Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34cdc2-mitotic B cyclin complex

Cell, Vol. 78, 813-822, September9, 1994, Copyright© 1994 by Cell Press

Temporal Order of S Phase and Mitosis in Fission Yeast Is Determined by the State of the p34Cd2-Mitotic B Cyclin Complex Jacqueline Hayles, Daniel Fisher, Alison Woollard, and Paul Nurse Cell Cycle Laboratory Imperial Cancer Research Fund Lincoln's Inn Fields London WC2A 3PX England

Summary We show here that the state of the p34C¢C2-p56Cd¢~ mitotic B cyclin complex determines whether a fission yeast cell undergoes S phase or mitosis. Mutants defective for p56 ~dc~ reset to G1 and rereplicate their DNA, while cells completely lacking the p34 c~¢2p56 °~c~3complex undergo multiple rounds of S phase. In contrast, formation of the p34~¢2-p56~¢~3 complex in G1 promotes cells inappropriately into mitosis. We propose that the temporal order of S phase and mitosis is maintained by the presence or absence of the p34cdc2-p56cdcl~ complex. Introduction Checkpoint or dependency controls acting during the cell cycle ensure that only one S phase takes place each cycle and that cells do not enter mitosis until DNA replication is complete. Together these checkpoints maintain the temporal order between S phase and mitosis, ensuring genome ploidy and integrity. During certain developmental processes, these dependencies can be disrupted. For example, during Drosphila embryonic and larval development (Smith and Orr-Weaver, 1991) and plant embryogenesis (Nagl et al., 1985), cells undergo repeated rounds of DNA replication in the absence of mitosis, and during red algae development, cells undergo multiple rounds of DNA replication followed by multiple mitoses (Golf and Coleman, 1990). The process of meiosis, when two nuclear divisions occur without an intervening S phase (John, 1990), can also be viewed as a departure from the normal temporal order of S phase and nuclear division. Certain chemical treatments ~nd mutations also lead to DNA replication without mitosis, possibly by acting on targets involved in this checkpoint control. For instance, treatment of mammalian cells with the antifungal agents trichostatin A and leptomycin B causes cells to arrest in G1 and G2, and recovery from this arrest is followed by rereplication of DNA in the absence of mitosis (Abe et al., 1991; Yoshida et al., 1990). Rereplication of DNA is also induced by staurosporine analog protein kinase inhibitors. In these cases, rereplication is induced to high levels in the presence of the drugs (Usui et al., 1991). A human cell line has been isolated that undergoes rereplication in the absence of G1, G2, or mitosis (Handeli and Weintraub, 1992). The function that is defective in this cell line is required both to inhibit S phase and to initiate mitosis, and

the cells appear to undergo complete rounds of replication rather than reinitiate replicons within a single S phase. The gene product involved in this mechanism has yet to be identified. In the fission yeast Schizosaccharomyces pombe, two genes, rum1 and cdc2, have been identified that are involved in the controls ensuring the dependencies between S phase and mitosis. Overproduction of the rum1 gene product induces multiple discrete rounds of S phase, whereas deletion of the rum I gene leads to entry into mitosis when DNA replication is blocked (Moreno and Nurse, 1994). Specific cdc2 mutations also result in DNA replication occurring without an intervening mitosis and in entry into mitosis when DNA replication is blocked (Broek et al., 1991; Enoch and Nurse, 1991). The cdc2 gene product is therefore not only required for the onset of S phase and mitosis but is also necessary for the temporal order of these two events. In this paper we describe the isolation of mutants altered in the dependency between S phase and mitosis. One of these mutants is defective in the cdc13 gene that encodes the mitotic B cyclin p56c~c13(Booher and Beach, 1988; Hagan et al., 1988). We show that this mutant and a previously isolated cdc13 mutant rereplicate after heat treatment. We also show that cells deleted for cdc13 (Moreno et al., 1989), which have no p34c~2-p56~la complex, undergo repeated rounds of S phase. Conversely, when the p34c~2-p56cdc~ complex is present at high levels in G1 cells arrested before START by the cdc10-129 mutation (Nurse et al., 1976), cells enter mitosis even though S phase is blocked. These results lead us to propose that lack of the p34c~2-p56c~13 complex in G2 resets the cell to G1 in readiness to enter S phase, while the presence of the complex in G1 results in premature entry into mitosis.

Results Isolation of Rereplicating Mutants To identify gene functions involved in the control of the temporal order of S phase and mitosis in fission yeast, we have screened for mutants that generate cells of higher ploidy as a consequence of rereplication. Using the procedure described by Broek et al. (1991) to enrich for mutants able to diploidize, we mutagenized a mam2 leul-32 h ~° strain, incubated the cells at 36°C for 1-4 hr, and then returned the cells to the permissive temperature (25°C). Any cell that rereplicated either during the heat pulse or immediately afterward would then be able to sporulate. The vegetative cells were destroyed and the spores plated out to form colonies. From these colonies, 1200 temperature-sensitive mutants were identified. Several classes of mutants were isolated, including cut (Hirano et al., 1986) and septation mutants (Nurse et al., 1976). Only three mutants generated cells that had a single enlarged nucleus, with no evidence of aberrant mitosis or septation, as would be expected for a mutant that had undergone rereplication. These mutants were crossed to representa-

Cell 814

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Table 1 Generation of Diploids in cdc13-9, cdc13-117, and Wild-Type Strains

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Formation of Diploid Colonies (%)

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0.5 85.5 99.0

1.0 1.6 1.7

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The percentage of diploid colonies formed at 25°C was counted following growth in EMM (Moreno et al., 1991) containing nitrogen at 25°C, EMM lackmg a nitrogen source at 36°C for 4 hr, and EMM iackmg a nitrogen source at 36°C for 4 hr followed by heat treatment at 49°C for 45 min.

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Figure 1. FACS Analysis of cdc13-9, cdc13.117,and Wild-Type Cells after Nitrogen Starvation and Heat Treatment (a) Samples from cdc13-9cells were taken during exponential growth in EMM (exp), after 4 hr nitrogen starvation at 36°C (4h 36 -N), and after 3 hr in EMM at 25°C following nitrogen starvation at 36°C (+3h 25). Cells replicate thetr DNA by 3 hr at 25°C, as seen by a change from a 2C to a 4C peak. Standards (stds) are 1C, 2C, and 4C DNA, shown for all experiments. (b) Samples of cdc13-117cells were taken during exponential growth in EMM (exp), after 4 hr nitrogen starvation at 36°C (4h 36 -N), after 4 hr mtrogen starvation at 36°C followed by heat treatment at 49°C for45 min (+HS), after 2 hrat 25°C in EMM following the heat treatment (+2h 25 +HS), and after 4 hr at 25°C in EMM following the heat treatment (+4h 25 +HS). (c) Wild-type cells were sampled as described in (b). There was no change from a 2C to a 4C peak, showing that rereplication dtd not occur. (d) Dtploid cdc13-117cellssampled during exponentialgrowth at 25°C (exp), after 4 hr mtrogen starvabon at 36°C (4h 36 -N), and after 8 hr at 25°C in EMM following the heat treatment (+8h 25 +HS). After 8 hr incubation at 25°C, the DNA content had increased from 4C to 8C.

tive cdc mutants and were found to be alleles of two previously identified cdc genes, cdc2 and cdc13. Mutants of cdc2 have previously been shown to induce rereplication after nitrogen starvation and heat treatment (Broek et al., 1991). In this screen we isolated a further two cdc2 alleles as well as a cdc13 mutant that rereplicates. These two genes, cdc2 and cdc13, e n c o d e proteins that interact to form a c o m p l e x whose activity regulates entry into mitosis (Nurse, 1990).

Mutants of cdc13 Mitotic B Cyclin Undergo Rereplication We analyzed the new cdc13-9 mutant and the previously identified cdc13-117 mutant (Nurse et al., 1976) to investigate the role of mitotic B cyclin in the d e p e n d e n c y of S phase upon the previous mitosis. The mutant, cdc13-9, is a temperature-sensitive lethal that arrests in G2 when incubated at 36°C. On return of this mutant to the permissive t e m p e r a t u r e of 25°C, s o m e of the cells undergo an extra round of S phase without an intervening mitosis. As with rereplicating mutants of cdc2 (Broek et al., 1991), a higher proportion of cells undergoing DNA rereplication can be generated if the cdc13-9 mutant is nitrogen starved for 4 hr at 3 6 ° C before returning to 25°C. This treatment results in the formation of 8 5 % diploids from an initially haploid strain (Table 1). Fluorescence-activated cell scan (FACS) analysis shows that rereplication occurred by 3 hr after shift to 2 5 ° C (Figure la). During these 3 hr, no cells with mitotic spindles were observed, and there was no increase in cell number, showing that rereplication occurred without mitosis or cell division (data not shown). The only previously identified cdc13 temperature-sensitive mutant, cdc13-117, could also be induced to form diploids (Table 1) and to rereplicate (Figure lb), but only if the cells were heat treated to a higher t e m p e r a t u r e (see legend to Figure lb). A diploid cdc13-117 strain could be induced to b e c o m e tetraploid by a similar regime (Figure ld). No rereplication or diploid induction was seen in a wild-type control (Figure l c ; Table 1). Therefore, temperature-sensitive cdc13 mutants can be induced to undergo an extra S phase without mitosis when nitrogen starved and heat treated. The effect of this treatment is unclear, but, as suggested for the previously iso-

Temporal Order of S Phase and Mitosis 815

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Figure 2. A cdc13,J Strain Rereplicates Its DNA to Give Elongated Cells with Highly Enlarged Nuclei (a) A diploid cdc13/I strain (cdc13A::ura4+l

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(Moreno et al., 1989). FACS samples were analyzed at 6, 8, 11, 14, 17, and 19 hr after inoculation of the spores into EM M with added adenine and leucme (250 rag/I) but without uracil. All of the cells in the wild-type culture had a 2C DNA content by 14-19 hr. The cdc13zt culture showed a 4C peak at 11 hr, and during the period from 14-19 hr, discrete peaks corresponding to 2C, 4C, 8C, 16C, and 32C were observed. Note the log scale for DNA content. (b) Samples were taken of germinated cdc13A spores at 14 hr after inoculation and stained with DAPI. Elongated cells with a large nucleus and ungerminated spores with a small nucleus can be seen. (c) FACS analysis of cdc13zl strain after plasmid loss. Samples were taken as follows: at O hr, 70% of the cells have a 1C DNA content; at 4 hr, the elongated cells have a DNA content of 2C and 4C; at 8 hr, the elongated cells have an 8C DNA content; at 16 hr, the elongated cells have a DNA content of 32C or above.

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lated cdc2 mutants (Broek et al., 1991), it may result in a more complete denaturation of the thermolabile p56 ~d~13.

Cells Lacking the Mitotic B Cyclin p56 °~c13 Undergo Multiple Rounds of S Phase The genes c d c 2 and cdc13 e n c o d e c o m p o n e n t s of the p34CdCLp56cdc13complex, w h o s e activity is responsible for mitotic onset, The fact that heat treatment of temperaturesensitive mutants of either cdc2 or cdc13 can induce DNA rereplication suggests that disruption of the p34 ° ~ L p56 c~a c o m p l e x m a y be responsible for r e p r o g r a m m i n g

a G2 cell to enter G1 and undergo S phase. This possibility was tested by germinating spores deleted for cdc13. A diploid strain with one copy of cdc13 deleted and replaced with ura4 was sporulated and the spores inoculated into minimal m e d i u m lacking uracil. Only spores deleted for cdc13 (cdc13zl) were able to germinate and grow (Moreno et al., 1989). These spores proceeded to G2 and then rereplicated their DNA (Figure 2a). In both the wild-type control and the cdc13/I strain, S phase occurred 4 - 8 hr after inoculation (Figure 2a; Nurse and Thuriaux, 1977). In both cultures, some ura- spores germinated and under-

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Figure 3. p34'~c2and p56~c'3 Protein Levels and State of the p34C~C2-p56=~c13Complex during Rereplication (a) A Western blot of the p34C=2-p56C~°~complex precipitated from soluble extracts of wild-type cells (lane 1) and cdc13/I cells (lane 2) probed with anti-p56~ 3 SP4 (top panel) and anti-p34~2 PN24 (bottom panel), p34c~2 ts present in both precipitates, but p56~ 3 is only detected in precipitates from the wild-type extracts, showing that there is no complex present in the cdc13A strain. (b) A Western blot of the p34~d~-p56~ complex precipitated from soluble extracts of wild-type (lane 1) and cdc13-9 (lane 2) cells probed with SP4 (top panel) and PN24 (bottom panel) p34~2 was detected in precipitates from both extracts, but p56~ 3 was only seen at a low level in cdc13-9 extracts compared with that seen in wild-type extracts, suggesting that in cdc13-9 the complex is less stable or present at a lower level. Some proteolysis of p56~d°~occurred during the sample preparatton, and several bands reactive to the antibody can be seen. (c) Western blots of insoluble and soluble protein extracts from 972 wtld type, cdc13-117, and cdc2-L7 probed wtth SP4 (top panels) and PN24 (bottom panels). Lane 1, insoluble, exponential growth; lane 2, insoluble, after heat treatment; lane 3, soluble, exponential growth; lane 4, soluble, after heat treatment. Quantification showed that in wild-type cell extracts after heat treatment, soluble p34c~c2(lane 4) was reduced to 70% of the amount found in exponentially growing cells (lane 3).

went S phase but became arrested as small cells with 2C DNA content. By 19 hr of growth, about a third of the cells in the cdc13/I culture had attained DNA contents of 32C or above. After staining the nucleus with DAPI, we observed cells to be highly elongated with giant nuclei (Figure 2b). During the period of 11-19 hr, the wild-type cells undergo approximately three generations of growth. In the cdc13,d strain over this period, there are also approximately three doublings in the DNA content from 4C to 32C. This suggests that the cells rereplicate with around normal cell cycle timing and that the dependency of S phase initiation upon cell mass increase is still intact. Cells were examined at intervals of 1 hr between 1217 hr both by immunofluorescence to check for the presence of spindles and by DAPI staining to see whether they partially enter mitosis before rereplicating. All the cells with a large nucleus had interphase microtubules, no spindles (data not shown), and no condensed chromosomes (data not shown; Figure 2b). Thus, there was no evidence that the cdc13A mutant underwent any aspect of mitosis before undergoing DNA rerepiication.

Confirmation that cells lacking p56 cd~73are induced to rereplicate their DNA was obtained using a cdc13/I strain kept viable by the cdc13 gene carried on an unstable plasmid. Cells losing the plasmid were unable to undergo mitosis and so produced highly elongated cells. This strain was nitrogen starved, which induces considerable plasmid loss, and then reinoculated into medium containing nitrogen at 32°C. The elongated cells were selected using FACS on the basis of increased forward scatter, and their DNA content was then estimated. 1As shown in Figure 2c, the DNA content of elongated cells lacking cdc13 was increased to 32C and above. We conclude that cells completely lacking the cdc13 gene are able to undergo multiple rounds of DNA 1replication.

Rereplication Is Associated with Lack of the p34CdC2-p56C~°~S Complex p56 cdc13is not detectable in extracts from the cdc13/I strain by Western blotting (data not shown; Moreno et al., 1989). These cells should not therefore have any p34cdc2-p56~cl~ complex. To confirm this, we used p13 ~U~7beads (Labbe

Temporal Order of S Phase and Mitos~s 817

et al., 1989) to precipitate the p34~--p56~d°~ complex from wild-type and cdc13/I cell extracts. The complex between p56 c ~ and p34 c~c2could be detected after precipitation followed by SDS-polyacrylamide gel electrophoresis and Western blotting in a wild-type extract (Figure 3a, lane 1) but not in a cdc13z~ extract (Figure 3a, lane 2). These results indicate that the cdc13/I cells lack p56 c~°~ and are unable to form a p 3 4 ~ - p 5 6 ~d~ complex. The complex is also unstable in the cdc13-9 mutant. Precipitation with p13 " ~ beads, followed by SDS-polyacrylamide gel electrophoresis and Western blotting, barely detected the complex in cdc13-9 extracts compared with wild-type extracts, even at the permissive temperatu re of 25°C (Figures 3b, lanes 1 and 2). p34 ¢dc2and p56 °d¢~ Are Present in Two Different Cellular Fractions Presence of the complex in the cdc13-9 mutant could not be monitored directly during the rereplication experiment because p56 cdc13is found mostly in an insoluble fraction after nitrogen starvation and heat treatment. In an earlier study, we considered the possibility that p56 cd~ was involved in the rereplication induced by heat treating cdc2 mutants. Because we showed that p56 c~73 disappeared from a wild-type extract that did not rereplicate, we considered it unlikely that p56 c~c~ destruction was specifically involved in this phenomenon (Broek et al., 1991). We have now shown that p34°~2 is also present in a less-soluble cellular fraction and, as a consequence, we have reassessed the levels of p56 ~ and p34 ~c2 in wi Id-type, cdc13117, and cdc2-L7 mutants before and after heat treatment. Both p34 ~d~2and p56 ~c~3 were found in the insoluble and soluble extracts derived from exponentially growing cells of both mutants and wild type (Figure 3c, lanes 1 and 3). The p56 c~c~3found in the soluble fraction (Figure 3c, lane 3) is often observed as lower molecular weight bands rather than the full-length protein because the soluble fraction of p56 ~c~3 is more easily degraded during the extraction procedure than is p56 °d~3 from the insoluble fraction. After heat treatment we found that p56~d~ disappeared from the soluble extract of wild-type, cdc2-L7, and cdc13117 strains (Figure 3c, lanes 4) and that p34 c~c2 disappeared from the soluble extract of the cdc2-L7 and cdc13117 strains but not from wild-type soluble extracts (Figure 3c, lanes 4). Therefore, in situations in which cells can rereplicate, there is loss of soluble p34c~2 after heat treatment, thus confirming the previously reported results of Broek et al. (1991). However, we also found that both p56 °d°~ and p34 cd°2 persisted in the insoluble fraction of all three strains after heat treatment (Figure 3c, lanes 2). This shows that rereplication occurs in cells even though they still contain p34 cd:~.During rereplication, the insoluble forms of p34 ~ and p56 c ~ persist and the soluble forms of p34 ~ and p56 :d~ are not detected until around the time of S phase (Broek et al., 1991). Therefore, we have modified our earlier conclusion that rereplication is induced by loss of p34 ° ~ and propose that the effect of the heat treatment is to destroy the mitotic p34c~-p56 °~c~ complex that cannot be reestablished in the cdc2-L7 and cdc13-117 mutants. It is this failure to maintain the complex that leads to rereplicatien. This is correlated with the

loss of both p34 ~°~2and p56cd~ from the soluble fraction, and it is possible that in the wild-type strain after heat treatment, p56~ 3 is recruited from the insoluble fraction to reform the mitotic complex, thus inhibiting rereplication.

Overexpression of p34 cdc2and p56 cdcla Induces Premature Entry into Mitosis The data presented above show that lack of the p34 c~c2p56 c~c~ complex allows entry into S phase. The corollary of this is that if the complex was present in G1, it would promote entry into mitosis. To address this point, we have overexpressed p34 c~2 and p56 ~d~ in cells blocked in G1 using the cdc10-129 mutant. The cdc10-129 mutant, when incubated at the restrictive temperature (36°C), arrests in G1 before the cell cycle commitment point called START (Forsburg and Nurse, 1991a). Therefore, this experiment allows us to ask whether cells can enter mitosis from G1 if they contain elevated levels of the p34~c2-p56°~3 complex. A cdc10-129 mutant carrying an integrated copy of cdc2 expressed by the repressible nmtl promoter (Maundrell, 1993) was transformed with a plasmid carrying the cdc13 gene, also expressed by the repressible nmtl promoter. These cells were incubated in medium lacking thiamine to induce high level expression of both the cdc2 and cdc13 genes, and the culture was then shifted to 36°C. The cdc10-129 strain and cdc10-129 containing cdc2 and cdc13 with the nmtl promoter repressed were also shifted to 36°C. After 4 hr incubation at the restrictive temperature, all three strains had become blocked in G1 at START with a 1C DNA content (Figures 4a-4c, top panels). At this time, cells overexpressing cdc2 and cdc13 showed a high level of cells in mitosis (Figure 4c, bottom panel; Figure 4d) com pared with cells not overexpressing the two genes (Figures 4a and 4b, bottom panels; Figure 4d) or overexpressing either gene alone (data not shown). Cells overexpressing cdc2 and cdc13 entered mitosis even though they were arrested before S phase with a 1C DNA content (Figure 4c, top panel). They had a typical cut phenotype (Hirano et al., 1986) and had a mitotic spindle but no interphase microtubules. Precipitation with p13 ~u~1 beads followed by Western blotting showed that the p34~c2-p56 °~3 complex was present in cdc10-129 cells overexpressing the cdc2 and cdc13 genes (Figure 4e, lanes 3 and 4). In cdc10-129 cells at the restrictive temperature (Figure 4e, lane 2), only very low levels of the complex could be detected compared with the levels observed either in the strain overexpressing the two genes (Figure 4e, lanes 3 and 4) or in cdc10-129 cells grown at the permissive temperature (Figure 4e, lane 1). Western blotting of total cell extracts showed that p34 ~d~2was present in cdc10-129 cells at both temperatures (Figure 4e, lanes 5 and 6). However, only a low level of p56 ~dcl~was detected at the restrictive temperature (Figure 4e, lane 6), and this may be a factor contributing to the low level of p34c~2-p56 ~ complex detected in these cells. (Figure 4e, lane 5). Thus, high level expression of cdc2 and cdc13 leads to the formation of a complex that can promote entry into mitosis from the G1 phase of the cell cycle even when DNA replication is blocked.

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Time at 36°C (hours) Figure 4. Expression of the p34~'a~2-p56~c13 Complex in G1 Induces Mitosis (a) cdc10-129 cells shifted to the restrictive temperature for 4 hr. (Top) FACS analysis demonstrating a uniform cell cycle arrest with a 1C DNA content. (Bottom) DAPI-stamed elongated cells blocked in G1 with no cells in mitosis. (b) cdc10-129 cells containing cdc2 and cdc13 constructs with the nmtl promoter repressed, Top and bottom panels are as described in (a). (c) cdc10-129 cells containing cdc2 and cdc13 constructs with the nmtl promoter derepressed. The top panel is as described in (a); the bottom panel shows cells entering a premature mitosis from G1. Cells with a cut phenotype are clearly visible.

Temporal Order of S Phase and Mitos~s 819

Discussion We have shown that in fission yeast the presence or absence of the p 3 4 ~ L p 5 6 c~c7~mitotic B cyclin complex plays a crucial role in determining whether a cell undergoes S phase or mitosis. It is well established that activated p 3 4 ~ L p 5 6 c"c~3protein kinase in G2 brings about mitosis in eukaryotic cells (Nurse, 1990). Here we show that disruption of the complex in G2 reprograms fission yeast cells to enter G1 at a point where they can undergo S phase. This has been demonstrated by showing that heat treatment of temperature-sensitive cdc2 (Broek et al., 1991) or cdc13 mutants results in G2 cells undergoing rereplication and becoming diploid. More strikingly, cells deleted for cdc13 and thus completely lacking the p34~2-p56 ~"~ complex arrest initially in G2 and then undergo multiple rounds of DNA replication. Such cells are locked into a rereplicating state and become highly polyploid, with DNA contents of 32C or above. Previously, we proposed that the state of p34 ~ 2 determined whether a cell was in G1 and would undergo S phase or whether it was in G2 and would undergo mitosis. We now extend this hypothesis to suggest that it is the presence of the p 3 4 ~ L p 5 6 ~ 3 mitotic B cyclin complex that defines a cell in G2 and that the absence of this complex defines the cell in G I . Our hypothesis (Figure 5) suggests that disruption of the complex at any stage of the cell cycle promotes cells to enter G1, from which they can proceed to S phase. During the eukaryotic cell cycle, resetting to G1 is brought about at exit from mitosis when B cyclin is degraded and the p34c"~Lmitotic B cyclin complex disrupted. This puts the cell in a state of preparation for S phase and prevents entry into mitosis, thus maintaining the dependency between S phase and mitosis. In S. pombe it is not clear what state of p34 =~2 in G1 promotes S phase, but it may involve complexing with a G1 cyclin as in the budding yeast. However, the cyclin encoded by p u c l ÷, which shows similarity to the budding yeast G1 cyclin CLN3 (Forsburg and Nurse, 1991b), appears to have more of a role in regulating sexual development than progression through G 1 (Forsburg and Nurse, 1994). It is also not clear how rum1 overexpression interacts with the p34=~L p56 ~ complex to bring about rereplication (Moreno and Nurse, 1994); this is currently under investigation. Fission yeast cells blocked in G1 before START have reduced levels of the p 3 4 ~ L p 5 6 ~ complex. If both these pro-

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F~gure5. Model of the Role of p34~CLMItot~cB Cyclin Complex m Defining the TemporalOrder of S Phase and Mitosis The p34~°Lp56'~'3 complexis presentdunng G2 and when activated promotesentry into mitosis.At the end of mitosis,p56~c~3is degraded, the p34~2 kinase ~sreactivated,and cells enter GI. Disruptionof the complex in G2 resetsthe cells to G1 and allows p34~°2 to take up a form suitablefor entry into S phase.This may involveassociationwith a Gl-type cyclin, as shown by a question mark. The presenceof the p34~CLp56~ complexdunng G1 promotesentry into mitosis.

teins are expressed to a high level in Gl-blocked cells, the p34c~Lp56 c~13complex is formed and cells are promoted into mitosis. This demonstrates that G1 cells can be reprogrammed to become G2 cells and can enter mitosis if a high enough level of the complex is generated. This provides support for the view that the presence of the p34 c~2p56 c~1~ complex defines a cell in G2 from which it can proceed into mitosis. Our experiments show that the major events of S phase and mitosis can be induced inappropriately by manipulating the p 3 4 ~ L p 5 6 ~c~s complex. It is possible that there are other more subtle events of S phase and mitosis that are not induced by these changes in the state of the complex, although the fact that the diploids generated by heat treatment are viable suggests that these events are not crucial for the cell. In budding yeast, the presence of the CLB mitotic B cyclins have been shown to down-regulate the G1 CLN cyclins and thus prevent progression toward S phase (Amon et al., 1993), while the initial increase in the level of CLB2 protein requires the CLN1 and CLN2 cyclins (Amon et al., 1994). In contrast with our results reported here, overexpression of CLB cyclins does not promote entry into mitosis from G1 (Amon et al., 1994) and deletion of the CLB genes does not appear to induce rereplication (Fitch et al., 1992), although in cells lacking CLB1, CLB2, CLB3, and CLB4, there is possibly a slow increase in DNA

(d) Percent of mitotic cells in cdc10-129 (open squares),cdc10-129 with cdc2 and cdc13 and the nmtl promoter repressed(closed diamonds), and cdc10-129 with cdc2 and cdc13 and the nmtl promoterderepressed(closedcircles)at hourlytime pointsafter shift to the restrictivetemperature (36°C). By 4 hr, all the cells are arrestedin G1, as determinedby FACS analysis(top panels in [a]-[c]). The mitotic index drops to less than 50/0 by 3-5 hr, as expected in cdc10-129 cells alone and in those cells with plasmid but with the promoter repressed. In contrast, cdc10-129 cells overexpressmgcdc2 and cdc13 show an increase in the number of mitotic cells to nearly 60% after 4 hr. The failure of the remaining cells to enter mitosis is explained by loss of the cdc13 plasmid from about 30% of the cells. (e) A Western blot of the p34C~CLp56`dc1~complexprecipitatedfrom cdclO-129 cells grownat the permissivetemperature(25°C) (lane 1), cdc10-129 cells after 4 hr at the restrictivetemperature(36°C) (lane 2), cdc10.129 cells overexpressingcdc2 and cdc13 at the permissivetemperature(lane 3), and cdc10-129 cells overexpressmgcdc2 and cdc13 after 4 hr at 36°C (lane 4). Both p34~c2 and p56~°'3 are detected in precipitatesfrom cdc10-129 extracts at 25°C and in extracts from strains overexpressingcdc2 and cdc13. Very little p56~°~ was detected in cdc10-129 extracts at 36°C, suggestingthat the complex is only present at low levels in G1 cells. Some proteolysisof p56~claoccurred during sample preparation, and severalbands reactiveto the antibodycan be seen. A Western blot of total p56c~laand p34~ in cdci0-129 cells at the permissivetemperature is shown in lane 5 and after 4 hr at 36°C is shown in lane 6. p34~c2 is present at both temperatures,but only a low level of p56~ 3 is detected at 36°C comparedwith 25°C.

Cell 820

content (Richardson et al., 1992). The difference between the two yeasts may be because of differences in organization of their cell cycles. During the Saccharomyces cerevisiae cell cycle, a short spindle is initiated around the time of S phase and is present until later in the cell cycle when subsequent events of mitosis are initiated. Six B-type cyclins have been identified that have overlapping functions for progress into S phase and mitosis (Epstein and Cross, 1992; Fitch et al., 1992; Richardson et al., 1992; Schwob and Nasmyth, 1993). It is possible that the overlapping of S phase and mitosis and the overlapping functions of the C L B genes involved in both processes preclude the type of mechanism we have proposed here for fission yeast. However, the proposal that the state of the p34 ~ L mitotic B cyclin complex determines whether a cell is in G1 or G2 may have relevance for other eukaryotes. In certain developmental situations, such as meiosis or endoreduplication, the disruption of the d e p e n d e n c y between S phase and nuclear division may also be brought about by changes in the state of the p 3 4 ~ L m i t o t i c B cyclin complex. When repeated rounds of S phase occur during Drosophila embryogenesis, no B cyclin is detected (Lehner and O'Farrell, 1990), while B cyclin from clams and Xenopus persists between the first and second meioses (Hunt et al., 1992; Kobayashi et al., 1991) when S phase is suppressed. Recently, it has been shown that ablation of c - m o s in Xenopus oocytes restores an S phase between the first and second meiotic divisions (Furuno et al., 1994). Mos has previously been shown to be required for the accumulation of B cyclin in mouse eggs (O'Keefe et al., 1991), and B cyclin has been shown to be a substrate for the Mos kinase (Roy et al., 1990). These observations are consistent with the presence of the p34cdcLmitotic B cyclin complex preventing S phase and promoting nuclear division, while the absence of the complex inhibits mitosis and puts the cell into a G1 state from which it can proceed to S phase. In many organisms the presence of the p34°d~Lmitotic B cyclin c o m p l e x promotes entry into mitosis whereas exit from mitosis is brought about by loss of the complex due to mitotic B cyclin destruction. Therefore, it is possible that the presence or absence of the p34cd°Lmitotic B cyclin complex may provide a more general mechanism for maintaining the temporal order between S phase and mitosis in other eukaryotes as well as in fission yeast. Experimental Procedures S. pombe Methods All strains were derived from 972h-, 975h+, and 968h 9°wtld-typestrains using standard genetical procedures (Leupold, 1970). All media and growth conditions are as described by Moreno et al. (1991) unless otherwise stated. The selection screen for rereplicating mutants using the leul.32 mare2 h9°strain has previously been described (Broek et al., 1991). Mutants obtained after the selection process were stained with DAPI (4,6,dtamidmo-2-phenylindole) as prevtously described (Moreno et al., 1991) and screened visually after incubation at the restrictive temperature (36°C) for 4 hr followed by a shift to the permissive temperature (25°C) for 3 hr. The protocols for generating diploid cells by heat treatment and scoring of diplotd colontes are described in Broek et al. (1991) Cells were prepared for FACS and stamed with propldium iodide as previously described (Sazer and Sherwood, 1990).

For cell number determination, cells were fixed as previously described (Moreno et al., 1991). A diploid cdc13/t strain (cdc13,d::ura4+lcdc13 ÷ ade6-M210/ade6M216 leu1.32/leu 1-32 ura4-D18/ura4.D18 h÷/h-) and a wild-type diploid strain (cdc13+lcdc13 + ade6-704/ade6 + leu1-32/leu1+ ura4-294/ura4 + h÷/h-) were sporulated and the ura ÷ spores germinated m Edinburgh mimmal medium (EMM) with adenine and leucine as described in Moreno et al. (1989). Before inoculation into growth medium, large diploid spores were removed by elutriatton (Aves et al., 1985). This reduced the dtploids in the culture to less than 5%. Cells deleted for cdc13 were kept viable by the multicopy plasmtd pSM2cdc13. The culture was grown to midexponential growth in EMM, washed three ttmes with EMM lacking a nitrogen source (EMM-N) and incubated for 17 hr at 25°C in EMM-N to induce plasmid loss. The culture was then elutriated to remove the elongated cells (Ayes et al., 1985), and the small cells were inoculated mto EMM at 32°C. Samples were taken at 0 hr, 4 hr, 8 hr, and 16 hr after inoculation. The cdc2 gene in pREP5 (pREP1 [Maundrell, 1993] with the LEU2+ gene replaced with sup3-5) was integrated by selecting for white colonies in a cdc10-129 ade6-704 leul-32 mutant (Moreno et al., 1991); the cdc13 gene in pREP41 was transformed into cdc10-129 ade6-704 leul-32 cdc2 + int pREP5 cdc2 using the lithium acetate method (Moreno et al., 1991) and maintained as a multtcopy plasmtd. The cells were grown in the presence of thtamine to repress expresston from the nmtl promoter and then washed three times in EM M and incubated for 18 hr at 25°C to allow expression of cdc2 and cdc13. The cells were then shifted up to the restrictive temperature (36°C), and samples were taken for FACS analysis, mitobc index, and precipitation wtth p13s"~ beads at hourly time points. Cells were prepared for DAPI staining and for immunofluorescence using methanol fixation as prevtously described (Moreno et al., 1991). The tat1 antibody (a gift from K. Gull) and a goat anti-mouse Texas red-conjugated secondary antibody (Jackson Immunoresearch Laboratories) were used to visualize tubulin. Plasmid Construction A Ndel site was made at the first ATG of the cdc13 open reading frame using Bio-Rad Mutagene. The cdc13 Ndel fragment was then cloned into pREP41, allowing expression from the nmtl promoter. For expression of cdc13 from the SV40 promoter, a cdc13 BamHI-Sall fragment, from whtch the sequence 5' to the open reading frame had been deleted (Hagan et al., 1988), was cloned into pSM2 (Moreno et al., 1991). Protein Preparation Total protein extracts were prepared as descnbed by Moreno et al. (1991). Total protein extract from the cdc13• strain was prepared 18 hr after inoculation. Spores were removed by elutriat=on and the enlarged cells used to make the extract. The soluble and insoluble fracttons of p34~2 and p56~dc~3were prepared from1 x 108cellsof972,cdc13-117,andcdo2.L7mexponential growth at 25°C or after heat treatment at 49°C as previously described (Broek et al., 1991 ; Moreno et al, 1991). After cell breakage, the glass beads were washed with 600 p_lof HE buffer (Simanis and Nurse, 1986) plus 1% Triton X-100 (HET), and the supernatant was centrifuged at 1400 x g for 2 mm at 4°C to remove cell debris. The supernatant was removed and the pellet washed with 100 Id of HET, and the extract was centrtfuged at 310,000 x g in a TL100 rotor for 15 mm at 4°C. The supernatant (soluble fraction) was added to an equal volume of 2 x sample buffer. The remaining pellet (insoluble fraction) was washed and resuspended to the same volume as the soluble fractton, in 1 x sample buffer. Precipitations Using p13~"cl Beads For precipitation of the p34C~Lp56~c~ complex, p13"cl was coupled to Affi-Gel 15 (Bio-Rad). The complex was precipitated from 1 ml of total cell extract (2 mg/ml) with 30 ~1 of p13s"cl beads (5 mg/ml). The beads were mcubated for 2 hr at 4°C, washed five ttmes with 0.8 ml of HB buffer, and finally resuspended in HB buffer with an equal volume of 2x sample buffer (Moreno et al., 1991). Electrophoresis and Western Blotting Procedures Protein extracts and precipitates were electrophoresed using SDS-

Temporal Order of S Phase and Mitosis 821

polyacrylamide gel electrophoresis (Laemmh, 1970). Western blotting was carried out using Immobilon (Millipore) probed with SP4 (Moreno et al., 1989) or PN24 (Simanis and Nurse, 1986). The anti-cdc2 antibody (PN24) was raised to the C-terminal 6 amino acids of p34 ~2, and the anti-cdcl 3 antibody was raised to the whole protein. Proteins were detected using horseradish peroxidase-conjugated anti-rabbit secondary antibody visualized using ECL (Amersham).

Acknowledgments Correspondence should be addressed to J. H. We thank all our colleagues in the Cell Cycle Laboratory of the Imperial Cancer Research Fund, in particular, Matthew O'Connell, Bode Stern, Karim Labib, and Fred Chang, for discussions and help. We thank the Welfcome Trust for support to D. F. and The Royal Society for support to P. N. Received June 28, 1994; revised August 8, 1994.

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