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J Cell Biol 1997 Apr 07;1371:183-92. doi: 10.1083/jcb.137.1.183.
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A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle.

Hua XH , Yan H , Newport J .

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.

PubMed ID: 9105046
PMC ID: PMC2139856
Article link: J Cell Biol
Grant support: [+]

Species referenced: Xenopus
Genes referenced: cdk2 mcm3 mmut orc2

Article Images: [+] show captions
References [+] :
Bell, Yeast origin recognition complex functions in transcription silencing and DNA replication. 1994, Pubmed