Intra-S-Phase Checkpoint Activation by Direct CDK2 Inhibition

Abstract

To ensure proper progression through a cell cycle, checkpoints have evolved to play a surveillance role in maintaining genomic integrity. In this study, we demonstrate that loss of CDK2 activity activates an intra-S-phase checkpoint. CDK2 inhibition triggers a p53-p21 response via ATM- and ATR-dependent p53 phosphorylation at serine 15. Phosphorylation of other ATM and ATR downstream substrates, such as H2AX, NBS1, CHK1, and CHK2 is also increased. We show that during S phase when CDK2 activity is inhibited, there is an unexpected loading of the minichromosome maintenance complex onto chromatin. In addition, there is an increased number of cells with more than 4N DNA content, detected in the absence of p53, suggesting that rereplication can occur as a result of CDK2 disruption. Our findings identify an important role for CDK2 in the maintenance of genomic stability, acting via an ATM- and ATR-dependent pathway.

We thank all members of the High Throughput Screening Laboratory for kinase assays and initial studies of CDK2 inhibitor; Song Cho, Scott Frank, and Christian Toouli for critical reading of the manuscript; and Larry L'Italien for p53 siRNA validation.

This work was supported in part by the Schering-Plough Research Institute.