Limits...
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.

Show MeSH

Related in: MedlinePlus

High concentrations of cdk2–cyclin E inhibits both DNA  replication and the binding of MCM3 to chromatin. (A) Purified  egg cytosol containing 1 μM of added cdk2–cyclin E was preincubated for 30 min. After this incubation, membrane and sperm  (1,000/μl) were added to the extract. As shown, by using phase  optics (left), these sperm formed intact nuclei, and by fluorescent  staining (right), the DNA decondensed. (B) Interphase cytosol  was preincubated alone (− cdk2–cyclin E), or with 1 μM of cdk2– cyclin E (cdk2–Cyclin E) for 30 min. After this membrane and  1,000 sperm/μl were added. The autoradiogram shows 32P incorporated into DNA during 15 min pulses starting at indicated  times after sperm addition. In the absence of cyclin E, replication  occurred normally, while in the presence of cyclin E it was  strongly inhibited at all time points. (C) cdk2–cyclin E was added  to interphase cytosol to the final concentrations indicated. After  a 30-min incubation, 1,000 sperm/μl were added to each reaction,  the reactions were incubated for another 30 min, and then the  sperm were pelleted. The pellets were resuspended in SDS-PAGE  sample buffer. MCM3 bound to the sperm chromatin was determined by Western blotting using anti-MCM3 antibody. As shown,  MCM3 binding to chromatin was inhibited as the concentration  of cdk2–cyclin E increased from 0.12 to 1.0 μM. (D) Interphase  cytosol was first incubated with 1,000 sperm/μl for 30 min. 1 μM  of cdk2–cyclin E was then added together with membrane. Photographs show a typical nucleus that formed under these conditions. (E) Interphase cytosol was first incubated with 1,000  sperm/μl for 30 min. After this, membrane was added with or  without 1 μM of cdk2–cyclin E and DNA replication was assayed  as in B. Replication in both extracts was identical, demonstrating  that late addition of cdk2–cyclin E does not inhibit chromatin  from replicating. (F) Sperm was added to cytosol to a final concentration of 1,000 sperm/μl. After a 30-min incubation, the indicated amounts of cdk2–cyclin E were added, and the reaction was  incubated for another 30 min. At the end of the incubation the  sperm were pelleted and assayed for MCM3 by Western blotting  as described in C. As shown, late addition of cdk2–cyclin E does  not inhibit binding of MCM3 to chromatin. Bars: (A and D) 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139856&req=5

Figure 2: High concentrations of cdk2–cyclin E inhibits both DNA replication and the binding of MCM3 to chromatin. (A) Purified egg cytosol containing 1 μM of added cdk2–cyclin E was preincubated for 30 min. After this incubation, membrane and sperm (1,000/μl) were added to the extract. As shown, by using phase optics (left), these sperm formed intact nuclei, and by fluorescent staining (right), the DNA decondensed. (B) Interphase cytosol was preincubated alone (− cdk2–cyclin E), or with 1 μM of cdk2– cyclin E (cdk2–Cyclin E) for 30 min. After this membrane and 1,000 sperm/μl were added. The autoradiogram shows 32P incorporated into DNA during 15 min pulses starting at indicated times after sperm addition. In the absence of cyclin E, replication occurred normally, while in the presence of cyclin E it was strongly inhibited at all time points. (C) cdk2–cyclin E was added to interphase cytosol to the final concentrations indicated. After a 30-min incubation, 1,000 sperm/μl were added to each reaction, the reactions were incubated for another 30 min, and then the sperm were pelleted. The pellets were resuspended in SDS-PAGE sample buffer. MCM3 bound to the sperm chromatin was determined by Western blotting using anti-MCM3 antibody. As shown, MCM3 binding to chromatin was inhibited as the concentration of cdk2–cyclin E increased from 0.12 to 1.0 μM. (D) Interphase cytosol was first incubated with 1,000 sperm/μl for 30 min. 1 μM of cdk2–cyclin E was then added together with membrane. Photographs show a typical nucleus that formed under these conditions. (E) Interphase cytosol was first incubated with 1,000 sperm/μl for 30 min. After this, membrane was added with or without 1 μM of cdk2–cyclin E and DNA replication was assayed as in B. Replication in both extracts was identical, demonstrating that late addition of cdk2–cyclin E does not inhibit chromatin from replicating. (F) Sperm was added to cytosol to a final concentration of 1,000 sperm/μl. After a 30-min incubation, the indicated amounts of cdk2–cyclin E were added, and the reaction was incubated for another 30 min. At the end of the incubation the sperm were pelleted and assayed for MCM3 by Western blotting as described in C. As shown, late addition of cdk2–cyclin E does not inhibit binding of MCM3 to chromatin. Bars: (A and D) 10 μm.

Mentions: Although the level of cdk2–cyclin E activity does not vary significantly during the embryonic cell cycle (Fang and Newport, 1991; Rempel et al., 1995; Howe and Newport, 1996), the experiments presented above demonstrate that the actual concentration of kinase surrounding chromatin will vary over 200-fold depending on whether the cell cycle is at the end of mitosis (no nucleus) or S phase (intact nucleus). To investigate whether this gradient of kinase activity can participate in regulating DNA replication, E. coli–produced recombinant Xenopus cdk2 and cyclin E proteins were added to extracts to increase the cytosolic pool of cdk2–cyclin E surrounding chromatin before nuclear assembly. Specifically, cdk2–cyclin E was added to purified egg cytosol to a final concentration of 1 μM, or 16 times the normal cytosolic concentration. This extract was incubated for 30 min, and then membranes and sperm chromatin (1,000/μl) were added to the extract. Based on microscopic observations, the added chromatin and membrane assembled into normal nuclei, and the DNA decondensed (Fig. 2 A). In control extracts lacking exogenous cdk2– cyclin E, pulse labeling with radioactive dATP demonstrated that replication initiated, as usual, after a 20–30 min lag (Newport, 1987) continued for 30 min and then stopped (Fig. 2 B, − cdk2–cyclin E). By contrast, extracts which were preincubated with cdk2–cyclin E 30 min before addition of sperm and membranes failed to show any significant DNA replication (Fig. 2 B, Early Addition). This experiment demonstrates that if the concentration of cdk2–cyclin E present in the cytosol during nuclear assembly is 16-fold higher than normal, replication is strongly 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)

High concentrations of cdk2–cyclin E inhibits both DNA  replication and the binding of MCM3 to chromatin. (A) Purified  egg cytosol containing 1 μM of added cdk2–cyclin E was preincubated for 30 min. After this incubation, membrane and sperm  (1,000/μl) were added to the extract. As shown, by using phase  optics (left), these sperm formed intact nuclei, and by fluorescent  staining (right), the DNA decondensed. (B) Interphase cytosol  was preincubated alone (− cdk2–cyclin E), or with 1 μM of cdk2– cyclin E (cdk2–Cyclin E) for 30 min. After this membrane and  1,000 sperm/μl were added. The autoradiogram shows 32P incorporated into DNA during 15 min pulses starting at indicated  times after sperm addition. In the absence of cyclin E, replication  occurred normally, while in the presence of cyclin E it was  strongly inhibited at all time points. (C) cdk2–cyclin E was added  to interphase cytosol to the final concentrations indicated. After  a 30-min incubation, 1,000 sperm/μl were added to each reaction,  the reactions were incubated for another 30 min, and then the  sperm were pelleted. The pellets were resuspended in SDS-PAGE  sample buffer. MCM3 bound to the sperm chromatin was determined by Western blotting using anti-MCM3 antibody. As shown,  MCM3 binding to chromatin was inhibited as the concentration  of cdk2–cyclin E increased from 0.12 to 1.0 μM. (D) Interphase  cytosol was first incubated with 1,000 sperm/μl for 30 min. 1 μM  of cdk2–cyclin E was then added together with membrane. Photographs show a typical nucleus that formed under these conditions. (E) Interphase cytosol was first incubated with 1,000  sperm/μl for 30 min. After this, membrane was added with or  without 1 μM of cdk2–cyclin E and DNA replication was assayed  as in B. Replication in both extracts was identical, demonstrating  that late addition of cdk2–cyclin E does not inhibit chromatin  from replicating. (F) Sperm was added to cytosol to a final concentration of 1,000 sperm/μl. After a 30-min incubation, the indicated amounts of cdk2–cyclin E were added, and the reaction was  incubated for another 30 min. At the end of the incubation the  sperm were pelleted and assayed for MCM3 by Western blotting  as described in C. As shown, late addition of cdk2–cyclin E does  not inhibit binding of MCM3 to chromatin. Bars: (A and D) 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139856&req=5

Figure 2: High concentrations of cdk2–cyclin E inhibits both DNA replication and the binding of MCM3 to chromatin. (A) Purified egg cytosol containing 1 μM of added cdk2–cyclin E was preincubated for 30 min. After this incubation, membrane and sperm (1,000/μl) were added to the extract. As shown, by using phase optics (left), these sperm formed intact nuclei, and by fluorescent staining (right), the DNA decondensed. (B) Interphase cytosol was preincubated alone (− cdk2–cyclin E), or with 1 μM of cdk2– cyclin E (cdk2–Cyclin E) for 30 min. After this membrane and 1,000 sperm/μl were added. The autoradiogram shows 32P incorporated into DNA during 15 min pulses starting at indicated times after sperm addition. In the absence of cyclin E, replication occurred normally, while in the presence of cyclin E it was strongly inhibited at all time points. (C) cdk2–cyclin E was added to interphase cytosol to the final concentrations indicated. After a 30-min incubation, 1,000 sperm/μl were added to each reaction, the reactions were incubated for another 30 min, and then the sperm were pelleted. The pellets were resuspended in SDS-PAGE sample buffer. MCM3 bound to the sperm chromatin was determined by Western blotting using anti-MCM3 antibody. As shown, MCM3 binding to chromatin was inhibited as the concentration of cdk2–cyclin E increased from 0.12 to 1.0 μM. (D) Interphase cytosol was first incubated with 1,000 sperm/μl for 30 min. 1 μM of cdk2–cyclin E was then added together with membrane. Photographs show a typical nucleus that formed under these conditions. (E) Interphase cytosol was first incubated with 1,000 sperm/μl for 30 min. After this, membrane was added with or without 1 μM of cdk2–cyclin E and DNA replication was assayed as in B. Replication in both extracts was identical, demonstrating that late addition of cdk2–cyclin E does not inhibit chromatin from replicating. (F) Sperm was added to cytosol to a final concentration of 1,000 sperm/μl. After a 30-min incubation, the indicated amounts of cdk2–cyclin E were added, and the reaction was incubated for another 30 min. At the end of the incubation the sperm were pelleted and assayed for MCM3 by Western blotting as described in C. As shown, late addition of cdk2–cyclin E does not inhibit binding of MCM3 to chromatin. Bars: (A and D) 10 μm.
Mentions: Although the level of cdk2–cyclin E activity does not vary significantly during the embryonic cell cycle (Fang and Newport, 1991; Rempel et al., 1995; Howe and Newport, 1996), the experiments presented above demonstrate that the actual concentration of kinase surrounding chromatin will vary over 200-fold depending on whether the cell cycle is at the end of mitosis (no nucleus) or S phase (intact nucleus). To investigate whether this gradient of kinase activity can participate in regulating DNA replication, E. coli–produced recombinant Xenopus cdk2 and cyclin E proteins were added to extracts to increase the cytosolic pool of cdk2–cyclin E surrounding chromatin before nuclear assembly. Specifically, cdk2–cyclin E was added to purified egg cytosol to a final concentration of 1 μM, or 16 times the normal cytosolic concentration. This extract was incubated for 30 min, and then membranes and sperm chromatin (1,000/μl) were added to the extract. Based on microscopic observations, the added chromatin and membrane assembled into normal nuclei, and the DNA decondensed (Fig. 2 A). In control extracts lacking exogenous cdk2– cyclin E, pulse labeling with radioactive dATP demonstrated that replication initiated, as usual, after a 20–30 min lag (Newport, 1987) continued for 30 min and then stopped (Fig. 2 B, − cdk2–cyclin E). By contrast, extracts which were preincubated with cdk2–cyclin E 30 min before addition of sperm and membranes failed to show any significant DNA replication (Fig. 2 B, Early Addition). This experiment demonstrates that if the concentration of cdk2–cyclin E present in the cytosol during nuclear assembly is 16-fold higher than normal, replication is strongly 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