A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle.
Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2-cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2-cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2-cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2-cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2-cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2-cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.
Hua
X H
XH
Biology Department, University of California, San Diego, La Jolla 92093-0347, USA.
Yan
H
H
Newport
J
J
D000818:Animals; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000502:physiology; D002843:Chromatin / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism / Q000494:pharmacology; D003600:Cytosol / Q000737:chemistry; D004261:DNA Replication / Q000502:physiology*; D005260:Female; D016193:G1 Phase / Q000502:physiology; D016195:G2 Phase / Q000502:physiology; D008297:Male; D009865:Oocytes / Q000166:cytology / Q000201:enzymology; D011485:Protein Binding / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D018741:Replication Origin / Q000502:physiology; D016196:S Phase / Q000502:physiology*; D013094:Spermatozoa / Q000737:chemistry; D014981:Xenopus; D029867:Xenopus Proteins
D018797:Cell Cycle Proteins; D002843:Chromatin; D016213:Cyclins; D029867:Xenopus Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases