Cyclin A-CDK activity during G1 phase impairs MCM chromatin loading and inhibits DNA synthesis in mammalian cells

Abstract

Progression through the mammalian cell division cycle is regulated by the sequential activation of cyclin-dependent kinases, CDKs, at specific phases of the cell cycle. Cyclin A-CDK2 and cyclin A-CDK1 phosphorylate nuclear substrates during S and G(2) phases, respectfully. However, the DNA helicase complex, MCM2-7, is loaded onto the origin of replications in G(1), prior to the normally scheduled induction of cyclin A. It has previously been shown that cyclin A-CDKs phosphorylate MCM2 and MCM4 in vitro, thereby diminishing helicase activity. Thus, in this study we hypothesize that, in vivo, cyclin A-CDK activity during G(1) would result in an inhibition of progression into the S phase. To test this, we establish an in vivo method of inducing cyclin A-CDK activity in G(1) phase and observe that activation of cyclin A-CDK, but not cyclin E-CDK complexes, inhibit DNA synthesis without affecting other G(1) events such as cyclin D synthesis, E2F activation and cdc6 loading onto chromatin. We further report that the mechanism of this S phase inhibition occurs, at least in part, through impaired loading of MCM onto chromatin, presumably due to decreased levels of cdt1 and premature phosphorylation of MCM by cyclin A-CDK. In addition to providing in vivo confirmation of in vitro predictions regarding cyclin A-CDK phosphorylation of the MCM complex, our results provide insight into the cellular effects of unscheduled cyclin A-CDK activity in mammalian cells.