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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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Nuclei assembled in the presence of high cdk2–cyclin E  fail to initiate DNA replication when the cytosol is diluted. (A) Interphase cytosol was first incubated with 1 μM of cdk2–cyclin E for  30 min. After this preincubation, sperm chromatin (1,000 sperm/μl)  and membrane were added. Aliquots were removed and incubated with [32P]dATP to determine early replication (left). As expected, high cdk2–cyclin E concentration blocked replication. The  remainder of the mixture was incubated for a further 60 min and  then diluted with 4 vol of fresh extract containing both cytosol  and membrane but lacking cdk2–cyclin E. The diluted reaction  was then divided in half, and new sperm chromatin (1,000/μl) was  added to one half. Radioactively labeled dATP was then added  to both reactions, and DNA replication was assayed after a further 60-min incubation. Nuclei assembled in the presence of high  cdk2–cyclin E concentrations failed to replicate after dilution of  the extract (− new sperm) while new nuclei added to such a diluted extract replicated normally (+ new sperm). (B) After dilution, an aliquot was taken from the sample “+ new sperm,” and  bio-dUTP was added. After 1 h of incubation, the nuclei were  spun onto a coverslip, stained with straptavidine-conjugated Texas  red, and mounted with Hoechst. Five nuclei were visualized in  this field (left), and four of them had bio-dUTP incorporated  (right).
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Figure 5: Nuclei assembled in the presence of high cdk2–cyclin E fail to initiate DNA replication when the cytosol is diluted. (A) Interphase cytosol was first incubated with 1 μM of cdk2–cyclin E for 30 min. After this preincubation, sperm chromatin (1,000 sperm/μl) and membrane were added. Aliquots were removed and incubated with [32P]dATP to determine early replication (left). As expected, high cdk2–cyclin E concentration blocked replication. The remainder of the mixture was incubated for a further 60 min and then diluted with 4 vol of fresh extract containing both cytosol and membrane but lacking cdk2–cyclin E. The diluted reaction was then divided in half, and new sperm chromatin (1,000/μl) was added to one half. Radioactively labeled dATP was then added to both reactions, and DNA replication was assayed after a further 60-min incubation. Nuclei assembled in the presence of high cdk2–cyclin E concentrations failed to replicate after dilution of the extract (− new sperm) while new nuclei added to such a diluted extract replicated normally (+ new sperm). (B) After dilution, an aliquot was taken from the sample “+ new sperm,” and bio-dUTP was added. After 1 h of incubation, the nuclei were spun onto a coverslip, stained with straptavidine-conjugated Texas red, and mounted with Hoechst. Five nuclei were visualized in this field (left), and four of them had bio-dUTP incorporated (right).

Mentions: To test these predictions, cdk2–cyclin E was added to purified cytosol and incubated for 30 min. After this, sperm chromatin (final 1,000/μl) and membranes were added. Aliquots of this sample were removed, incubated with radioactively labeled dATP, and then assayed for DNA replication. As expected, nuclei assembled in the presence of high cdk2–cyclin E failed to initiate replication during the subsequent 60 min incubation (Fig. 5 A, left). To test the effects of dilution on this inhibition, extracts were diluted with 4 vol of untreated extract. This addition dilutes cdk2– cyclin E remaining in the cytosol but should not dilute cdk2– cyclin E that has been compartmentalized within nuclei. The result of this experiment showed that nuclei preassembled in extracts initially containing high cdk2–cyclin E concentrations, failed to initiate DNA replication upon dilution (Fig. 5 A, right, top, − new sperm). This observation is consistent with the prediction that once cdk2–cyclin E is compartmentalized in nuclei, dilution of cdk2–cyclin E remaining in the cytosol will not restore replication.


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)

Nuclei assembled in the presence of high cdk2–cyclin E  fail to initiate DNA replication when the cytosol is diluted. (A) Interphase cytosol was first incubated with 1 μM of cdk2–cyclin E for  30 min. After this preincubation, sperm chromatin (1,000 sperm/μl)  and membrane were added. Aliquots were removed and incubated with [32P]dATP to determine early replication (left). As expected, high cdk2–cyclin E concentration blocked replication. The  remainder of the mixture was incubated for a further 60 min and  then diluted with 4 vol of fresh extract containing both cytosol  and membrane but lacking cdk2–cyclin E. The diluted reaction  was then divided in half, and new sperm chromatin (1,000/μl) was  added to one half. Radioactively labeled dATP was then added  to both reactions, and DNA replication was assayed after a further 60-min incubation. Nuclei assembled in the presence of high  cdk2–cyclin E concentrations failed to replicate after dilution of  the extract (− new sperm) while new nuclei added to such a diluted extract replicated normally (+ new sperm). (B) After dilution, an aliquot was taken from the sample “+ new sperm,” and  bio-dUTP was added. After 1 h of incubation, the nuclei were  spun onto a coverslip, stained with straptavidine-conjugated Texas  red, and mounted with Hoechst. Five nuclei were visualized in  this field (left), and four of them had bio-dUTP incorporated  (right).
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Figure 5: Nuclei assembled in the presence of high cdk2–cyclin E fail to initiate DNA replication when the cytosol is diluted. (A) Interphase cytosol was first incubated with 1 μM of cdk2–cyclin E for 30 min. After this preincubation, sperm chromatin (1,000 sperm/μl) and membrane were added. Aliquots were removed and incubated with [32P]dATP to determine early replication (left). As expected, high cdk2–cyclin E concentration blocked replication. The remainder of the mixture was incubated for a further 60 min and then diluted with 4 vol of fresh extract containing both cytosol and membrane but lacking cdk2–cyclin E. The diluted reaction was then divided in half, and new sperm chromatin (1,000/μl) was added to one half. Radioactively labeled dATP was then added to both reactions, and DNA replication was assayed after a further 60-min incubation. Nuclei assembled in the presence of high cdk2–cyclin E concentrations failed to replicate after dilution of the extract (− new sperm) while new nuclei added to such a diluted extract replicated normally (+ new sperm). (B) After dilution, an aliquot was taken from the sample “+ new sperm,” and bio-dUTP was added. After 1 h of incubation, the nuclei were spun onto a coverslip, stained with straptavidine-conjugated Texas red, and mounted with Hoechst. Five nuclei were visualized in this field (left), and four of them had bio-dUTP incorporated (right).
Mentions: To test these predictions, cdk2–cyclin E was added to purified cytosol and incubated for 30 min. After this, sperm chromatin (final 1,000/μl) and membranes were added. Aliquots of this sample were removed, incubated with radioactively labeled dATP, and then assayed for DNA replication. As expected, nuclei assembled in the presence of high cdk2–cyclin E failed to initiate replication during the subsequent 60 min incubation (Fig. 5 A, left). To test the effects of dilution on this inhibition, extracts were diluted with 4 vol of untreated extract. This addition dilutes cdk2– cyclin E remaining in the cytosol but should not dilute cdk2– cyclin E that has been compartmentalized within nuclei. The result of this experiment showed that nuclei preassembled in extracts initially containing high cdk2–cyclin E concentrations, failed to initiate DNA replication upon dilution (Fig. 5 A, right, top, − new sperm). This observation is consistent with the prediction that once cdk2–cyclin E is compartmentalized in nuclei, dilution of cdk2–cyclin E remaining in the cytosol will not restore replication.

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
Related in: MedlinePlus