Cyclin-dependent kinase 4/6 activity is a critical determinant of pre-replication complex assembly.

Abstract

Cyclin-dependent kinases (CDKs) are important in regulating cell cycle transitions, particularly in coordinating DNA replication. Although the role of CDK2 activity on the replication apparatus has been extensively studied, the role of CDK4/6 in DNA replication control is less understood. Through targeted inhibition of CDK4/6 activity, we demonstrate that CDK4/6 kinase activity promotes cdc6 and cdt1 expression, and pre-replication complex (pre-RC) assembly in cycling cells. Conversely, CDK2 inhibition had no effect on the pre-RC assembly. The inhibition of pre-RC assembly is dependent on a functional retinoblastoma (RB) protein, which mediates downstream effects. As such, CDK4/6 inhibition has minimal effect on the replication apparatus in the absence of RB. The requirement of CDK4/6 was further interrogated using cells lacking D-type cyclins, in which replication complexes form normally, and correspondingly CDK4/6 inhibition had no effect on cell cycle or replication control. However, in the absence of D-type cyclins, CDK2 inhibition resulted in the attenuation of cdc6 and cdt1 levels, suggesting overlapping roles for CDK4/6 and CDK2 in regulating replication protein activity. Finally, CDK4/6 inhibition prevented the accumulation of cdc6 and cdt1 as cells progressed from mitosis through the subsequent G(1). Combined, these studies indicate that CDK4/6 activity is important in regulating the expression of these critical mediators of DNA replication.