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Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases

Nature volume 412, pages 355–358 (2001)Cite this article

A Corrigendum to this article was published on 04 December 2003

Abstract

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs). In the budding yeast, Saccharomyces cerevisiae, inactivation of CDKs during late mitosis involves degradation of B-type cyclins as well as direct inhibition of cyclin–CDK complexes by the CDK-inhibitor protein Sic1 (refs 1,2,3). Several striking similarities exist between Sic1 and Cdc6, a DNA replication factor essential for the formation of pre-replicative complexes at origins of DNA replication4,5,6,7,8,9. Transcription of both genes is activated during late mitosis by a process dependent on Swi5 (ref. 10). Like Sic1, Cdc6 binds CDK complexes in vivo11,12 and downregulates them in vitro11. Here we show that Cdc6, like Sic1, also contributes to inactivation of CDKs during late mitosis in S. cerevisiae. Deletion of the CDK-interacting domain of Cdc6 does not inhibit the function of origins of DNA replication during S phase, but instead causes a delay in mitotic exit; this delay is accentuated in the absence of Sic1 or of cyclin degradation. By contributing to mitotic exit and inactivation of CDKs, Cdc6 helps to create the conditions that are required for its subsequent role in the formation of pre-replicative complexes at origins of DNA replication.

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Figure 1: Cdc6 promotes Clb2-associated kinase inhibition by a mechanism independent of Clb2 degradation.
Figure 2: Exit from mitosis is delayed in Δ 47cdc6 mutant cells compared with CDC6 wild-type cells.
Figure 3: The 47 amino-terminal truncation in CDC6 confers a strong mitotic delay to Δ hct1 cells.
Figure 4: Δ sic1 Δ 47cdc6 double-mutant cells are defective in the exit from mitosis.

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Acknowledgements

We thank J. Correa, K. Labib, S. Moreno, L. Pelloquin and the ASP laboratory for many discussions, and K. Labib for critically reading this manuscript. We are grateful to R. Basco, J. Correa, J. Diffley, S. Ufano and C. Vázquez for yeast strains and anti-Pgk antibody. We also thank N. Skinner for editing the text. This research was supported by CICYT and PGC grants to A.B.

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Author notes

  1. Arturo Calzada and Maria Sacristán: These authors contributed equally to this work

  2. Correspondence and requests for materials should be addressed to A.B.

Authors and Affiliations

  1. Departamento de Microbiología y Genética, Instituto de Microbiología-Bioquímica e Instituto de Biología Molecular y Celular del Cáncer, Campus Miguel de Unamuno, Universidad de Salamanca, Salamanca, CSIC 37007, Spain

    Arturo Calzada, Maria Sacristán, Elisa Sánchez & Avelino Bueno

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  1. Arturo Calzada
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Corresponding author

Correspondence to Avelino Bueno.

Supplementary information

Figure 1

(JPG 24.7 KB)

Activation of chromosomal DNA replication origins in D47cdc6 cells. Left and middle panels, two-dimensional gels of NcoI digested DNA from asynchronous cultures of wild-type or D47cdc6 strains hybridized with a 5 kb NcoI fragment containing ARS1. A drawing of how replication intermediates distribute on (N/N) two-dimensional gels.

Figure 2

(JPG 110 KB)

A nocodazole block-and-release experiment with wild-type, D47cdc6 and Dhct1 strains. Samples taken at the indicated intervals were processed for DNA content analysis (a), Clb2-associated H1 kinase activity (b and e, left panel), Westerns analysis (c, d and e, middle panel) and mitotic spindle analysis (e, right panel).

Figure 3

(JPG 62.2 KB)

Genetic interactions among cdc14-1 and cdc23-1 ts alleles and the D47cdc6 amino-terminal truncation. Ten-fold dilutions of wild-type, D47cdc6, cdc14-1, cdc23-1, cdc14-1 D47cdc6 and cdc23-1 D47cdc6 isogenic S. cerevisiae strains from cultures in log phase at 22°C were inoculated in YEPD Petri dishes and incubated at the indicated temperatures.

Figure 4

(JPG 25.2 KB)

SIC1 rescues the late-in-mitosis defect observed in D47cdc6 Dhct1 cells. (a), Clb2-associated H1 kinase assays and Western analysis in asynchronous D47cdc6 Dhct1 GAL1-10:SIC1 cells repressed (lane 1) or induced (lane 3) for SIC1 expression. Nocodazole samples (lanes 2 and 4) and untagged Clb2 control (C) are shown for reference. (b), Extended spindles analysis of the same strain when SIC1 is off and on.

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Calzada, A., Sacristán, M., Sánchez, E. et al. Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases. Nature 412, 355–358 (2001). https://doi.org/10.1038/35085610

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