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.

Genetic and biochemical observations have partially characterized the events which potentiate DNA for replication during S phase of the eukaryotic cell cycle. In yeast, the multiprotein complex ORC binds to specific DNA sequences, and this association is essential for converting these sites into origins used during DNA replication (Bell et al., 1993; Rao et al., 1995; Donovan and Diffley, 1996). Although metazoan origin sequences have not yet been rigorously defined, it is likely that the metazoan homologues of the yeast ORC proteins serve a similar function (Gavin et al., 1995; Carpenter et al., 1996). Recent biochemical experiments using cell-free extracts derived from Xenopus eggs have demonstrated that both cdc6 and the MCM family of proteins only associate with chromatin after the metazoan ORC complex has bound to the chromatin (Coleman et al., 1996). Furthermore, this study showed that the association of MCM with chromatin was dependent on the prebinding of the cdc6 protein. Cdc6 is a 61-kD protein that appears to be essential for generating active origins (Bueno and Russell, 1992; Kelly et al., 1993; Liang et al., 1995; Nishitani and Nurse, 1995; Piatti et al., 1995; Coleman et al., 1996), and the MCM proteins form a multisubunit complex that is both essential for DNA replication (Hennessy et al., 1991; Yan et al., 1991, 1993; Dalton and Whitebread, 1995) and likely involved in limiting replication to a single round per cell cycle (Tye, 1994; Kubota et al., 1995; Chong et al., 1995; Madine et al., 1995a,b).

A number of studies have shown that activation of DNA replication at S phase is dependent on cdk2 kinase activity. For example, Xenopus extracts which normally replicate exogenously added chromatin templates efficiently, fail to do so after removal of cdk2 kinase activity (Fang and Newport, 1991; Jackson et al., 1995). Precisely how cdk2 contributes to the activation of replication at the molecular level is currently unknown. Interestingly, a number of excellent experiments demonstrate that cdk activity also functions to limit replication to a single round each cell cycle. For example, using the fission yeast Schizosaccharomyces Pombe as a model system, it was found that cells containing certain temperature sensitive mutations in either the cdc2 protein or cyclin B initiate a second round of DNA replication without first entering mitosis (Brock et al., 1991; Hayles et al., 1994; Moreno and Nurse, 1994; CorreaBordes and Nurse, 1995). Similarly, Drosophila embryonic cells lacking cyclin A undergo endoreduplication (Sauer et al., 1995). It has also been shown that when synchronized rat fibroblasts are treated with inhibitors known to block cdc2 activity, multiple rounds of replication occur in the absence of mitosis (Usui et al., 1991). Together these results argue strongly that during G2 of the cell cycle cdk kinase activity is essential for blocking endoreduplication, and that this mechanism is evolutionarily conserved between all eukaryotic cell types.

More recently, it has been proposed that the absence of cdk kinase activity at the end of mitosis generates a permissive period during which proteins essential for potentiating DNA for replication, such as cdc6 and MCM, can associate with DNA and that the activation of cdk during late G1 inhibits further association (Dahmann et al., 1995, Piatti et al., 1996). Based on this model the periodic oscillation of cdk activity could allow potentiation to occur during early G1 and inhibit subsequent potentiation at all other times during the cell cycle. Although the model is both appealing and consistent with the cyclic oscillation of cdc2 activity during the somatic cell cycle, it is not easily reconciled with the conditions found during the early embryonic cell cycles of many species. In particular, during the first 12 cell cycles of Xenopus embryogenesis, DNA replication is limited to a single round per cell cycle, yet, cdk2–cyclin E activity is constant during the entire cell cycle (Fang and Newport, 1991; Rempel et al., 1995; Howe and Newport, 1996). Clearly, based on the model described, the presence of cdk2 throughout the embryonic cell cycle should inhibit associations between chromatin and proteins involved in replication, thereby blocking DNA replication. In considering this inconsistency it has been proposed that in eggs the distribution of cdk2 between different cellular compartments might provide a means for reconciling the apparent conflict between the somatic and embryonic situation (Su et al., 1995). Specifically, if cdk2 is actively transported into nuclei, the actual concentration of kinase surrounding chromatin in the cytoplasm during mitosis might be significantly lower than the concentration of kinase surrounding this same chromatin during S phase when it is contained within nuclei. As such, the cell cycle–dependent compartmentalization of cdk2 could generate a gradient of cdk2 activity which would allow essential replication proteins to bind to chromatin at the end of mitosis immediately before nuclei form and prevent further binding after nuclei form.

To test this possibility we have increased the amount of cdk2–cyclin E activity surrounding chromatin in Xenopus extracts before the formation of nuclei. Our results demonstrate that under these conditions the ORC complex binds to chromatin normally. However, MCM3 fails to associate, and after nuclear formation, DNA replication does not initiate. Overall, our results strongly suggest that the cell cycle–dependent compartmentalization of active cdk2 kinase within nuclei participates in regulating DNA replication during the cell cycle.

Materials And Methods

Preparation of Fractionated Interphase Cytosol and Membrane

The egg extracts used in these studies were prepared by fractionating crude interphase egg extracts (Fang and Newport, 1991) in a centrifuge (TL100; Beckman Instruments, Inc., Fullerton, CA) at 55,000 rpm for 90 min. The cytosol was collected and rapidly frozen in liquid nitrogen. The membrane fraction was diluted in 1.5 ml of egg lysis buffer (ELB; Fang and Newport, 1991) containing 10 μg/ml of aprotinin and leupeptin. Following this it was layered on top of a 0.5 ml sucrose cushion (8.7% in ELB) and spun at 20,000 rpm for 20 min. The membrane was aliquoted, rapidly frozen in liquid nitrogen, and stored at −80°C.

Isolation of Fusion Proteins

pGEX-KG containing Xenopus cyclin A was transformed into TOPP 3 Escherichia coli bacteria strain (Strategene, La Jolla, CA). The construction and transformation of pGST–cyclin E and pGST-cdk2 has been described previously (Guadagno and Newport, 1996). Recombinant proteins were expressed and purified from soluble fractions by affinity chromatography on glutathione–sepharose beads as described (Solomon et al., 1990). Fractions that contain recombinant protein were concentrated and buffer exchanged as previously described (Guadagno and Newport, 1996).

P13 Precipitation and Western Blot

P13 was coupled to CNBr-activated Sepharose beads as described in Dunphy et al. (1988). Packed P13 beads (20 μl) were incubated with 50 μl of diluted supernatant or resuspended pellet fractions at 4°C for 1 h. The P13 beads were then pelleted, washed extensively with wash buffer (Fang and Newport, 1991), and eluted with SDS-PAGE sample buffer. The samples were fractionated on 10% SDS-PAGE gels and then analyzed by standard Western blot techniques (Harlow and Lane, 1988) using appropriate antibodies and ECL reagents (Amersham Corp., Chicago, IL).

Fractionation Experiments

In the cyclin E transport assay, 10 μl of extract was incubated with 2 μl of membrane fraction and various concentrations of demembraned sperm for 60 min. After this the samples were layered on a 15% sucrose cushion in a capillary tube and spun in a microfuge fitted with a horizontal rotor for 10 min at 4°C. Cyclin–cdk complexes in both the supernatant and the pellet fractions were precipitated with p13 beads and subjected to Western blot analysis.

To pellet the sperm, 10 μl of sample was diluted fivefold with ELB, layered onto a 17% sucrose cushion, and centrifuged for 10 min at 14,000 rpm at 4°C. The supernatant and pellet fractions were then mixed with SDS sample buffer. One fifth of the supernatant and all of the pellet fraction were loaded on 10% SDS-PAGE gel and processed for Western blotting.

DNA Replication Assay

DNA replication was assayed by incorporation of [γ-32P]dATP. Pulse or continuous labeling was carried out as described by Kornbluth et al. (1992). DNA replication was also assayed by incorporation of biotinlabeled dUTP. 1 μl of Bio-dUTP was added to 200 μl of extract at the beginning of the reaction. After a 1-h incubation, 20-μl aliquots were taken and chilled on ice for 10 min. The samples were then diluted and fixed with 800 μl of ELB with 2 mM EGS for 20 min at room temperature. After this the samples were layered on PBS containing 25% sucrose and spun onto polylysine-treated coverslips. The coverslips coated with nuclei were washed, fixed with 3.7% formaldehyde in PBS for 10 min, and then washed another three times. Nonspecific binding was blocked by incubating with PBS containing 10% FCS and 0.1% Triton X-100 for 10 min. The coverslips were then rinsed and incubated for 1 h with PBS containing 10% FCS and 1:100 dilution of streptavidine-conjugated Texas red. After this, the coverslips were rinsed and mounted with 25% glycerol with 0.01% Hoechst.

Immunofluorescence Microscopy

Immunofluorescence staining of MCM3 protein on sperms was performed as described in Yan and Newport (1995). The fixed sperms were first stained with rabbit anti-MCM3 antibody (1:1,000 in PBS) for 1 h, washed, and then stained with goat–anti rabbit rhodamine conjugate (Sigma Chemical Co., St. Louis, MO 1:100 in PBS). The samples were mounted with Hoechst solution (0.01% in ELB) and viewed with a fluorescence microscope.

Results

When sperm chromatin is added to Xenopus egg extracts prepared in the presence of the protein synthesis inhibitor cycloheximide, the chromatin is first encapsulated into nuclei and then efficiently replicated once (Lohka and Masui, 1984; Blow and Laskey, 1986; Newport, 1987). The absence of further rounds of replication demonstrates that the mechanisms which limit replication to a single round per cell cycle are active in extracts. Moreover, because such extracts lack cyclins A, B1, and B2, cdc2 kinase is inactive. This demonstrates that unlike yeast cells, inhibition of multiple rounds of DNA replication in egg extracts does not require mitotic cdc2 kinase activity. However, these extracts do contain cdk2–cyclin E kinase activity (Fang and Newport, 1991). Because cdk2–cyclin E normally accumulates within nuclei, it is possible that the concentrations of cdk2 surrounding chromatin in the cytosol and the nucleus differ and that this difference might participate in regulating DNA replication.

To pursue this possibility we have determined whether the ckd2–cyclin E concentration in nuclei is higher than that present in the cytosol. To do this, different numbers of sperm chromatin were added to a fixed volume of egg extract. After a 60-min incubation, during which the chromatin was both assembled into nuclei and replicated, the nuclei were separated from the surrounding cytosol by sedimentation through a 15% sucrose cushion. The resulting nuclear pellet was then assessed for cdk2–cyclin E content by Western blot analysis using both anti-cdk2 and anti– cyclin E antibodies. The results of these experiments (Fig. 1, A and B) demonstrated that the amount of cdk2–cyclin E complex present in the nuclear pellet increased linearly up to 2,000 sperm/μl. No further increase in cdk2–cyclin E was observed at concentrations above 2,000 sperm/μl, indicating that all of the endogenous cdk2–cyclin E complex initially present in the cytosol had been transported into the newly formed nuclei. This conclusion is supported by direct comparison of the cdk2–cyclin E in both the cytosol and nuclear fractions in an extract containing 2,000 nuclei/μl (Fig. 1 C). It is clear that under these conditions the vast majority (>95%) of the cyclin E is nuclear, and relatively little (<5%) remains in the cytosol. Based on the observation that nuclear cdk2–cyclin E is readily released from interphase nuclei treated with mild detergent (0.1% Triton X-100), the majority of the cdk2–cyclin E transported into nuclei does not appear to be tightly associated with chromatin (results not shown).

This experiment allows us to calculate the difference in cdk2–cyclin E concentration present initially in the cytosol, before nuclei form, relative to the concentration of cdk2– cyclin E inside nuclei. To do this, the average diameter of nuclei formed in the extract was measured from microscopic observations using beads of defined size as a reference standard. This nuclear diameter (17 ± 2 μm) was used to calculate nuclear volume. Based on these calculations, 2,000 nuclei occupy a volume of 5 × 10−3 μl. Because all of the cdk2–cyclin E initially present in 1 μl of cytosol is transported into 2,000 nuclei, these results demonstrate that the cdk2–cyclin E activity initially present in the cytosol is concentrated 200-fold after transport into nuclei. Previous studies have estimated that the cdk2–cyclin E concentration in egg cytosol is 0.06 μM (van Renterghem et al., 1994), whereas our results demonstrate that the concentration of cdk2–cyclin E within nuclei is 12.0 μM. Therefore, before the assembly of a nuclear membrane around sperm chromatin, mitotic chromosomes, or the DNA in permeablized nuclei, the chromatin will be surrounded by levels of cdk2–cyclin E kinase activity 200-fold lower than will surround it once the chromatin has acquired a nuclear envelope.

High Cytoplasmic cdk2–cyclin E Levels Block DNA Replication

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.

To determine whether chromatin which is preincubated in normal extract becomes refractive to the inhibition of replication by high cdk2–cyclin concentrations, sperm chromatin (1,000 μl) was preincubated in purified egg cytosol (i.e., lacking membranes) for 30 min. After this the extract was split into two aliquots. To one aliquot, membranes alone were added, while to the other, both cyclin E–cdk2 (final concentration 1 μM) and membranes were added. Again, nuclei assembled normally in both samples, and the encapsulated DNA decondensed (Fig. 2,D). Importantly, under these conditions, DNA replication was identical in both samples (Fig. 2 E). This experiment demonstrates that addition of 1 μM cdk2–cyclin E, by itself, does not inhibit DNA replication. Rather, the time of addition of the kinase relative to the addition of sperm chromatin determines whether replication is inhibited. Replication is blocked if cdk2–cyclin E is present in the cytosol at high concentrations before chromatin addition. However, replication occurs normally if chromatin is exposed to cytosol before the addition of cdk2–cyclin E.

High Cytosolic Concentrations of cdk2–cyclin E Blocks Binding of MCM3 to Chromatin

The results above suggest that high concentrations of cytosolic cdk2–cyclin E may inhibit proteins required for DNA replication from associating with chromatin. To examine this possibility, we investigated how the association of MCM3 with chromatin is affected by cytosolic cdk2–cyclin E concentration. Specifically, different concentrations of cdk2–cyclin E protein were added to pure cytosol and incubated for 30 min, and then sperm chromatin was added to the cytosol. After another 30 min incubation the sperm chromatin was separated from the cytosol by centrifugation through a 17% sucrose cushion, and MCM3 binding to chromatin was analyzed on Western blots using anti- Xenopus MCM3 antibody as a probe. The results from these experiments showed quite clearly that the association of MCM3 with chromatin decreased as cdk2–cyclin E concentration increased (Fig. 2,C). Little decrease was observed at cdk2–cyclin E concentrations twofold higher than endogenous levels (0.12 μM), a 50% reduction was observed at fourfold higher levels (0.25 μM), an 80% reduction occurred at eightfold higher levels (0.5 μM), and a 90% reduction occurred at 16-fold higher than endogenous levels (1 μM). By contrast, when sperm chromatin was incubated first for 30 min in pure egg cytosol and then 1 μM cdk2–cyclin E was added to the extract, little decrease in the binding of MCM3 to chromatin was observed (Fig. 2 F). Thus, for at least one essential DNA replication protein, MCM3, high concentrations of cdk2–cyclin E blocks binding of the protein to chromatin. Importantly, these results also show that once MCM3 is bound to chromatin, increasing the concentration of cdk2–cyclin E, as would occur following nuclear formation, does not cause dissociation of the protein from chromatin.

With respect to how cdk2–cyclin E blocks the association of MCM with chromatin, it has been shown that the ORC complex binds to DNA before MCM and that this interaction is a prerequisite for MCM binding to chromatin (Coleman et al., 1996). Therefore, if high concentrations of cdk2–cyclin E modified ORC so as to prevent it from interacting with DNA, this modification would also prevent MCM from associating with chromatin. To test this possibility we used an anti-ORC2 antibody to examine whether binding of the ORC complex to chromatin was sensitive to cdk2–cyclin E concentration. The results of this experiment showed quite clearly that concentrations of exogenously added cdk2–cyclin E which completely inhibited MCM binding had no effect on the association of ORC with DNA (Fig. 3). Therefore, the inhibitory effects of cdk2-cyclin E on the interaction of MCM with chromatin appear to involve a step downstream of the association of ORC with DNA.

Inhibition of DNA Replication Requires cdk2–cyclin E Activity

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.

If cdk2–cyclin E inhibited the association of MCM with chromatin by sequestering activities needed for mediating this association, then addition of a kinase inactive cdk2– cyclin E complex should also inhibit this association. To test this possibility, different concentrations of a kinase defective cdk2, cdk2K33R, and cyclin E were added to extracts. After a 30 min incubation, sperm chromatin was added to these extracts, and after a further 30 min incubation, the association of MCM with this chomatin was determined (Fig. 4 B). The results from this experiment quite clearly showed that the kinase inactive cdk2–cyclin E complex did not inhibit MCM from binding to chromatin. This result in combination with the experiment above demonstrates that it is the catalytic activity of cdk2, not the amount of cdk2– cyclin E complex, which prevents MCM from associating with chromatin.

Generation of Pseudo G1 and G2 Nuclei in the Same Extract

The results presented above show that chromatin incubated in cytosol containing high concentrations of cdk2– cyclin E fails to replicate when it assembles into nuclei. Further, our results show that cdk2–cyclin E is concentrated within nuclei as a result of nuclear transport. This sequence of events makes two predictions. First, it predicts that chromatin assembled into nuclei in extracts initially containing high concentrations of cdk2–cyclin E will not replicate if the cytosol is diluted. This will occur because the cdk2–cyclin E is already compartmentalized within nuclei and no longer subject to dilution. The second prediction is that fresh nuclei added to this diluted cytosol would replicate. This would occur because the new chromatin would be assembled into nuclei in cytosol containing low concentrations of cdk2–cyclin E.

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.

Importantly, we find that chromatin that is added to the extracts after dilution replicates normally (Fig. 5,A, right, bottom, + new sperm). To show that under these conditions replication was exclusive to the chromatin added after dilution, biotin-dUTP was added to the extract. After this, the extract was incubated for 30 min, and then the nuclei were fixed and stained with streptavidin conjugated to the fluorophor Texas red. Nuclei which had replicated (Fig. 5,B, right, Texas red positive) were then counted. Similarly, the DNA-specific fluorophor bisbenzimide was added to the extract to visualize and count the total number of nuclei in the extract (Fig. 5 B, left). The result of these observations demonstrated that the nuclei fell into two distinct classes; those that had replicated (80%) and those that failed to replicate (20%). Importantly, the number of nuclei that replicated was identical to the number of nuclei added to the extract after dilution, and the number of nuclei that failed to replicate was identical to the number of nuclei assembled before dilution. Thus, by controlling the time of addition of chromatin relative to transport of cdk2–cyclin E, we can generate an extract which contains two sets of nuclei, one set representing pseudo G2 nuclei which are inhibited for replication and one set representing G1 nuclei which are able to carry out a single round of replication.

Like Cyclin E, Cyclin A also Inhibits DNA Replication and MCM3 Binding

In somatic cells, cyclin E is degraded during S phase of the cell cycle, and cyclin A becomes the predominant cyclin subunit associated with cdk2 (Dulic et al., 1992; Koff et al., 1992). Therefore, we have examined whether cyclin A, like cyclin E, can block replication in egg extracts. The extracts used in this study were all made in the presence of the protein synthesis inhibitor cycloheximide and lacked cyclin A. Therefore, recombinant E. coli produced GST–Xenopus cyclin A was added to the extract. This added protein rapidly associated with the endogenous pool of cdc2 to form active kinase (data not shown). Like cdk2–cyclin E, cdc2– cyclin A complex was efficiently transported into nuclei (data not shown; Pines and Hunter, 1991).

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.

Cyclin A has been shown to be involved in both DNA replication and initiation of mitosis (Strausfeld et al., 1994, Jackson et al., 1995). Therefore, the inhibition of replication by cyclin A might occur because addition of cyclin A initiated mitosis. However, several pieces of evidence argue against this possibility. First, if cyclin A induced mitosis at 66 nM, it should do so independent of its order of addition relative to sperm. However, when cyclin A was added after sperm (late addition), DNA replication occurred normally (Fig. 6 C). Second, the morphological organization of both nuclei and DNA in extracts with 66 nM or higher cyclin A was examined by fluorescent microscopy. Nuclei formed, and the DNA within these nuclei decondensed in extracts pretreated with cyclin A at concentrations up to 100 nM. In extracts pretreated with 200 nM cyclin A, mitosis was initiated based on the observation that the added sperm chromatin condensed into metaphase chromosomes and failed to form nuclei (data not shown). Thus, these results show that the inhibition of replication by addition of moderate concentrations of cyclin A does not occur because cyclin A causes the extract to enter mitosis.

Together, our experiments demonstrate that at the physiological concentration of cyclin A expected to be present in the nuclei of somatic cells during late S phase and G2, cyclin A would not cause prebound MCM3 to be released from DNA. However, this concentration of cyclin A could strongly inhibit the reassociation of MCM3 with DNA once it had been released (Todorov et al., 1995) as a result of a single round of replication. As such, cdk2–cyclin A at this concentration, could block further rounds of DNA replication.

Discussion

The experimental results presented in this report strongly suggest that the transport of cdk2–cyclin E kinase complexes into nuclei may play a critical role in negatively regulating DNA replication during the cell cycle. In support of this we have shown that cdk2–cyclin E kinase is rapidly transported into nuclei formed in egg extracts. Further, by quantitating both the volume of nuclei and the amount of cdk–cyclin E transported per nucleus we show that this compartmentalization generates a cell cycle dependent gradient of cdk2–cyclin E within eggs. Before nuclear formation, the cdk2–cyclin E concentration in the cytosol is 200fold lower than the concentration of cdk2–cyclin E present in nuclei. Our results strongly suggest that the low concentration of cdk2–cyclin E kinase present in the cytosol at the end of mitosis and before nuclei form allows MCM proteins to associate with chromatin, thereby potentiating DNA for replication. After nuclear formation, our data suggests that MCM proteins released during replication (Chong et al., 1995; Todorov et al., 1995) would be inhibited from rebinding chromatin due to the high cdk2 activity present in nuclei. In support of this, we have shown that increasing the concentration of cdk2–cyclin E in the cytosol 16-fold does not block assembly of sperm chromatin into nuclei but does inhibit both replication and the binding of MCM3. Importantly, we have also shown that this inhibition is dependent on the order of addition of cdk2–cyclin E relative to sperm chromatin. If cytosol is preincubated with cdk2– cyclin E before chromatin addition, MCM3 does not bind to chromatin, and DNA replication fails to occur. However, if MCM3 is allowed to bind to chromatin first, the subsequent addition of cdk2–cyclin E does not displace this bound MCM3, and DNA replication occurs normally. We have also shown that moderately high concentrations of cdc2–cyclin A kinase, like cdk2–cyclin E, prevents MCM3 from associating with chromatin and inhibits DNA replication but cannot displace prebound MCM3 from chromatin. This demonstrates that once MCM is bound to chromatin it cannot be displaced from the chromatin by increasing either cdk2–cyclin E or cdc2–cyclin A concentrations. Overall, these results suggest that during the embryonic cell cycle MCM proteins can only bind to and potentiate chromatin for DNA replication between the end of mitosis and before the assembly of chromatin into nuclei.

With respect to how cdk2–cyclin E blocks the binding of MCM3 to chromatin, a recent report in combination with the work presented here suggests that cdk2–cyclin E does not inhibit the binding of MCM to chromatin by directly phosphorylating MCM. Specifically, Madine et al. (1995a) have shown that chromatin added to egg extracts depleted of MCM proteins form nuclei but do not replicate. However, when MCM proteins are added back to these extracts, the MCM complex is transported into preexisting nuclei and rescues DNA replication. If MCM proteins were substrates of cdk2 we would expect that the very high cdk2–cyclin E concentration within nuclei before addition of MCM would modify the added MCM as it entered nuclei, thereby blocking replication. Because this does not occur, direct phosphorylation of MCM by cdk2 does not appear to be critical for inhibiting its association with DNA. An alternative possibility would be that cdk2 kinase phosphorylates and inhibits protein factors which are required for the association of MCM with chromatin. It has recently been shown that both the multiprotein ORC complex and cdc6 protein must bind to chromatin before MCM proteins will associate with chromatin (Coleman et al., 1996). Our results demonstrate that cdk2–cyclin E inhibits the interaction of MCM3 with chromatin at a step downstream from the association of the ORC complex with chromatin. Specifically, we find that cdk2 concentrations which prevent MCM3 binding do not inhibit the association of ORC with chromatin. This suggests that proteins which both bind to chromatin after ORC and are required for the association of MCM with chromatin, such as cdc6, may be inactivated by cdk2 kinase activity. Interestingly, unlike the rescue of DNA replication by MCM, addition of cdc6 to nuclei formed in cdc6- depleted extracts does not rescue DNA replication (Coleman et al., 1996). It is quite possible that the high activity of cdk2 kinase in nuclei prevents cdc6 from binding to chromatin and this, in turn, prevents MCM from associating with chromatin.

A Role for cdk2 in Regulating DNA Replication during the Somatic Cell Cycle

Because cdk2–cyclin E is constituitively active during the embryonic cell cycle (Fang and Newport, 1991), our results suggest that MCM proteins can only bind to chromatin and potentiate this template for DNA replication before nuclear formation, when the local concentration of cdk2 kinase activity surrounding chromatin is relatively low. However, in somatic cells cdk2 kinase is inactive during early G1 (Koff et al., 1991; Lew et al., 1991). Therefore, during early G1 of the somatic cell cycle, MCM proteins should be able to enter nuclei and bind to chromatin. The activation of cdk2–cyclin E at mid-G1 would be expected to block such binding during the remainder of the cell cycle. As such, the delayed activation of cdk2 kinase activity following mitosis may act to create a temporal window between the end of mitosis and mid-G1 during which DNA is potentiated for replication. Consistent with this hypothesis, it has recently been shown that in S. cerivisiae, cdc6 synthesis can only potentiate DNA replication between anaphase and the activation of the metazoan equivalent of cdk2–cyclin E kinase, cdk1–Clb 5 and 6, at late G1 (Piatti et al., 1996). The authors proposed that the activation of Cdk1-Clb kinases at late G1 inhibited formation of replication competent DNA beyond this point. Our results strongly suggest that cdk2 activation at this time blocks replication by inhibiting the association of MCM with chromatin. This study also found that cdc6 associated with cdk1-Clb kinases, leaving open the question of whether cdk1-Clb complexes might inhibit cdc6 by sequestering the protein or by phosphorylating it. Our observation that the association of MCM3 with chromatin is sensitive to cdk2– cyclin E kinase activity and insensitive to the amount of cdk2–cyclin E complex present (Fig. 4) suggests that it is the kinase activity of cdk2 which blocks the association of MCM with chromatin.

A Relationship between cdk2-dependent Inhibition of Replication and Licensing Factor

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.

For somatic cells in particular, the compartmentalized inhibitor mechanism is mechanistically more conservative and less restrictive than the licensing factor model. For example, in somatic tissues such as the liver, cells can remain in Go of the cell cycle for one or more years before entering G1 phase and then replicating their DNA. The “licensing” model would require that the license, which only associates with DNA at mitosis, remain completely stable during the one or more years that these cells are in Go. Given the natural lifetime of proteins, it is likely that some of the license would be degraded during this period. As such, some of the DNA in these cells would be unable to replicate following release from Go. By contrast, if proteins essential for potentiating replication, such as MCM3, are synthesized shortly after cells exit Go, they could enter nuclei, bind to chromatin, and potentiate a round of replication during a well defined temporal window in which cdk2–cyclin kinases are inactive. Thus, the inhibitor model predicts that potentiation of chromatin for a single round of replication and inhibition of endoreduplication are controlled by the normal oscillations of cdk2–cyclin E and A activity, which occur during the cell cycle. We believe that the tight temporal linkage between the licensing event, the initiation of DNA replication, and the inhibition of re-replication supported by this model may provide a more conservative and controlled means of ensuring that all DNA is replicated once per cell cycle than the mitosis-dependent licensing mechanism.

Although our results strongly suggest that the accumulation of cdk2 kinase within nuclei can regulate the potentiation of chromatin for replication, it remains to be determined whether this is the only mechanism carrying out this function. Several observations indicate that this may not be the case. Specifically, we find that when Cip is added to extracts to inactivate cdk2 kinase activity before nuclear formation, MCM binding to chromatin is rescued (Fig. 4 A). However, after nuclear formation, inactivation of cdk2 by addition of Cip does not allow MCM to bind to chromatin (Hua, X., unpublished observations). This could occur because the accumulated cdk2 within nuclei rapidly activates a system that prevents MCM binding, and that once activated this system functions independently of cdk2. Alternatively, it may indicate that a second system exists to prevent MCM association, and that the activity of this second system is independent of the cdk2 concentration within nuclei. Resolving between these two possibilities will be an important focus of future investigation. However, given the essential nature of limiting replication to a single round per cell cycle, redundant cdk2-dependent and -independent mechanisms may be necessary to ensure cell viability.

Acknowledgments

The authors would like to thank Johannes Walter and Jeff Stack for encouragements and many helpful discussions, John Howe for sharing reagents, and Randy Poon and Tony Hunter for cdk2-K33R expression plasmid.

This work was supported by a grant (GM 44656) from the National Institutes of Health to J. Newport.

References

References
Bell
SP
,
Kobayashi
R
,
Stillman
B
Yeast origin recognition complex functions in transcription silencing and DNA replication
Science (Wash DC)
1993
262
1844
1849
[PubMed]
Blow
JJ
Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor
J Cell Biol
1993
122
993
1002
[PubMed]
Blow
JJ
,
Laskey
RA
Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs
Cell
1986
47
577
587
[PubMed]
Blow
JJ
,
Laskey
RA
A role for the nuclear envelope in controlling DNA replication within the cell cycle
Nature (Lond)
1988
332
546
548
[PubMed]
Broek
D
,
Bartlett
R
,
Crawford
K
,
Nurse
P
Involvement of p34cdc2in establishing the dependency of S-phase on mitosis
Nature (Lond)
1991
349
388
393
[PubMed]
Bueno
A
,
Russel
P
Dual functions of Cdc6: a yeast protein required for DNA replication also inhibits nuclear division
EMBO (Eur Mol Biol Organ) J
1992
11
2167
2176
[PubMed]
Carpenter
PB
,
Mueller
PR
,
Dunphy
WG
Role for a Xenopus Orc2-related protein in controlling DNA replication
Nature (Lond)
1996
379
357
360
[PubMed]
Chong
JPJ
,
Mahbubani
HM
,
Khoo
C-Y
,
Blow
JJ
Purification of an MCM-containing complex as a component of the DNA replication licensing system
Nature (Lond)
1995
375
418
421
[PubMed]
Coleman
TR
,
Carpenter
PB
,
Dunphy
WG
The XenopusCdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts
Cell
1996
87
53
63
[PubMed]
Correa-Bordes
J
,
Nurse
P
p25rum1 orders S phase and mitosis by acting as an inhibitor of the p34cdc2 mitotic kinase
Cell
1995
83
1001
1009
[PubMed]
Dahmann
C
,
Diffley
JF
,
Nasmyth
KA
S phase-promoting cyclin dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state
Curr Biol
1995
5
1257
1269
[PubMed]
Dalton
S
,
Whitebread
L
Cell cycle-regulated nuclear import and export of cdc47, a protein essential for initiation of DNA replication in budding yeast
Proc Natl Acad Sci USA
1995
92
2514
2518
[PubMed]
Donovan
S
,
Diffley
JF
Replication origins in eukaroytes
Curr Opin Genet Dev
1996
6
203
207
[PubMed]
Dulic
V
,
Lees
E
,
Reed
SI
Association of human cyclin E with a periodic G1–S phase protein kinase
Science (Wash DC)
1992
257
1958
1961
[PubMed]
Dunphy
WG
,
Brizuela
L
,
Beach
D
,
Newport
JW
The Xenopuscdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis
Cell
1988
54
423
431
[PubMed]
Fang
F
,
Newport
JW
Evidence that the G1–S and G2–M transitions are controlled by different cdc2 proteins in higher eukaryotes
Cell
1991
66
731
742
[PubMed]
Gavin
KA
,
Hidaka
M
,
Stillman
B
Conserved initiator proteins in eukaryotes
Science (Wash DC)
1995
270
1667
1671
[PubMed]
Guadagno
TM
,
Newport
JW
Cdk2 kinase is required for entry into mitosis as a positive regulator of Cdc2-cyclin B kinase activity
Cell
1996
84
73
82
[PubMed]
Harlow, E., and D. Lane. 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 474–504.
Hayle
J
,
Fisher
D
,
Woollard
A
,
Nurse
P
Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34cdc2– mitotic B cyclin complex
Cell
1994
78
813
822
[PubMed]
Hennessy
KM
,
Lee
A
,
Chen
E
,
Botstein
D
A group of interacting yeast DNA replication genes
Genes Dev
1991
5
958
969
[PubMed]
Howe
JA
,
Newport
JW
A developmental timer regulates degradation of cyclin E1 at the midblastula transition during Xenopusembryogenesis
Proc Natl Acad Sci USA
1996
93
2060
2064
[PubMed]
Jackson
PK
,
Chevalier
S
,
Philippe
M
,
Kirschner
MW
Early events in DNA replication require cyclin E and are blocked by p21CiP1
J Cell Biol
1995
130
755
769
[PubMed]
Kelly
TJ
,
Martin
GS
,
Forsburg
SL
,
Stephen
RJ
,
Russo
A
,
Nurse
P
The fission yeast cdc18 gene product couples S phase to START and mitosis
Cell
1993
74
371
382
[PubMed]
Koff
A
,
Giordano
A
,
Desai
D
,
Yamashita
K
,
Harper
JW
,
Elledge
S
,
Nishimoto
T
,
Morgan
DO
,
Franza
BR
,
Roberts
JM
Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle
Science (Wash DC)
1992
257
1689
1694
[PubMed]
Kornbluth
S
,
Smythe
C
,
Newport
JW
In Vitro cell cycle arrest induced by using artificial DNA templates
Mol Cell Biol
1992
12
3216
3223
[PubMed]
Kubota
Y
,
Takisawa
H
Determination of initiation of DNA replication before and after nuclear formation in Xenopusegg cell free extracts
J Cell Biol
1993
123
1321
1331
[PubMed]
Kubota
Y
,
Mimura
S
,
Nishimoto
S
,
Takisawa
H
,
Nojima
H
Identification of the yeast MCM3-related protein as a component of XenopusDNA replication licensing factor
Cell
1995
81
601
609
[PubMed]
Leno
GH
,
Downes
CS
,
Laskey
RA
The nuclear membrane prevents replication of human G2 nuclei but not G1 nuclei in Xenopusegg extract
Cell
1992
69
151
158
[PubMed]
Liang
C
,
Weinreich
M
,
Stillman
B
ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome
Cell
1995
81
667
676
[PubMed]
Lohka
MJ
,
Masui
Y
Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs
J Cell Biol
1984
98
1222
1230
[PubMed]
Madine
MA
,
Khoo
C-Y
,
Mills
AD
,
Laskey
RA
MCM3 complex required for cell cycle regulation of DNA replication in vertebrate cells
Nature (Lond)
1995a
375
421
424
[PubMed]
Madine
MA
,
Khoo
C-Y
,
Mills
AD
,
Musahl
C
,
Laskey
RA
The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopusegg extracts
Curr Biol
1995b
5
1270
1279
[PubMed]
Moreno
S
,
Nurse
P
Regulation of progression through the G1 phase of the cell cycle by the rum1+ gene
Nature (Lond)
1994
367
236
242
[PubMed]
Newport
J
Nuclear reconstitution in vitro: stages of assembly around protein-free DNA
Cell
1987
48
205
217
[PubMed]
Nishitani
H
,
Nurse
P
p65-cdc18 plays a major role in controlling the initiation of DNA replication in fission yeast
Cell
1995
83
397
405
[PubMed]
Piatti
S
,
Lengauer
C
,
Nasmyth
K
Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a ‘reductional' anaphase in the budding yeast Saccharomyces cerevisiae.
EMBO (Eur Mol Biol Organ) J
1995
14
3788
3799
[PubMed]
Piatti
S
,
Bohm
T
,
Cocker
JH
,
Diffley
JFX
,
Nasmyth
K
Activation of S-phase promoting CDKs in late G1 defines a “point of no return” after which Cdc6 synthesis cannot promote DNA replication in yeast
Genes Dev
1996
10
1516
1531
[PubMed]
Pines
J
,
Hunter
T
Human cyclins A and B1 are differentially located in the cell and undergo cell cycle–dependent nuclear transport
J Cell Biol
1991
115
1
17
[PubMed]
Rao
H
,
Stillman
B
The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators
Proc Natl Acad Sci USA
1995
92
2224
2228
[PubMed]
Rempel
RE
,
Sleight
SB
,
Maller
JL
Maternal Xenopus Cdk2-cyclin E complexes function during meiotic and early embryonic cell cycles that lack a G1 phase
J of Biol Chem
1995
270
6843
6855
[PubMed]
Sauer
K
,
Knoblich
JA
,
Richardson
H
,
Lehner
CF
Distinct modes of cyclin E/cdc2c kinase regulation and S-phase control in mitotic and endoreduplication cycles of Drosophilaembryogenesis
Genes Dev
1995
9
1327
1339
[PubMed]
Solomon
MJ
,
Glotzer
M
,
Lee
TH
,
Philippe
M
,
Kirschner
MW
Cyclin activation of p34cdc2
Cell
1990
63
1013
1024
[PubMed]
Strausfeld
UP
,
Howell
M
,
Rempel
R
,
Maller
JL
,
Hunt
T
,
Blow
JJ
Cip 1 blocks the initiation of DNA replication in Xenopusextracts by inhibition of cyclin-dependent kinases
Curr Biol
1994
4
876
883
[PubMed]
Su
TT
,
Follette
PJ
,
O'Farrell
PH
Qualifying for the license to replicate
Cell
1995
81
825
828
[PubMed]
Todorov
IT
,
Attaran
A
,
Kearsey
SE
BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication
J Cell Biol
1995
129
1433
1445
[PubMed]
Tye
B-K
The MCM2-3-5 proteins: are they replication licensing factors?
Trends Cell Biol
1994
4
160
166
[PubMed]
Usui
T
,
Yoshida
M
,
Abe
K
,
Osada
H
,
Isono
K
,
Beppu
T
Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-25a
J Cell Biol
1991
115
1275
1282
[PubMed]
Van Renterghem
B
,
Browning
MD
,
Maller
JL
Regulation of mitogen-activated protein kinase activation by protein kinases A and C in a cellfree system
J Biol Chem
1994
269
24666
24672
[PubMed]
Yan
H
,
Newport
JW
An analysis of the regulation of DNA synthesis by cdk2, cip1, and licensing factor
J Cell Biol
1995
129
1
15
[PubMed]
Yan
H
,
Gibson
S
,
Tye
BK
Mcm2 and Mcm3, two proteins important for ARS activity, are related in structure and function
Genes and Dev
1991
5
944
957
[PubMed]
Yan
H
,
Merchant
AM
,
Tye
BK
Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast
Genes Dev
1993
7
2149
2160
[PubMed]

Please address all correspondence to John Newport, Biology Department 0347, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093. Tel.: (619) 534-3423; Fax: (619) 534-0555.