ATP-dependent activation of p21WAF¹/CIP¹-associated Cdk2 by Cdc6

When cells progressing in mid-S phase are damaged with a base-modifying chemical, they arrest in S phase long after the CHK1 checkpoint signal fades out, partly because of p53-mediated long-lasting induction of the cyclin-dependent kinase inhibitor p21WAF¹/CIP¹. We have recently found that enforced expression of Cdc6, the assembler of prereplicative complexes, markedly advances recovery from the prolonged S-phase arrest and reactivation of Cdk2 despite the presence of a high level of induced p21. Here, we report that Cdc6 protein can activate p21-associated Cdk2 in an ATP-dependent manner in vitro. Consistently, Cdc6 mutated for ATPase or a putative cyclin binding motif is no longer able to activate the Cdk2 in vitro or promote reinitiation of S-phase progression and reactivation of Cdk2 in vivo. These results reveal the never anticipated function of Cdc6 and redefine its role in the control of S-phase progression in mammalian cells.