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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 - J. Cell Biol. (1997)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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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High concentration of cdk2–cyclin E doesn't inhibit  ORC2 from binding to chromatin. 500 nM of cdk2–cyclin E was  added either before (early cdk2–cyclin E) or 30 min after (late cdk2– cyclin E) sperm addition. Chromatin fractions were extracted with  ELB containing 0.1% NP-40 and spun through a sucrose cushion  containing 0.1% NP-40. The pellet fractions were analyzed for  MCM3 and ORC2 content by Western blotting with specific antibodies. MCM3 binding to chromatin is inhibited by early addition  of cdk2–cyclin E, while ORC2 binding is unaffected.
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Figure 3: High concentration of cdk2–cyclin E doesn't inhibit ORC2 from binding to chromatin. 500 nM of cdk2–cyclin E was added either before (early cdk2–cyclin E) or 30 min after (late cdk2– cyclin E) sperm addition. Chromatin fractions were extracted with ELB containing 0.1% NP-40 and spun through a sucrose cushion containing 0.1% NP-40. The pellet fractions were analyzed for MCM3 and ORC2 content by Western blotting with specific antibodies. MCM3 binding to chromatin is inhibited by early addition of cdk2–cyclin E, while ORC2 binding is unaffected.

Mentions: With respect to how cdk2–cyclin E blocks the association of MCM with chromatin, it has been shown that the ORC complex binds to DNA before MCM and that this interaction is a prerequisite for MCM binding to chromatin (Coleman et al., 1996). Therefore, if high concentrations of cdk2–cyclin E modified ORC so as to prevent it from interacting with DNA, this modification would also prevent MCM from associating with chromatin. To test this possibility we used an anti-ORC2 antibody to examine whether binding of the ORC complex to chromatin was sensitive to cdk2–cyclin E concentration. The results of this experiment showed quite clearly that concentrations of exogenously added cdk2–cyclin E which completely inhibited MCM binding had no effect on the association of ORC with DNA (Fig. 3). Therefore, the inhibitory effects of cdk2-cyclin E on the interaction of MCM with chromatin appear to involve a step downstream of the association of ORC with DNA.


A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle.

Hua XH, Yan H, Newport J - J. Cell Biol. (1997)

High concentration of cdk2–cyclin E doesn't inhibit  ORC2 from binding to chromatin. 500 nM of cdk2–cyclin E was  added either before (early cdk2–cyclin E) or 30 min after (late cdk2– cyclin E) sperm addition. Chromatin fractions were extracted with  ELB containing 0.1% NP-40 and spun through a sucrose cushion  containing 0.1% NP-40. The pellet fractions were analyzed for  MCM3 and ORC2 content by Western blotting with specific antibodies. MCM3 binding to chromatin is inhibited by early addition  of cdk2–cyclin E, while ORC2 binding is unaffected.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139856&req=5

Figure 3: High concentration of cdk2–cyclin E doesn't inhibit ORC2 from binding to chromatin. 500 nM of cdk2–cyclin E was added either before (early cdk2–cyclin E) or 30 min after (late cdk2– cyclin E) sperm addition. Chromatin fractions were extracted with ELB containing 0.1% NP-40 and spun through a sucrose cushion containing 0.1% NP-40. The pellet fractions were analyzed for MCM3 and ORC2 content by Western blotting with specific antibodies. MCM3 binding to chromatin is inhibited by early addition of cdk2–cyclin E, while ORC2 binding is unaffected.
Mentions: With respect to how cdk2–cyclin E blocks the association of MCM with chromatin, it has been shown that the ORC complex binds to DNA before MCM and that this interaction is a prerequisite for MCM binding to chromatin (Coleman et al., 1996). Therefore, if high concentrations of cdk2–cyclin E modified ORC so as to prevent it from interacting with DNA, this modification would also prevent MCM from associating with chromatin. To test this possibility we used an anti-ORC2 antibody to examine whether binding of the ORC complex to chromatin was sensitive to cdk2–cyclin E concentration. The results of this experiment showed quite clearly that concentrations of exogenously added cdk2–cyclin E which completely inhibited MCM binding had no effect on the association of ORC with DNA (Fig. 3). Therefore, the inhibitory effects of cdk2-cyclin E on the interaction of MCM with chromatin appear to involve a step downstream of the association of ORC with DNA.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH