Cyclin A2-Cyclin-Dependent Kinase 2 Cooperates with the PLK1-SCF^β-TrCP1-EMI1-Anaphase-Promoting Complex/Cyclosome Axis To Promote Genome Reduplication in the Absence of Mitosis

Abstract

Limiting genome replication to once per cell cycle is vital for maintaining genome stability. Inhibition of cyclin-dependent kinase 1 (CDK1) with the specific inhibitor RO3306 is sufficient to trigger multiple rounds of genome reduplication. We demonstrated that although anaphase-promoting complex/cyclosome (APC/C) remained inactive during the initial G₂ arrest, it was activated upon prolonged inhibition of CDK1. Using cellular biosensors and live-cell imaging, we provide direct evidence that genome reduplication was associated with oscillation of APC/C activity and nuclear-cytoplasmic shuttling of CDC6 even in the absence of mitosis at the single-cell level. Genome reduplication was abolished by ectopic expression of EMI1 or depletion of CDC20 or CDH1, suggesting the critical role of the EMI1-APC/C axis. In support of this, degradation of EMI1 itself and genome reduplication were delayed after downregulation of PLK1 and β-TrCP1. In the absence of CDK1 activity, activation of APC/C and genome reduplication was dependent on cyclin A2 and CDK2. Genome reduplication was then promoted by a combination of APC/C-dependent destruction of geminin (thus releasing CDT1), accumulation of cyclin E2-CDK2, and CDC6. Collectively, these results underscore the crucial role of cyclin A2-CDK2 in regulating the PLK1-SCF^β-TrCP1-EMI1-APC/C axis and CDC6 to trigger genome reduplication after the activity of CDK1 is suppressed.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank Michele Pagano, Roger Tsien, and David Turner for generous gifts of reagents. Many thanks are due to Anita Lau, Nelson Lee, and Nicole Ng for technical assistance and members of the Poon laboratory for useful comments.

This work was supported in part by the Research Grants Council grants HKUST6415/05 M, HKUST6439/06 M, and 662007 to R.Y.C.P.