Cdk1 and Cdk2 have overlapping functions in both S phase progression and centrosome duplication

To explore the redundancy of Cdk1 and Cdk2 during S phase, we disrupted the chicken CDK2 gene to generate cdk1as/cdk2 ^−/− mutants (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200702034/DC1). Ablation of Cdk2 had little effect on DT40 WT cells, but retarded the cellular proliferation in the cdk1as background even in the absence of 1NM-PP1 (Fig. 2, A and B). Accordingly, cdk1as/cdk2 ^−/− cells that were synchronized in G1 phase by elutriation took approximately 2 h longer than cdk1as cells to initiate S phase (compare the FACS histogram in Fig. 2 C with Fig. 1 D). Nonetheless, the double-mutant cells were still able to complete S phase and accumulated in G2 phase even in the presence of a low dose (1 μM) of 1NM-PP1 (Fig. 2 D). A tenfold higher dose of the inhibitor blocked the asynchronous cell cycle both in G1 and G2, as judged by the histogram of DNA content (Fig. 2 D). This suggests that Cdk1 is responsible for S phase control in the absence of Cdk2.

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Figure 2.

Effects of a Cdk2 deletion on cdk1as cells. (A) Growth curve of cdk1as/cdk2 ^−/− cells compared to the indicated controls. (B) Cell cycle distribution of the indicated cell lines, calculated from BrdU pulse labeling, phosphohistone H3 staining, and PI staining experiments. (C) Cell cycle analysis of cdk1as/cdk2 ^−/− cells, following synchronization in G1 phase by elutriation. (D) Cell cycle analysis of cdk1as/cdk2 ^−/− and cdk1as/cdk2 ^−/+ cells treated for 8 h with the indicated concentration of 1NM-PP1. The control was not exposed to 1NM-PP1. (E) Centrosomes detected by γ-tubulin immunofluorescence (green color) and counterstained with DAPI (blue color) in fixed cdk1as and cdk1as/cdk2 ^−/− cells (bar, 10 μm), at the indicated times after 1 μM or 10 μM 1NM-PP1 addition. (F) Average number of centrosomes per cell (n = 50) plotted against time after 10 μM 1NM-PP1 addition.

We next analyzed the centrosomes by γ-tubulin staining in arrested cdk1as and cdk1as/cdk2 ^−/− cells. 8 h after Cdk1 inhibition, cdk1as cells contained two separated centrosomes, and the centrosome number appeared to double subsequently during 8-h intervals (Fig. 2, E and F). A similar separation and doubling of the centrosomes occurred in cdk1as/cdk2 ^−/− cells that were treated with 1 μM 1NM-PP1 (Fig. 2 E). However, the centrosomes no longer duplicated in cdk1as/cdk2 ^−/− treated with 10 μM 1NM-PP1 (Fig. 2, E and F). This phenotypic difference between cdk1as and cdk1as/cdk2 ^−/− cells indicates that Cdk1 and Cdk2 share overlapping functions in the control of both DNA replication and centrosome duplication.

Cdk1 activity is required to initiate but not to sustain DNA synthesis throughout S phase, in cells lacking Cdk2

We aimed to analyze the S phase functions of Cdk1 in cdk1as/cdk2 ^−/− cells in closer detail, by examining cell cycle progression. We collected the G1 fraction of cells, treated them with 10 μM 1NM-PP1, and analyzed the subsequent progression through the cell cycle (Fig. 3, A and B). The G1 fraction of cdk1as/cdk2 ^−/+ cells was able to increase their DNA content (Fig. 3 A), and uptake BrdU (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200702034/DC1) in the presence of 1NM-PP1. In contrast, the same dose of the inhibitor completely abolished the cell cycle progression and BrdU uptake in cdkas/cdk2 ^−/− cells (Fig. 3 B; Fig. S4). These observations indicate functional redundancy between Cdk1 and Cdk2 for the initiation of S phase.

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Figure 3.

Effects of Cdk1 inhibition on DNA replication and cyclin synthesis in cdk1as/cdk2^−/− and cdk1as/cdk2^−/+ cells. (A) Cdk1 inhibition does not prevent S phase in cdk1as/cdk2 ^−/+ cells. Cells synchronized in G1 by elutriation were incubated in 10 μM 1NM-PP1, and samples were taken every 2 h for cell cycle analysis, as in Fig. 1 D. (B) Cdk1 inhibition in cdk1as/cdk2 ^−/− cells inhibits entry into S phase. The same experiment as in A was performed. (C) Inhibition of Cdk1 blocks the initiation of DNA synthesis in cdk1as/cdk2 ^−/− cells. Asynchronous cultures of cdk1as/Cdk2 ^−/+ or ^−/− cells were exposed to a 10-min pulse of BrdU and subjected to cell cycle analysis as in Fig. 1 C (top panels). BrdU labeled cells were chased with BrdU-free medium containing 10 μM 1NM-PP1 for a further 7 h (pulse-chase analysis shown in bottom panel). The X-axis of the dot blot shows the content of DNA on a linear scale; the Y axis shows the BrdU uptake on a log scale. Cells in the gate area represent the G1 fraction, of which the percentage is shown. (D) Time course of cyclin synthesis. Cells synchronized in G1 phase were incubated in the presence or absence of 10 μM 1NM-PP1. Cell extracts were analyzed by SDS-PAGE and immunoblotting at the indicated time points.

To confirm this notion, we examined the cell cycle progression in asynchronous populations of cdk1as/cdk2 ^−/+ and cdk1as/cdk2 ^−/− cells using pulse-chase BrdU labeling. After 10 min of BrdU exposure, G1, S, and G2 fractions of the cells were clearly distinguishable by dot blot analysis of BrdU/PI double staining (Fig. 3 C). BrdU was subsequently removed from the medium and the cells were further incubated in the presence of 10 μM 1NM-PP1. Both the G1 and S phase population of cdk1as/cdk2 ^−/+ cells were able to complete DNA synthesis, and the entire population of the cells was blocked in the G2 phase. Conversely, the G1 fraction of the homozygous cdk1as/cdk2 ^−/− mutants did not initiate DNA synthesis, whereas the mid S phase cells were shifted toward G2 (Fig. 3 C). Thus, although ongoing DNA synthesis during S phase does not appear to require Cdk1/2 activity, either Cdk1 or Cdk2 activity is essential to initiate DNA replication.

To further explore this G1 arrest induced by Cdk1 inhibition in cdk1as/cdk2 ^−/− cells, we measured the levels of cyclin A and B in cells synchronized in G1 by elutriation (Fig. 3 D). Both cyclins accumulated even after addition of 10 μM 1NM-PP1, suggesting that Cdk1/2 inhibition does not interfere with the expressions of genes required for the G1/S transition.

Inhibition of Cdks in G2 phase does not result in endoreplication in DT40 or HeLa cells

Hayles et al. (1994) demonstrated that artificial inactivation and reactivation of Schizosaccharomyces pombe Cdc2 in G2 phase is sufficient to reset the cell cycle and to initiate a further round of DNA replication, without previous chromosome segregation. To investigate whether a similar reversible inhibition of Cdk activity is also sufficient to induce endoreplication in vertebrate cells, we synchronized cdk1as/cdk2 ^−/− cells in G2 phase by addition of 1 μM 1NM-PP1 for 6 h, and subsequently increased the inhibitor concentration to 10 μM for an additional hour to completely block Cdk1 activity. Afterward, we washed the inhibitor out with excess medium, and monitored the cell cycle progression by measuring DNA content by using FACS analysis (Fig. 4 A). Surprisingly, unlike S. pombe, this inhibition and reactivation of Cdk1 in G2 phase did not result in endoreplication, and all cells initiated the next round of replication only after mitosis (Fig. 4 A; note the absence of cells containing DNA >4N). This observation is in marked contrast with a previous study by Itzhaki et al. (1997), who showed that a human fibroblast cell line continued to increase in ploidy, after conditional inhibition of Cdk1 expression.

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Figure 4.

Analysis of DNA endoreplication and Mcm binding to chromatin after Cdk1 and Cdk1/2 inhibition in DT40 and HeLa cells. (A) Inhibition of Cdk1 does not trigger endoreplication in cdk1as/cdk2 ^−/− cells. The cells were treated with 1 μM 1NM-PP1 for 6 h to allow accumulation in G2 phase, followed by addition of 10 μM 1NM-PP1 for an additional hour (1–10 μM block). The inhibitor was washed out in excess medium and the cells were further incubated without the inhibitor for 14 h. At 2, 6, and 14 h after wash out, cell cycle analysis was performed. (B) Evaluation of origin licensing by analyzing chromatin binding of Mcm2-4 in cdk1as/cdk2 ^−/− cells. Asynchronous populations (lanes 1 and 4), and cells synchronized in G2 by a 12-h incubation in 1 μM 1NM-PP1, and then further treated with 10 μM 1NM-PP1 (lanes 2 and 5), or with 10 μM 1NM-PP1, and 50 μM Roscovitine (lanes 3 and 6) are shown. The indicated samples were subjected to chromatin fractionation and immunoblot analysis of the soluble (left) and chromatin (right) fraction. (C) Cell cycle analysis of HeLa cells released from a double thymidine block. At 7 h after release, 100 μM Roscovitine or DMSO was added to the cells, which were incubated for additional 2 h (9 and 9+Ros). Progression into the next G1 phase was inhibited by simultaneous addition of 100 ng/ml Nocodazole. (D) Lack of origin licensing in HeLa cells following Roscovitine treatment in G2. Cells were prepared as in C. Samples were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Note the absence of Cdt1 and Mcms in the chromatin fraction after Cdk1 inhibition in lane 9.

To confirm that Cdk1 inactivation does not activate replication origins of cdk1as/cdk2 ^−/− cells, we analyzed the recruitment of Mcm proteins, essential components of the pre-RC, to chromatin. In G2 phase, Mcm2–7 are excluded from chromatin, and need to be loaded as a hexameric ring structure onto the DNA to license origins for a new round of DNA replication. In correlation with the results in Fig. 4 A, we could not detect Mcm2, 3, and 4 in the chromatin fraction of cdk1as/cdk2 ^−/− cells that were synchronized in G2 by1 μM 1NM-PP1, and then further treated with 10 μM 1NM-PP1 to fully block Cdk1 (Fig. 4 B, lane 5). Moreover, additional inhibition with the general Cdk inhibitor Roscovitine also failed to induce Mcm2–4 binding to chromatin (Fig. 4 B, lane 6). Thus, inhibition of both Cdk1 and Cdk2 during G2 phase may not be sufficient for origin licensing in chicken DT40 cells.

Previous studies suggested that the general kinase inhibitor DMAP induces Mcm loading onto chromatin in the G2 phase of HeLa cells (Coverley et al., 1996). We repeated the same experiments by inhibiting Cdks more specifically by treating HeLa cells with Roscovitine. For this purpose, we synchronized the cells in early S phase by a double thymidine block, released them, and analyzed the chromatin binding of both the licensing factor Cdt1 and Mcms, as cells progressed through S phase. After 7 h after release, when most cells were in G2 phase (Fig. 4 C), Mcms had been largely displaced from the chromatin (Fig. 4 D, lane 8). At this time, we added Roscovitine to the cells, while a control sample was left without Cdk inhibition (Fig. 4, C and D; compare “9” with “9+Ros”). Both samples were treated with Nocodazole, an inhibitor of spindle formation, to avoid the entry in the next G1 phase. In accordance with the results obtained with chemical genetics in DT40 cells (Fig. 4, A and B), Roscovitine did not appear to induce Mcm loading onto chromatin in the G2 population of HeLa cells (Fig. 4 D, lane 10). In conclusion, Cdk inhibition in G2 phase is not necessarily sufficient to induce origin licensing in chicken and human cells.

Inhibition of Cdk1as in mitotic cells causes endoreplication

Vassilev et al. (2006) recently observed an induction of endoreplication in Nocodazole arrested human cells treated with a Cdk1 inhibitor. We wished to know whether Cdk1 inhibition by the chemical genetics method also triggers endoreplication in mitotic DT40 cells. To avoid a prolonged treatment with Nocodazole, we first synchronized cdk1as cells in G2 using 1NM-PP1, and then released them into mitosis by removing the inhibitor, while adding Nocodazole to obtain cells that were briefly arrested in prometaphase (see diagram in Fig. 5 A). In contrast to the cells arrested in G2 (Fig. 4 C), the mitotic cdk1as cells exhibited DNA replication without completing mitosis, after addition of 10 μM 1NM-PP1 (Fig. 5 B). Cdk1 inhibition also led to rapid dephosphorylation of Histone H3, indicative of decondensation of chromosomes (Fig. 5 C). We also observed the quick dephosphorylation of the APC/C subunit Cdc27 (Fig. 5 D), which is hyperphosphorylated by Cdk1 in mitosis (Rudner and Murray, 2000). Moreover, we found that the APC/C was activated in response to mitotic Cdk1 inhibition, as judged by the rapid degradation of cyclin A upon addition of the inhibitor (Fig. 5 E). Nocodazole arrested cdk1WT cells showed no such response to 1NM-PP1 and remained unchanged in mitosis (unpublished data). These data suggest that inhibition of endoreplication may be carried out differently in G2 and M phase.

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Figure 5.

Inhibition of Cdk1 in mitosis causes endoreplication depending on proteolysis. (A) Experimental design. After a 7-h exposure to 10 μM 1NM-PP1 we released the cells from a G2 arrest into medium containing Nocodazole. After a 1-h incubation in Nocodazole, Cdk1 was again inhibited by the addition of 10 μM 1NM-PP1. (B) Endoreplication after inhibition of Cdk1 in mitosis. cdk1as cells arrested in prometaphase as described in A were incubated with 10 μM 1NM-PP1 and pulse labeled with BrdU at the indicated time points. Dot blot analysis is displayed as in Fig. 1 E. (C) Analysis of Histone H3 phosphorylation in samples treated as described in A. Cells were fixed at 10 min after the second Cdk1 inhibition or without inhibition, and stained with both PI (X-axis, linear scale) and anti phospho-Ser10 Histone H3 antibodies (Y-axis, log scale). (D) Analysis of Cdc27 phosphorylation by immunoblotting in cells synchronized in G2 phase or mitosis as described in A. 15 min after release from the 1NM-PP1 block into mitosis (lane M) Cdc27 shifts upwards due to hyper-phosphorylation by Cdk1. It remains phosphorylated after 1 h in Nocodazole (lane 1, Noc 1h). Following Cdk1 inhibition by addition of 10 μM 1NM-PP1, Cdc27 is rapidly de-phosphorylated and shifts back to its G2 form within minutes (see lane 10′). (E) Immunoblot analysis of cyclin A and Histone H2A levels in Nocodazole arrested cdk1as cells at the indicated times after the second Cdk1 block. (F) Endoreplication initiated by the second Cdk1 inhibition is blocked by MG132. The same experiment as in B in the presence of the proteasome inhibitor MG132 is shown. (F) MG132 blocks origin licensing induced by the second Cdk1 inhibition. Chromatin fractionation of cdk1as cells arrested in mitosis as shown in A, before (lane 1 and 4) and 2 h after 1NM-PP1 addition (lane 2 and 5). To inhibit the proteasome, cells had been incubated for 1 h with MG132 before the 1NM-PP1 inhibitor was added (lane 3 and 6). Mcm and Histone H2A levels were measured by SDS PAGE and immunoblotting.

Endoreplication induced by Cdk1 inhibition in mitosis depends on proteolysis

A possible explanation for the differential effects of Cdk1 inhibition on DNA synthesis in G2 and M phase could be the activation of APC/C mediated proteolysis during mitosis but not G2 phase (compare cyclin stability in Fig. 1 F and Fig. 5 E). To test this hypothesis, we treated cdk1as cells that were synchronized in mitosis as shown in Fig. 5 A with the proteasome inhibitor MG132 before Cdk1 inhibition. We found that MG132 prevented the induction of DNA synthesis by 1NM-PP1 (compare Fig. 5 F with Fig. 5 B). Furthermore, MG132 suppressed the induction of Mcm loading on chromatin after Cdk1 inhibition (Fig. 5 G, compare lane 5 with lane 6). These results suggest that Cdk1 inhibition is not sufficient to allow origin licensing and endoreplication, unless proteolysis is activated.

Geminin needs to be degraded in human cells to allow origin licensing

To verify these results from DT40 cells in a human cell line, we analyzed Nocodazole-arrested HeLa cells treated with Roscovitine. We found that this Cdk inhibitor initiated origin licensing, as judged by Cdt1 and Mcm2 loading onto chromatin, as previously described by Ballabeni et al. (2004). Inhibition of the proteasome by MG132 abolished the Roscovitine induced chromatin binding of Mcm2 and Cdt1 in HeLa cells (Fig. 6 A, lane 3 and lane 6), confirming our previous results with DT40 cells (Fig. 5, F and G).

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Figure 6.

Causal relationship of Geminin destruction and origin licensing in mitotic HeLa cells after Cdk1 inhibition. (A) Chromatin fractionation of Nocodazole (Noc) arrested cells with the indicated additions. MG132 was added to mitotic cells after a 12-h incubation in Nocodazole. 50 μM Roscovitine was added 1 h later, and the cells were incubated for an additional 2 h. Shown are the immunoblots of soluble and chromatin fractions with the indicated antisera. (B) Degradation of APC/C substrates Cdc20 and Aurora kinase A after Roscovitine treatment of Nocodazole arrested HeLa cells. Cell lysates before or 2 h after 50 μM Roscovitine treatment were analyzed by immunoblotting with the indicated antibodies. (C) Chromatin fractionation of Nocodazole-arrested HeLa cells after transient expression of the indicated cDNAs. The cells were pre-synchronized by a double thymidine block, and transient transfections of either GFP or the destruction box deficient mutants of cyclin B or Geminin were performed, after release from the second thymidine block. Nocodazole was added 7 h after the release, and samples were taken after 12 h further incubation. The soluble and chromatin fractions were probed by immunoblotting with the indicated antisera. Note the absence of Cdt1 and Mcm3 in the Roscovitine treated samples containing the indestructible Geminin mutant (lane 12).

The proteolysis dependence of origin licensing and endoreplication induced by Cdk1 inhibition, suggests that proteins other then cyclins need to be degraded to allow pre-RC formation. Geminin, which is an APC/C substrate and a licensing inhibitor (McGarry and Kirschner, 1998; Wohlschlegel et al., 2000), is a good candidate to account for this proteolysis requirement after Cdk inhibition. Accordingly, we found that Roscovitine triggered the destruction of both cyclin B1 and Geminin (Fig. 6 A, lane 2), whereas MG132 treatment stabilized these proteins (Fig. 6 A, lane 3). Fig. 6 B shows that the APC/C targets Aurora kinase A and Cdc20 are also degraded upon Cdk inhibition during mitosis.

To test if the inhibition of Geminin degradation is sufficient to prevent Roscovitine-induced origin licensing, we investigated the effects of ectopic expression of a Geminin mutant that is resistant to APC/C dependent degradation, on mitotic HeLa cells treated with Roscovitine (Benjamin et al., 2004). We transiently expressed this stable Geminin mutant in HeLa cells, after release from a double thymidine block. As a control experiment, we expressed GFP or an APC/C-resistant mutant of mouse cyclin B1 in HeLa cells treated in the same manner. The transfected cells were arrested in prometaphase by Nocodazole treatment. In the control samples, licensing was initiated by the addition of Roscovitine, as judged by the chromatin loading of Mcms and Cdt1 (Fig. 6 C, lane 8 and lane 10). In contrast, expression of the stable Geminin mutant inhibited loading of Cdt1 and Mcm onto chromatin in response to Roscovitine treatment (Fig. 6 C, lane 12). In fact, we observed that even overexpression of WT Geminin in mitosis partially inhibited Mcm loading (unpublished data). We conclude that Geminin needs to be targeted for degradation by the APC/C, even after Cdk inactivation, to allow for origin licensing and endoreplication in vertebrate cells.

Redundant and specific functions of Cdk/cyclin complexes in the cell cycle

Our phenotypic comparison of cdk1as and cdk1as/cdk2 ^−/− cells reveals redundant as well as specific roles of vertebrate Cdk1 in the control of DNA replication, centrosome duplication, and mitosis (Fig. 7 A). We show that the deletion of Cdk2 renders both the initiation of DNA replication and centrosome duplication dependent on Cdk1 (Fig. 2 and Fig. 3). This suggests that Cdk1 and Cdk2 share an essential function in the control of S phase. Our findings also suggest that the centrosome cycle and the cell cycle can be uncoupled simply by blocking entry into mitosis, and that either Cdk1 or 2 activity is required to drive this cell cycle–independent centrosome amplification.

In principle, the experiment in Fig. 3 C shows that mid S phase cells do not need Cdk1/2 activity to continue replication. However, the dynamics of S phase completion appear to be changed after Cdk1 inhibition in cdk1as/cdk2 ^−/− cells. A detailed analysis of late origin firing and the dynamics of replication elongation will be necessary to determine the roles of Cdk1/2 during ongoing replication.

The functional overlap of Cdk1 and Cdk2 does not include the mitotic functions of Cdk1, which cannot be compensated by Cdk2. We also found that neither Cdk1 nor Cdk2 appear to control the events of early G1 phase such as initiation of de novo cyclin synthesis (Fig. 3 D), which is likely to be triggered by Cdk4/6. Moreover, we cannot exclude at this point that either kinase carries out other specific functions that evade our phenotypic analysis.

It remains to be addressed, which of the different cyclins is primarily responsible for the S phase functions of Cdk1. A-type and B-type cyclins, the predominant binding partners of Cdk1as in the DT40 cells (Fig. S3 E) can both initiate DNA replication (Strausfeld et al., 1996; Moore et al., 2003). Cyclin A is the more likely candidate for this function, because cyclin B/Cdk1 is rapidly exported from the nucleus during S phase (Yang et al., 1998) and kept inactive by the Wee1 kinase (Chow et al., 2003). However, a recent study suggests that cyclin E is also capable of binding and activating Cdk1, especially after deletion of Cdk2 (Aleem et al., 2005). The precise functions of the individual cyclins during S phase need to be addressed in future studies.

Differential control of endoreplication during G2 and M phase

Our study points to fundamentally different mechanisms in the control of endoreplication before and after mitotic Cdk1 activation and entry into M phase. The three main players of licensing control in vertebrate appear to be Geminin and Cdk1 (and possibly Cdk2), as well as proteolysis pathways such as the degradation of Geminin and Cdt1. In the following section we will discuss our findings on the function of these players comparing G2 to M phase. A summary of this discussion is presented in Fig. 8 B.

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Figure 8.

Model of Cdk1 function in controlling DNA replication in vertebrate cells.

Geminin inhibits origin licensing independently of Cdks

Our results in Fig. 6 and Fig. 7 show that removal of Geminin is a necessity for origin licensing both in G2 and M phase, and that Geminin can act independently of Cdk activity. This idea is not supported by a previous study (Ballabeni et al., 2004), where Roscovitine treatment does not cause APC/C activation and Geminin degradation in mitotic HeLa cells. In contrast, we observed in consistence with previous studies (Listovsky et al., 2000) that the APC/C was rapidly activated upon mitotic Cdk1 inactivation causing the degradation of Geminin as well as other APC/C substrates (Fig. 6 A). We also confirmed that a degradation resistant Geminin mutant inhibited origin licensing, even after Cdk1 inactivation (Fig. 6 C). The discrepancy between our and Ballabeni et al.'s (2004) results may be due to the twofold lower doses of Roscovitine used in the previous study, which may trigger Geminin degradation only partially.

Effects of Cdk1 inhibition on endoreplication in G2 and M phase

We found that specific inhibition of both Cdk1 and Cdk2 initiated neither origin licensing nor DNA re-replication during G2 phase in DT40 (Fig. 4 B). The same result was obtained from Roscovitine-treated HeLa cells (Fig. 4 D). Paradoxically, Cdk1 inhibition in prometaphase rapidly triggered origin licensing and endoreplication (Fig. 5, B and G, lane 2 and lane 5; and Fig. 6 A). Accordingly, Cdk1 inhibition during G2 phase does not lead to APC/C activation and Geminin destruction, whereas in M phase Cdk1 inactivation results in the rapid removal of Geminin and other APC/C substrates (Fig. 6, A and B). The presence of the APC/C inhibitor Emi1 during G2 but not during M phase (Reimann et al., 2001; Margottin-Goguet et al., 2003) is a likely explanation for this difference in APC/C activity between the two different cell cycle phases. This idea is supported by a recent report by Machida and Dutta (2007), which demonstrates that depletion of the APC/C inhibitor Emi1 is sufficient to induce endoreplication in human cells.

In contrast to our results, the APC/C appears to be activated during G2 phase in Cdk1 depleted HT2-19 human cells and Drosophila cells (Hayashi, 1996; Laronne et al., 2003). This could explain the observed endoreplication in these cells after Cdk1 inactivation. The premature APC/C activation in G2 might be an effect of incomplete Cdk1 inactivation, which could allow Emi1 degradation, while not being sufficient to trigger mitosis. Alternatively, Emi1 levels might differ among different cell lines.

Cell culture, transfection, and synchronization

DT40 cells were cultured, and transfected as described previously (Sonoda et al., 1998). Takata et al. (1998) described the synchronization of DT40 cell by elutriation. 1NM-PP1 was obtained from Cellular Genomics and used at the indicated concentrations (1–10 μM). We used a 50-μM concentration of Roscovitine (Calbiochem), and a 5-μM concentration of MG-132 (Calbiochem). Cell cycle analysis of BrdU-pulsed and PI-stained samples was performed on a FACScan (Becton Dickinson) using CellQuest software (Takata et al., (1998). HeLa cells were synchronized in mitosis by incubation in 100 ng/ml Nocodazole, and in S phase by a double thymidine block. In brief, cells were incubated for 14 h in 2 mM thymidine, washed in PBS, released for 10 h, and then blocked for another 14 h in 2 mM thymidine. Transient transfection of HeLa cells by Lipofectamine (Invitrogen) was performed following the second thymidine block as described in the manufacturer's protocol. To target Geminin we transfected a QIAGEN-validated siRNA SI02653805 at a final concentration of 100 nM by oligofectamine (Invitrogen), following the manufacturer's instructions.

Preparation of total cell extracts and chromatin fractionation

For total cell extraction, 10⁶ DT40 cells or 10⁵ HeLa cells were lysed in 10 μl ECB buffer (50 mM Tris, pH 7.5, 120 mM NaCl, 0.5% NP-40, and 1 mM EDTA) containing 1 mM DTT and a 1:100 dilution of a protease inhibitor cocktail (Nakalai Tesque 25955-11). The extracts were incubated for 20 min on ice, sonicated, and suspended in 2× Laemmli buffer. For chromatin fractionation, we separated soluble and insoluble fractions as described by Ballabeni et al. (2004). 10⁶ DT40 or 10⁵ HeLa cells were first lysed for 20 min in 10 μl CSK buffer containing DTT and protease inhibitors as above. Following centrifugation, the supernatant was mixed with 2× Laemmili buffer, and the pellet was washed and resuspended in 10 μl CSK buffer, sonicated and suspended in 2× Laemmli buffer.

Immunoblotting, Immunoprecipitation, immunofluorescence, and Histone H1 kinase assays

Immunoblotting and immunoprecipitation, and histone H1 kinase assays were done as described earlier (Sonoda et al., 1998; Chow et al., 2003). Immunofluorescence of cyclin B was performed with formaldehyde fixed samples that were spun on a glass slide by centrifugation in a Cytospin centrifuge. α- and γ-tubulin immunofluorescence was performed as described earlier (Yamaguchi-Iwai et al., 1999).

Image acquisition and manipulation

All images were taken with an Olympus BX61 microscope, equipped with a Photometrics CoolSnap HQ camera, and Olympus Uplan/APO 100× lens (NA 1.35). Samples were mounted in Vectashield mounting medium with DAPI (Vector Laboratories, Inc.) and analyzed at room temperature. Image acquisition was performed using MetaMorph software.

Antibodies

Antibodies used in this study comprised the anti Cdk1 mouse monoclonal A17 antibody (Gannon et al., 1998), rabbit polyclonal anti Cdk1 antibody from Upstate Biotechnologies, rabbit polyclonal anti Cdk2 antibody from Abcam (ab-7954-1), anti-PSTAIRE (Poon et al., 1997), and rabbit polyclonal anti-phosphotyrosine15 Cdk1 antibody from Cell Signaling (Nr. 9111S). (06-923). Anti-chicken cyclin A (Maridor et al., 1993) and anti-chicken cyclin B2 (Gallant and Nigg, 1992) antibodies were gifts from E. Nigg's laboratory (MPI of Biochemistry, Munich, Germany); monoclonal anti-α-tubulin FITC conjugate (No. F2168), and γ-tubulin polyclonal (No. T3559) antibodies were obtained from Sigma-Aldrich; Mcm2, 3, 4, and Histone H2A polyclonal antibodies were obtained from Abcam (ab 4461-50, 4460-50, 3728-50, 13923-100, respectively); and anti–human Cdt1 antibody was a gift from H. Nishitani (Kyushu University, Fukuoka, Japan; Nishitani et al., 2001). The Cdt1 antibody used in Fig. 7 was purchased from Santa Cruz Biotechnology, Inc. (sc-28262). Aurora kinase A rabbit polyclonal antibodies were purchased from Abcam (ab12875), and to detect Cdc20 we used a mixture of two monoclonal anti-cdc20 antibodies from Santa Cruz Biotechnology, Inc. (SC1907 and SC1906). Human cyclin B1 monoclonal V152 antibodies (ab-72) and rabbit polyclonal anti-Geminin antibodies (ab-12147-50) were obtained from Abcam. For FACS analysis, ethanol-fixed cells were stained with anti-BrdU monoclonal antibody (BD Biosciences; Nr. 555627) and anti-phospho Ser10 rabbit polyclonal Histone H3 antibody (Upstate Biotechnology; Nr. 06-570). Alexa-labeled secondary antibodies were purchased from Molecular Probes, and HRP-labeled secondary antibodies were from Santa Cruz Biotechnology. Cdc27 was detected by a monoclonal antibody from Abcam (ab10538).

We would like to thank Erich Nigg, Hideo Nishitani, Michael Brandeis, and Stephan Geley for sharing plasmids and antibodies with us. We also thank Robert Fisher and Anindya Dutta for sharing unpublished results. Thanks to Julian Blow, Miguel Ferreira, Ciaran Morrison, Mathew Jones, and Julian Sale for critical reading of the manuscript.

Financial support was provided in part by CREST/JST, (Saitama, Japan) and the center of excellence (COE) grant for Scientific Research from the Ministry of Education, Culture, Sports and Technology. H. Hochegger was supported by a JSPS fellowship. We would like to thank all the members of the Takeda and Hunt labs for their support in this study.