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

Abstract

The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities. High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin 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 detected 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 directly. We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.

Mammalian Orc1 Protein Is Selectively Released from Chromatin and Ubiquitinated during the S-to-M Transition in the Cell Division Cycle

“…The affinity of Orc proteins for chromatin may depend on their phosphorylated state. For example, both XlOrc1 and XlOrc2 are hyperphosphorylated in metaphase-arrested eggs relative to activated eggs (5,55), and Xenopus ORCs can be selectively released from chromatin by incubating chromatin either in a metaphase extract (46) or with Cdc2/cyclin A (16,19). Thus, cyclin-dependent protein kinases may be responsible for altering the affinity of Orc1 for ORC-chromatin sites as cells transit from S to M phase.…”

“…In Xenopus, Orc proteins in activated eggs bind to sperm chromatin, whereas Orc proteins in meiotic eggs do not (7,16,19,46), and both Orc1 and Orc2 proteins are present on chromatin in cultured Xenopus cells during interphase but not during metaphase (44). In Drosophila, Orc2 remains bound to chromosomes throughout the cell cycle (36), whereas the amount of nuclear bound DmOrc1 is greatest during late G 1 and S phases (3), suggesting a cell cycle-dependent, differential association of DmOrc proteins with chromatin.…”

Decreased origin usage and initiation of DNA replication in haploinsufficient HCT116 Ku80+/- cells

“…Cdc6 is required for the assembly of the pre-RC and loading of the MCM complex, the putative DNA replication helicase (reviewed by Bell and Dutta, 2002). Cdk2 phosphorylates Mcm4p (Detweiler and Li, 1998) and Orc1p (Findeisen et al, 1999), members of the MCM and ORC complex, respectively, and is required to trigger replication (reviewed by Bell and Dutta, 2002 +/-by high-salt extraction of isolated nuclei and were immunoblotted for both subunits of Ku. The expression of p53 and p21 was examined to determine whether they were induced by the reduced Ku80 levels in Ku80 +/-cells.…”

The Mechanisms and Consequences of DNA Re-replication

“…Cdk can phosphorylate Mcm2 and Mcm4 in vivo and in vitro (133)(134)(135)(136)(137). In S. cerevisiae, MCM2-7 proteins are normally exported from nucleus in S phase and this fails to occur after Cdk inactivation.…”

“…This nuclear export of Mcm2-7 in S phase is one of the mechanisms to 17 inhibit re-replication (138). In Xenopus, Mcm4 is phosphorylated by Cdk and then loses the chromatin loading capacity (133,135,137,139). Mouse Mcm4/6/7 seems to lose its helicase activity after Cdk phosphorylation (136).…”