brewer, B. J. & Fangman, W. L. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51, 463–471 (1987)

  26. 26

    Takayama, Y. et al. GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast. Genes Dev. 17, 1153–1165 (2003)

  27. 27

    Masumoto, H., Sugino, A. & Araki, H. Dpb11 controls the association between DNA polymerases α and ε and the autonomously replicating sequence region of budding yeast. Mol. Cell. Biol. 20, 2809–2817 (2000)

  28. 28

    Edwards, S. et al. Saccharomyces cerevisiae DNA polymerase ε and polymerase σ interact physically and functionally, suggesting a role for polymerase ε in sister chromatid cohesion. Mol. Cell. Biol. 23, 2733–2748 (2003)

  29. 29

    Garcia, V., Furuya, K. & Carr, A. M. Identification and functional analysis of TopBP1 and its homologs. DNA Repair (Amst.) 4, 1227–1239 (2005)

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Acknowledgements

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.

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Correspondence to Hiroyuki Araki.

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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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