title: 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.
authors.affiliation: Department of Pharmacological Sciences at Saint Louis University, St. Louis, Missouri 63104, USA.
authors.forename: Leroy W
authors.initials: LW
authors.lastname: Wheeler
authors.forename: Nathan H
authors.initials: NH
authors.lastname: Lents
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Baldassare
mesh_terms: D000818:Animals; D016466:CHO Cells; D002843:Chromatin / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D019925:Cyclin A / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016193:G1 Phase; D006801:Humans; D019913:Nuclear Localization Signals; D021381:Protein Transport; D016196:S Phase
chemical_list: D002843:Chromatin; D019925:Cyclin A; D004268:DNA-Binding Proteins; D019913:Nuclear Localization Signals; D004247:DNA; D018844:Cyclin-Dependent Kinases