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Distinct Action of the Retinoblastoma Pathway on the DNA Replication Machinery Defines Specific Roles for Cyclin-Dependent Kinase Complexes in Prereplication Complex Assembly and S-Phase Progression

Department of Cell Biology and UC Cancer Center, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267,¹ Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599,² Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21287,³ Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267⁴

Received 9 January 2006/ Returned for modification 24 February 2006/ Accepted 26 July 2006

The retinoblastoma (RB) and p16ink4a tumor suppressors are believed to function in a linear pathway that is functionally inactivated in a large fraction of human cancers. Recent studies have shown that RB plays a critical role in regulating S phase as a means for suppressing aberrant proliferation and controlling genome stability. Here, we demonstrate a novel role for p16ink4a in replication control that is distinct from that of RB. Specifically, p16ink4a disrupts prereplication complex assembly by inhibiting mini-chromosome maintenance (MCM) protein loading in G₁, while RB was found to disrupt replication in S phase through attenuation of PCNA function. This influence of p16ink4a on the prereplication complex was dependent on the presence of RB and the downregulation of cyclin-dependent kinase (CDK) activity. Strikingly, the inhibition of CDK2 activity was not sufficient to prevent the loading of MCM proteins onto chromatin, which supports a model wherein the composite action of multiple G₁ CDK complexes regulates prereplication complex assembly. Additionally, p16ink4a attenuated the levels of the assembly factors Cdt1 and Cdc6. The enforced expression of these two licensing factors was sufficient to restore the assembly of the prereplication complex yet failed to promote S-phase progression due to the continued absence of PCNA function. Combined, these data reveal that RB and p16ink4a function through distinct pathways to inhibit the replication machinery and provide evidence that stepwise regulation of CDK activity interfaces with the replication machinery at two discrete execution points.

Published ahead of print on 14 August 2006.

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