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Biology Department, University of California, San Diego, La Jolla, California 92093-0347
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.
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