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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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Inhibition of MCM3  binding requires active cdk2– cyclin E kinase activity. (A)  500 nM of cdk2–cyclin E was  first incubated with interphase cytosol for 30 min  (cdk2–cyclin E). The reaction was then split into two  parts, and cip (final 600 nM)  was added to one half (+ cip). After a 15-min incubation, sperm  was added (final 5,000/μl) to both, and the reactions were carried  out for another 30 min. Chromatin-associated MCM3 was analyzed by Western blotting using anti-MCM3 antibody. Control  shows the amount of MCM3 bound to chromatin without cdk2– cyclin E treatment. When the kinase activity of cdk2–cyclin E  was blocked by cip, it could no longer inhibit MCM3 binding. (B)  Interphase cytosol was incubated either alone (control) or with  indicated concentrations of cdk2-K33R–cyclin E for 30 min. After this incubation, sperm chromatin was added to 5,000/μl. After  being incubated for another 30 min, the sperm was pelleted.  MCM3 bound to the sperm chromatin was assayed as in A. cdk2K33R–cyclin E, which is a kinase inactive complex, does not inhibit MCM3 from binding to chromatin.
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Figure 4: Inhibition of MCM3 binding requires active cdk2– cyclin E kinase activity. (A) 500 nM of cdk2–cyclin E was first incubated with interphase cytosol for 30 min (cdk2–cyclin E). The reaction was then split into two parts, and cip (final 600 nM) was added to one half (+ cip). After a 15-min incubation, sperm was added (final 5,000/μl) to both, and the reactions were carried out for another 30 min. Chromatin-associated MCM3 was analyzed by Western blotting using anti-MCM3 antibody. Control shows the amount of MCM3 bound to chromatin without cdk2– cyclin E treatment. When the kinase activity of cdk2–cyclin E was blocked by cip, it could no longer inhibit MCM3 binding. (B) Interphase cytosol was incubated either alone (control) or with indicated concentrations of cdk2-K33R–cyclin E for 30 min. After this incubation, sperm chromatin was added to 5,000/μl. After being incubated for another 30 min, the sperm was pelleted. MCM3 bound to the sperm chromatin was assayed as in A. cdk2K33R–cyclin E, which is a kinase inactive complex, does not inhibit MCM3 from binding to chromatin.

Mentions: In principle, increasing the cdk2–cyclin E concentration in the cytosol could inhibit DNA replication by two distinct mechanisms. The increased kinase activity of cdk2 could lead to the phosphorylation and inhibition of components required for assembly of MCM onto DNA. Alternatively, the increased concentration of cdk2–cyclin E protein complex could bind to and sequester activities required for mediating the association of MCM with chromatin (Piatti et al., 1996). Two experiments were performed to distinguish between these kinase- and complex-dependent possibilities. In one experiment a high concentration of cdk2– cyclin E was added to an extract, and the extract was then incubated for 30 min. After this the cdk2 kinase inhibitor Cip was added to the extract to inactivate cdk2–cyclin E kinase activity. Sperm chromatin was then added and assayed for MCM binding as described above. The result of this experiment (Fig. 4 A) demonstrated that the inactivation of cdk2–cyclin E kinase activity by Cip restores binding of MCM to chromatin. This result strongly suggests that cdk2 kinase activity itself, rather than the sequestration of factors by cdk2–cyclin E complex, is responsible for blocking the association of MCM with chromatin.


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)

Inhibition of MCM3  binding requires active cdk2– cyclin E kinase activity. (A)  500 nM of cdk2–cyclin E was  first incubated with interphase cytosol for 30 min  (cdk2–cyclin E). The reaction was then split into two  parts, and cip (final 600 nM)  was added to one half (+ cip). After a 15-min incubation, sperm  was added (final 5,000/μl) to both, and the reactions were carried  out for another 30 min. Chromatin-associated MCM3 was analyzed by Western blotting using anti-MCM3 antibody. Control  shows the amount of MCM3 bound to chromatin without cdk2– cyclin E treatment. When the kinase activity of cdk2–cyclin E  was blocked by cip, it could no longer inhibit MCM3 binding. (B)  Interphase cytosol was incubated either alone (control) or with  indicated concentrations of cdk2-K33R–cyclin E for 30 min. After this incubation, sperm chromatin was added to 5,000/μl. After  being incubated for another 30 min, the sperm was pelleted.  MCM3 bound to the sperm chromatin was assayed as in A. cdk2K33R–cyclin E, which is a kinase inactive complex, does not inhibit MCM3 from binding to chromatin.
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Figure 4: Inhibition of MCM3 binding requires active cdk2– cyclin E kinase activity. (A) 500 nM of cdk2–cyclin E was first incubated with interphase cytosol for 30 min (cdk2–cyclin E). The reaction was then split into two parts, and cip (final 600 nM) was added to one half (+ cip). After a 15-min incubation, sperm was added (final 5,000/μl) to both, and the reactions were carried out for another 30 min. Chromatin-associated MCM3 was analyzed by Western blotting using anti-MCM3 antibody. Control shows the amount of MCM3 bound to chromatin without cdk2– cyclin E treatment. When the kinase activity of cdk2–cyclin E was blocked by cip, it could no longer inhibit MCM3 binding. (B) Interphase cytosol was incubated either alone (control) or with indicated concentrations of cdk2-K33R–cyclin E for 30 min. After this incubation, sperm chromatin was added to 5,000/μl. After being incubated for another 30 min, the sperm was pelleted. MCM3 bound to the sperm chromatin was assayed as in A. cdk2K33R–cyclin E, which is a kinase inactive complex, does not inhibit MCM3 from binding to chromatin.
Mentions: In principle, increasing the cdk2–cyclin E concentration in the cytosol could inhibit DNA replication by two distinct mechanisms. The increased kinase activity of cdk2 could lead to the phosphorylation and inhibition of components required for assembly of MCM onto DNA. Alternatively, the increased concentration of cdk2–cyclin E protein complex could bind to and sequester activities required for mediating the association of MCM with chromatin (Piatti et al., 1996). Two experiments were performed to distinguish between these kinase- and complex-dependent possibilities. In one experiment a high concentration of cdk2– cyclin E was added to an extract, and the extract was then incubated for 30 min. After this the cdk2 kinase inhibitor Cip was added to the extract to inactivate cdk2–cyclin E kinase activity. Sperm chromatin was then added and assayed for MCM binding as described above. The result of this experiment (Fig. 4 A) demonstrated that the inactivation of cdk2–cyclin E kinase activity by Cip restores binding of MCM to chromatin. This result strongly suggests that cdk2 kinase activity itself, rather than the sequestration of factors by cdk2–cyclin E complex, is responsible for blocking the association of MCM with chromatin.

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