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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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Cyclin A inhibits MCM3 binding to chromatin and  blocks DNA replication. (A) Interphase cytosol was preincubated  alone (− CYCLIN A) or with 66 nM of cyclin A–GST fusion protein for 30 min. After this preincubation, membrane and sperm  (1,000 sperm/μl) were added. Equal volumes of these extracts  were removed at the indicated times and labeled with [32P]dATP  for 15 min. In extracts lacking added cyclin A, replication occurred normally, while in the presence of cyclin A replication was  strongly inhibited. (B) Interphase cytosol was preincubated with  or without 66 nM of cyclin A-GST for 30 min. Sperm nuclei were  then added to 5,000 sperm/μl. After a further 30-min incubation,  the samples were diluted fivefold with ELB and the sperm chromatin separated from the cytosol by centrifugation through a sucrose cushion. The cytosol (S) and chromatin (P) fractions were  analyzed for MCM3 content by Western blotting with antiMCM3 antibody. Preincubation of cytosol with cyclin A strongly  inhibited the subsequent association of MCM3 with chromatin.  (C) Interphase cytosol was first incubated with 1,000 sperm/μl for  30 min. This reaction was then divided in half, and 66 nM cyclin  A was added to one aliquot (+ CYCLIN A). Membrane was then  added to both halves, and DNA replication was assayed as in (A).  Under these conditions cyclin A did not inhibit DNA replication.  (D) Interphase cytosol was incubated with 5,000 sperm/μl for  30 min. Either ELB buffer (− CYCLIN A) or 67 nM of cyclin  A-GST (+ CYCLIN A) was then added to the reactions. After a  further 30-min incubation, samples were collected and analyzed  as in B. Under these conditions late addition of cyclin A did not  prevent MCM3 from binding to sperm chromatin. (E) The samples  were treated as in B above, however, instead of pelleting the sperm  after incubation, the chromatin-bound MCM3 was visualized by  staining with anti-MCM3 antibody, followed by rhodamine-labeled  anti–rabbit IgG. Early addition of cyclin A (+ CYCLIN A)  blocked the association of MCM3 with chromatin when compared  to controls lacking cyclin A (− CYCLIN A).
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Figure 6: Cyclin A inhibits MCM3 binding to chromatin and blocks DNA replication. (A) Interphase cytosol was preincubated alone (− CYCLIN A) or with 66 nM of cyclin A–GST fusion protein for 30 min. After this preincubation, membrane and sperm (1,000 sperm/μl) were added. Equal volumes of these extracts were removed at the indicated times and labeled with [32P]dATP for 15 min. In extracts lacking added cyclin A, replication occurred normally, while in the presence of cyclin A replication was strongly inhibited. (B) Interphase cytosol was preincubated with or without 66 nM of cyclin A-GST for 30 min. Sperm nuclei were then added to 5,000 sperm/μl. After a further 30-min incubation, the samples were diluted fivefold with ELB and the sperm chromatin separated from the cytosol by centrifugation through a sucrose cushion. The cytosol (S) and chromatin (P) fractions were analyzed for MCM3 content by Western blotting with antiMCM3 antibody. Preincubation of cytosol with cyclin A strongly inhibited the subsequent association of MCM3 with chromatin. (C) Interphase cytosol was first incubated with 1,000 sperm/μl for 30 min. This reaction was then divided in half, and 66 nM cyclin A was added to one aliquot (+ CYCLIN A). Membrane was then added to both halves, and DNA replication was assayed as in (A). Under these conditions cyclin A did not inhibit DNA replication. (D) Interphase cytosol was incubated with 5,000 sperm/μl for 30 min. Either ELB buffer (− CYCLIN A) or 67 nM of cyclin A-GST (+ CYCLIN A) was then added to the reactions. After a further 30-min incubation, samples were collected and analyzed as in B. Under these conditions late addition of cyclin A did not prevent MCM3 from binding to sperm chromatin. (E) The samples were treated as in B above, however, instead of pelleting the sperm after incubation, the chromatin-bound MCM3 was visualized by staining with anti-MCM3 antibody, followed by rhodamine-labeled anti–rabbit IgG. Early addition of cyclin A (+ CYCLIN A) blocked the association of MCM3 with chromatin when compared to controls lacking cyclin A (− CYCLIN A).

Mentions: To determine if cyclin A inhibited DNA replication and MCM3 binding, cytosol was preincubated with 66 nM of cyclin A (30 min), and then sperm chromatin was added and the extract incubated an additional 30 min. After this, the sperm were separated from the cytosol by centrifugation through sucrose, and chromatin-bound MCM3 was determined both from Western blots probed with antiMCM3 antibodies (Fig. 6 B) and immunofluorescent staining of the chromatin with this antibody (Fig. 6 E). The results of these experiments showed that in extracts preincubated with 66 nM cyclin A, both DNA replication in intact nuclei (Fig. 6 A) and MCM3 binding to chromatin (Fig. 6, B and E) were strongly inhibited. By contrast, when sperm were added to cytosol and incubated for 30 min before addition of 66 nM cyclin A, neither DNA replication (Fig. 6 C) nor MCM3 binding to chromatin (Fig. 6 D) was inhibited.


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)

Cyclin A inhibits MCM3 binding to chromatin and  blocks DNA replication. (A) Interphase cytosol was preincubated  alone (− CYCLIN A) or with 66 nM of cyclin A–GST fusion protein for 30 min. After this preincubation, membrane and sperm  (1,000 sperm/μl) were added. Equal volumes of these extracts  were removed at the indicated times and labeled with [32P]dATP  for 15 min. In extracts lacking added cyclin A, replication occurred normally, while in the presence of cyclin A replication was  strongly inhibited. (B) Interphase cytosol was preincubated with  or without 66 nM of cyclin A-GST for 30 min. Sperm nuclei were  then added to 5,000 sperm/μl. After a further 30-min incubation,  the samples were diluted fivefold with ELB and the sperm chromatin separated from the cytosol by centrifugation through a sucrose cushion. The cytosol (S) and chromatin (P) fractions were  analyzed for MCM3 content by Western blotting with antiMCM3 antibody. Preincubation of cytosol with cyclin A strongly  inhibited the subsequent association of MCM3 with chromatin.  (C) Interphase cytosol was first incubated with 1,000 sperm/μl for  30 min. This reaction was then divided in half, and 66 nM cyclin  A was added to one aliquot (+ CYCLIN A). Membrane was then  added to both halves, and DNA replication was assayed as in (A).  Under these conditions cyclin A did not inhibit DNA replication.  (D) Interphase cytosol was incubated with 5,000 sperm/μl for  30 min. Either ELB buffer (− CYCLIN A) or 67 nM of cyclin  A-GST (+ CYCLIN A) was then added to the reactions. After a  further 30-min incubation, samples were collected and analyzed  as in B. Under these conditions late addition of cyclin A did not  prevent MCM3 from binding to sperm chromatin. (E) The samples  were treated as in B above, however, instead of pelleting the sperm  after incubation, the chromatin-bound MCM3 was visualized by  staining with anti-MCM3 antibody, followed by rhodamine-labeled  anti–rabbit IgG. Early addition of cyclin A (+ CYCLIN A)  blocked the association of MCM3 with chromatin when compared  to controls lacking cyclin A (− CYCLIN A).
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Figure 6: Cyclin A inhibits MCM3 binding to chromatin and blocks DNA replication. (A) Interphase cytosol was preincubated alone (− CYCLIN A) or with 66 nM of cyclin A–GST fusion protein for 30 min. After this preincubation, membrane and sperm (1,000 sperm/μl) were added. Equal volumes of these extracts were removed at the indicated times and labeled with [32P]dATP for 15 min. In extracts lacking added cyclin A, replication occurred normally, while in the presence of cyclin A replication was strongly inhibited. (B) Interphase cytosol was preincubated with or without 66 nM of cyclin A-GST for 30 min. Sperm nuclei were then added to 5,000 sperm/μl. After a further 30-min incubation, the samples were diluted fivefold with ELB and the sperm chromatin separated from the cytosol by centrifugation through a sucrose cushion. The cytosol (S) and chromatin (P) fractions were analyzed for MCM3 content by Western blotting with antiMCM3 antibody. Preincubation of cytosol with cyclin A strongly inhibited the subsequent association of MCM3 with chromatin. (C) Interphase cytosol was first incubated with 1,000 sperm/μl for 30 min. This reaction was then divided in half, and 66 nM cyclin A was added to one aliquot (+ CYCLIN A). Membrane was then added to both halves, and DNA replication was assayed as in (A). Under these conditions cyclin A did not inhibit DNA replication. (D) Interphase cytosol was incubated with 5,000 sperm/μl for 30 min. Either ELB buffer (− CYCLIN A) or 67 nM of cyclin A-GST (+ CYCLIN A) was then added to the reactions. After a further 30-min incubation, samples were collected and analyzed as in B. Under these conditions late addition of cyclin A did not prevent MCM3 from binding to sperm chromatin. (E) The samples were treated as in B above, however, instead of pelleting the sperm after incubation, the chromatin-bound MCM3 was visualized by staining with anti-MCM3 antibody, followed by rhodamine-labeled anti–rabbit IgG. Early addition of cyclin A (+ CYCLIN A) blocked the association of MCM3 with chromatin when compared to controls lacking cyclin A (− CYCLIN A).
Mentions: To determine if cyclin A inhibited DNA replication and MCM3 binding, cytosol was preincubated with 66 nM of cyclin A (30 min), and then sperm chromatin was added and the extract incubated an additional 30 min. After this, the sperm were separated from the cytosol by centrifugation through sucrose, and chromatin-bound MCM3 was determined both from Western blots probed with antiMCM3 antibodies (Fig. 6 B) and immunofluorescent staining of the chromatin with this antibody (Fig. 6 E). The results of these experiments showed that in extracts preincubated with 66 nM cyclin A, both DNA replication in intact nuclei (Fig. 6 A) and MCM3 binding to chromatin (Fig. 6, B and E) were strongly inhibited. By contrast, when sperm were added to cytosol and incubated for 30 min before addition of 66 nM cyclin A, neither DNA replication (Fig. 6 C) nor MCM3 binding to chromatin (Fig. 6 D) was inhibited.

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