Distinct Action of the RB pathway on the DNA replication machinery defines specific roles for CDK-complexes in preRC assembly & S-phase progression

Department of Cell Biology and the UC Cancer Center, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599; Department of Medicine, Johns Hopkins University, Baltimore, MD 21287; Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

^* To whom correspondence should be addressed. Email: Erik.Knudsen{at}uc.edu.

	Abstract

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 to suppress aberrant proliferation and control genome stability. Here we demonstrate a novel role for p16ink4a in replication control that is distinct from RB. Specifically, p16ink4a disrupts pre-replication complex assembly by inhibiting MCM loading in G1, while RB was found to disrupt replication in S-phase through attenuation of PCNA function. This influence of p16ink4a on the pre-replication complexes was dependent on presence of RB and down-regulation of CDK-activity. Strikingly, inhibition of CDK2 activity was not sufficient to prevent the loading of MCM proteins onto chromatin, thus supporting a model wherein the composite action of multiple G1-CDK complexes regulates pre-replication complex assembly. Additionally, p16ink4a attenuated the levels of the assembly factors Cdt1 and Cdc6. Enforced expression of these two licensing factors was sufficient to restore the assembly of the pre-replication 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.