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 (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.
Findeisen
M
M
Division of Biology, University of Konstanz, Germany.
El-Denary
M
M
Kapitza
T
T
Graf
R
R
Strausfeld
U
U
D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D019925:Cyclin A / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D019927:Cyclin E; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D064148:Minichromosome Maintenance Complex Component 4; D009865:Oocytes; D051738:Origin Recognition Complex; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014020:Tissue Extracts / Q000378:metabolism; D014982:Xenopus laevis
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D019925:Cyclin A; D019926:Cyclin B; D056744:Cyclin B1; D019927:Cyclin E; D004268:DNA-Binding Proteins; D051738:Origin Recognition Complex; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014020:Tissue Extracts; D011494:Protein Kinases; C102333:histone H1 kinase; D018844:Cyclin-Dependent Kinases; C092802:MCM4 protein, S cerevisiae; D064148:Minichromosome Maintenance Complex Component 4