Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6,and MCM in egg extracts of Xenopus laevis

The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MC M) proteins onto chromatin to form the preinitiation complex. In Xenopus eg g extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that ch romatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-depe ndent kinase activities. High cyclin A-dependent kinase activity inhibits t he binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sper m chromatin, but has no effect on chromatin binding of control proteins. Cy clin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not d etected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM dire ctly. We conclude that chromatin-bound cyclin A kinase controls DNA replica tion by protein phosphorylation and chromatin release of Cdc6 and MCM, wher eas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.