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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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Comparison between the inhibitor and licensing models. In the inhibitor model, proteins required  for potentiating DNA replication (shaded circles) bind  to DNA when cdk2 activity is  low. In Xenopus eggs this occurs at the end of mitosis before nuclear formation, when  cdk2–cyclin E activity is dilute (1 and 2). In somatic  cells this would occur during  early G1 when cdk2–cyclin E  is inactive. After nuclear formation, cdk2–cyclin E (black  squares) is rapidly transported into nuclei and accumulates (4). Replication potentiating proteins which  enter the nucleus (black circles) are prevented from associating with DNA due to  the high nuclear concentration of cdk2–cyclin E. However, the nuclear cdk2 does  not displace potentiating  proteins which are prebound  to DNA. Similarly in somatic  cells, activation of nuclear cdk2–cyclin E or A kinases during late  G1 would block any further potentiation of DNA for replication.  During DNA replication the potentiating proteins are displaced  from DNA and prevented from rebinding DNA by the presence  of high concentrations of cdk2 kinase activity (5 and 6). In the licensing model, proteins (shaded circles) required for DNA replication associate with DNA at the end of mitosis (1 and 2). Because these proteins cannot enter the nucleus, enclosure of the  DNA within the nucleus blocks further licensing (3 and 4). As a  result of DNA replication the licensing proteins are converted to  an inactive state (5 and 6).
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Figure 7: Comparison between the inhibitor and licensing models. In the inhibitor model, proteins required for potentiating DNA replication (shaded circles) bind to DNA when cdk2 activity is low. In Xenopus eggs this occurs at the end of mitosis before nuclear formation, when cdk2–cyclin E activity is dilute (1 and 2). In somatic cells this would occur during early G1 when cdk2–cyclin E is inactive. After nuclear formation, cdk2–cyclin E (black squares) is rapidly transported into nuclei and accumulates (4). Replication potentiating proteins which enter the nucleus (black circles) are prevented from associating with DNA due to the high nuclear concentration of cdk2–cyclin E. However, the nuclear cdk2 does not displace potentiating proteins which are prebound to DNA. Similarly in somatic cells, activation of nuclear cdk2–cyclin E or A kinases during late G1 would block any further potentiation of DNA for replication. During DNA replication the potentiating proteins are displaced from DNA and prevented from rebinding DNA by the presence of high concentrations of cdk2 kinase activity (5 and 6). In the licensing model, proteins (shaded circles) required for DNA replication associate with DNA at the end of mitosis (1 and 2). Because these proteins cannot enter the nucleus, enclosure of the DNA within the nucleus blocks further licensing (3 and 4). As a result of DNA replication the licensing proteins are converted to an inactive state (5 and 6).

Mentions: It has been shown that when nuclei which have replicated once in a Xenopus extract are isolated, permeablized, and then added back to a second extract, these nuclei reinitiate replication (Blow and Laskey, 1988). By contrast, nuclei which are not permeablized before addition to a second extract fail to initiate replication. Similarly, when G2 nuclei, isolated from somatic tissue culture cells, are permeablized and then added to egg extracts, they replicate, whereas intact G2 nuclei do not (Leno et al., 1992). These observations have led to the proposal that a chromatin binding factor, called licensing factor, is essential for replication, and that the compartmental distribution of licensing factor between the nucleus and cytosol serves to limit replication to a single round per cell cycle (Blow and Laskey, 1988; Blow, 1993; Kubota and Takisawa, 1993). Specifically, the model predicts that licensing factor itself cannot enter nuclei. As such, the factor can only associate with and potentiate chromatin for DNA replication when the nuclear envelope is disassembled at mitosis. By contrast, our data strongly suggest that the compartmentalized accumulation of either cyclin E– or A–dependent cdk2 kinases within the nucleus may act to inhibit endoreduplication (Fig. 7). Our data are consistent with genetic observations implicating cdk kinases in blocking re-replication during the cell cycle (Broek et al., 1991; Hayle et al., 1994; Correa-Bordes and Nurse, 1995; Sauer et al., 1995). An advantage of this compartmentalized inhibitor model over the licensing model is that the binding of proteins required for one round of DNA replication is no longer restricted exclusively to mitosis. Rather, these associations can occur at times when cdk2–cyclin E– or A–dependent kinases are either dilute or inactive. During the early Xenopus embryonic cell cycle this would occur at the end of mitosis before nuclei have formed, while during the somatic cell cycle, this would occur during early G1, when cdk2–cyclin E kinase is inactive.


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)

Comparison between the inhibitor and licensing models. In the inhibitor model, proteins required  for potentiating DNA replication (shaded circles) bind  to DNA when cdk2 activity is  low. In Xenopus eggs this occurs at the end of mitosis before nuclear formation, when  cdk2–cyclin E activity is dilute (1 and 2). In somatic  cells this would occur during  early G1 when cdk2–cyclin E  is inactive. After nuclear formation, cdk2–cyclin E (black  squares) is rapidly transported into nuclei and accumulates (4). Replication potentiating proteins which  enter the nucleus (black circles) are prevented from associating with DNA due to  the high nuclear concentration of cdk2–cyclin E. However, the nuclear cdk2 does  not displace potentiating  proteins which are prebound  to DNA. Similarly in somatic  cells, activation of nuclear cdk2–cyclin E or A kinases during late  G1 would block any further potentiation of DNA for replication.  During DNA replication the potentiating proteins are displaced  from DNA and prevented from rebinding DNA by the presence  of high concentrations of cdk2 kinase activity (5 and 6). In the licensing model, proteins (shaded circles) required for DNA replication associate with DNA at the end of mitosis (1 and 2). Because these proteins cannot enter the nucleus, enclosure of the  DNA within the nucleus blocks further licensing (3 and 4). As a  result of DNA replication the licensing proteins are converted to  an inactive state (5 and 6).
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Figure 7: Comparison between the inhibitor and licensing models. In the inhibitor model, proteins required for potentiating DNA replication (shaded circles) bind to DNA when cdk2 activity is low. In Xenopus eggs this occurs at the end of mitosis before nuclear formation, when cdk2–cyclin E activity is dilute (1 and 2). In somatic cells this would occur during early G1 when cdk2–cyclin E is inactive. After nuclear formation, cdk2–cyclin E (black squares) is rapidly transported into nuclei and accumulates (4). Replication potentiating proteins which enter the nucleus (black circles) are prevented from associating with DNA due to the high nuclear concentration of cdk2–cyclin E. However, the nuclear cdk2 does not displace potentiating proteins which are prebound to DNA. Similarly in somatic cells, activation of nuclear cdk2–cyclin E or A kinases during late G1 would block any further potentiation of DNA for replication. During DNA replication the potentiating proteins are displaced from DNA and prevented from rebinding DNA by the presence of high concentrations of cdk2 kinase activity (5 and 6). In the licensing model, proteins (shaded circles) required for DNA replication associate with DNA at the end of mitosis (1 and 2). Because these proteins cannot enter the nucleus, enclosure of the DNA within the nucleus blocks further licensing (3 and 4). As a result of DNA replication the licensing proteins are converted to an inactive state (5 and 6).
Mentions: It has been shown that when nuclei which have replicated once in a Xenopus extract are isolated, permeablized, and then added back to a second extract, these nuclei reinitiate replication (Blow and Laskey, 1988). By contrast, nuclei which are not permeablized before addition to a second extract fail to initiate replication. Similarly, when G2 nuclei, isolated from somatic tissue culture cells, are permeablized and then added to egg extracts, they replicate, whereas intact G2 nuclei do not (Leno et al., 1992). These observations have led to the proposal that a chromatin binding factor, called licensing factor, is essential for replication, and that the compartmental distribution of licensing factor between the nucleus and cytosol serves to limit replication to a single round per cell cycle (Blow and Laskey, 1988; Blow, 1993; Kubota and Takisawa, 1993). Specifically, the model predicts that licensing factor itself cannot enter nuclei. As such, the factor can only associate with and potentiate chromatin for DNA replication when the nuclear envelope is disassembled at mitosis. By contrast, our data strongly suggest that the compartmentalized accumulation of either cyclin E– or A–dependent cdk2 kinases within the nucleus may act to inhibit endoreduplication (Fig. 7). Our data are consistent with genetic observations implicating cdk kinases in blocking re-replication during the cell cycle (Broek et al., 1991; Hayle et al., 1994; Correa-Bordes and Nurse, 1995; Sauer et al., 1995). An advantage of this compartmentalized inhibitor model over the licensing model is that the binding of proteins required for one round of DNA replication is no longer restricted exclusively to mitosis. Rather, these associations can occur at times when cdk2–cyclin E– or A–dependent kinases are either dilute or inactive. During the early Xenopus embryonic cell cycle this would occur at the end of mitosis before nuclei have formed, while during the somatic cell cycle, this would occur during early G1, when cdk2–cyclin E kinase is inactive.

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