Fission yeast cells with a temperature-sensitive Orp1 protein, a component of the origin recognition complex, cannot perform DNA replication at the restrictive temperature. Seventy percent of orp1-4 cells arrest with a 1C DNA content, whereas 30% proceed to mitosis (`cut'). The arrest depends upon the checkpoint Rad proteins and, surprisingly, the Chk1 protein, which is thought to act only from late S phase. The arrested cells maintain a 1C DNA content, as judged by flow cytometry, and the early origin ars3001has not been initiated, as judged by 2D gel analysis. We show that in G1-arrested orp1-4 cells, Wee1 phosphorylates and inactivates Cdc2. Activation of Chk1 occurs earlier than Cdc2 phosphorylation, indicating a novel role for Chk1, namely to induce and/or maintain Cdc2 phosphorylation upon checkpoint activation in G1. We also show that commitment to cutting occurs already in early G1 phase.
Introduction
The maintenance of genomic integrity is important for the survival of eukaryotic cells. Checkpoint mechanisms prevent cell cycle transitions until previous events have been completed or damaged DNA has been repaired. Checkpoint pathways and proteins are evolutionarily conserved from yeast to man, underlining their importance in maintaining genomic integrity. In fission yeast several checkpoint pathways monitor the status of the DNA and arrest the cell cycle in response to DNA damage or inhibition of DNA replication. The so-called checkpoint Rad proteins, Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1,are required for damage sensing and processing and are involved both in the DNA damage checkpoint pathway and in responding to replication arrest. There are two known effector kinases downstream of the checkpoint Rad proteins, Chk1 and Cds1. Chk1 is phosphorylated in response to DNA damage induced in late S or G2 in a rad3-dependent manner(Walworth et al., 1993;Walworth and Bernards, 1996;Martinho et al., 1998;Tanaka and Russell, 2001). Phosphorylation of Chk1 correlates with Chk1-dependent checkpoint activation,but the exact significance of the phosphorylation for either Chk1 kinase activity or for checkpoint regulation is as yet unclear. Cds1 is activated as a part of the intra-S checkpoint when the DNA is damaged during S phase or when DNA replication is inhibited by hydroxyurea or by a DNA polymerase defect(Murakami and Okayama, 1995;Lindsay et al., 1998). Activation of either kinase leads to inhibition of Cdc2 activity by maintaining the inhibitory phosphorylation of Tyr15(O'Connell et al., 1997;Rhind et al., 1997;Rhind and Russell, 1998;Wan and Walworth, 2001;Liu et al., 2002). Tyr15 of Cdc2 is phosphorylated by Wee1 and Mik1, and dephosphorylated in preparation for mitosis by Cdc25 (reviewed by Berry and Gould, 1996)). In the rest of the paper we shall refer to Tyr15 phosphorylation when discussing phosphorylation of Cdc2p.
Much less is known about checkpoints in the G1 phase of the cell cycle. This is mostly because G1 in fission yeast is very short under standard laboratory growth conditions, rendering the investigation of the existence and mechanism of such a checkpoint(s) difficult. However, several cell cycle mutants arrest in the G1 phase of the cell cycle (i.e. before any DNA replication has occurred and without proceeding to mitosis). This arrest might be due to checkpoint activation, thereby providing a means to investigate G1 checkpoint(s) in fission yeast. We have used the orp1-4 replication initiation mutant, which at the restrictive temperature either enters mitosis in the absence of DNA replication (`cut' phenotype) or arrests with a 1C DNA content without entering mitosis (`arrest' phenotype)(Grallert and Nurse, 1996). When the orp1-4 mutant is incubated at the restrictive temperature,about 70% of the cells arrest, whereas 30% cut(Grallert and Nurse, 1996). In the complete absence of orp1+ function, in the orp1Δ mutant, all of the cells cut.
The orp1 gene encodes one of the components of the fission yeast origin recognition complex (ORC). The ORC binds the replication origins throughout the cell cycle and serves as a landing pad for other proteins essential for the initiation of replication (reviewed byLeatherwood, 1998). One of these is the Cdc18 protein which, together with Cdt1(Nishitani et al., 2000), is required for loading of the MCM proteins (minichromosome maintenance) onto the origins, thus forming the pre-replication complex (preRC).
In this work we have studied both the arrest and the cut phenotypes of the orp1-4 mutant. We show that orp1-4 cells arrest in G1 owing to a checkpoint that requires the checkpoint Rad proteins and Chk1, but not Cds1. Cdc2 is phosphorylated by Wee1 in G1 after activation of Chk1 and the arrest of orp1-4 cells depends on this phosphorylation. We also analyse the cutting and show that commitment to cutting occurs before S phase.
Materials and Methods
Fission yeast strains and methods
All our strains are congenic with the Schizosaccharomyces pombe972h- strain. Strains used in this study are listed inTable 1. All basic growth and media conditions were as described (Moreno et al., 1991). The rum1Δ and ste9Δstrains have been described (Moreno et al., 1994; Yamaguchi et al.,1997; Kitamura et al.,1998; Edwards et al.,1999; Wright et al.,1998). Since they are sterile, the orp1-4 rum1Δ and orp1-4 ste9Δ strains were made by protoplast fusion(Sipiczki and Ferenczy, 1977). Experiments in liquid culture were performed using EMM medium, and exponential cultures were grown to a density of 2-4×106 cells/ml at the start of each experiment.
2D gel analysis
Purified genomic DNA was digested with HindIII and KpnI and analysed as described before (Muller et al., 2000). After blotting, the membrane was probed with a 32P-labelled fragment within the 3kb HindIII-KpnI fragment of the rDNA repeat of S. pombe(Sanchez et al., 1998).
Protein extracts and western blots
Protein extracts for western blotting were made by TCA extraction, as described previously (Caspari et al.,2000). For western blot analysis the following antibodies were used: anti-phosphotyrosine Cdc2 (Sigma C0228) at a dilution of 1:400;anti-pSTAIRE against Cdc2 (Santa Cruz sc-53) at a dilution of 1:2000; anti-HA(Babco) at a dilution of 1:1000); anti-myc (PharMingen 9E10) at a dilution of 1:1000; and anti-α-tubulin (Sigma T5168) at a dilution of 1:20,000. The secondary antibodies were either HRP or AP conjugates, used at a dilution of 1/5000. Detection was performed using the enhanced chemiluminescence procedure(NEN ECL kit). Cdc2 and phosphorylated Cdc2 was measured using ECF detection(Amersham) and quantified with a phosphoimager and the Image Quant software. Chk1 and Wee1 phosphorylation was followed by Super Signal (Pierce)detection.
Cds1 kinase activity
Cds1 was immunoprecipitated and Cds1 kinase activity was measured using MBP as substrate as described (Lindsay et al.,1998). Quantification of the kinase activity was performed using a phosphoimager and the ImageQuant software.
Flow cytometry and microscopy
About 107 cells were spun down for each sample and fixed in 70%ethanol before storing at 4°C. Samples were processed for flow cytometry as described (Sazer and Sherwood,1990), stained with Sytox Green (Molecular Probes S-7020, http://pingu.salk.edu/fcm/protocols/ycc.html)and analysed with a Becton-Dickinson FACSCalibur machine. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining, and Calcofluor was used for cell wall and septum staining as described previously(Moreno et al., 1991).
Synchronous cells
Partially synchronous cells were selected from exponentially growing cultures by lactose gradient centrifugation on 10-30% gradients, as described(Carr et al., 1995).
Results
orp1-4 cells have not initiated DNA replication at the arrest point
Several pieces of evidence suggest that orp1-4 cells arrest in G1(Grallert and Nurse, 1996). However, it could not be excluded that they initiate DNA replication and arrest at an early stage, since flow cytometry analysis cannot distinguish cells in G1 from early S phase cells that have synthesised a small fraction of their DNA. In order to investigate where the cells arrest, we analysed replication initiation at the early replication origin ars3001, which is located in the rDNA repeats. Together with the K-repeat origin this is the first known origin to be replicated as the cells enter S phase(Kim and Huberman, 2001). In the positive control, which is orp1-4 cells at the permissive temperature, the ars3001 origin was both used as an origin, evidenced by a bubble-arc, and replicated passively, evidenced by a Y-arc(Fig. 1, top panel). These signals are due to a small percentage (less than 10%) of cells that are in S phase at any one time. On continued incubation at the restrictive temperature the bubble arc disappears and the Y-arc becomes much weaker and is hardly discernible after 3 hours. If all the cells had accumulated in early S phase,the replication signals would be expected to increase rather than decrease. The strong decrease is evidence that orp1-4 cells have not initiated DNA replication at the arrest point.
The G1 arrest of orp1-4 cells is checkpoint dependent
About 70% of the orp1-4 mutant cells arrest prior to DNA replication at the restrictive temperature. We combined orp1-4 with a number of checkpoint mutations. Mutation of any of several checkpoint rad genes in orp1-4 abolished the arrest(Fig. 2A), demonstrating that it is checkpoint dependent. Consistently, phosphorylation of Rad9, a marker of checkpoint activation (Caspari et al.,2000), was clearly induced in orp1-4 cells(Fig. 2B). As a positive control, the extent of Rad9 phosphorylation was the same as that in a cdc17ts mutant at the restrictive temperature(Fig. 2B), conditions that are known to require checkpoint activation for arrest. We conclude that arrest of the orp1-4 mutant is checkpoint dependent.
The orp1-4 rad double mutants appear to enter mitosis (cut)earlier than the orp1-4 single mutant(Fig. 2A). To verify that the double mutants cut earlier and not only more extensively, we selected synchronous orp1-4, orp1-4 rad26 and orp1-4 cdc18-K46 cells after lactose gradient centrifugation and followed cutting at the restrictive temperature. Both double mutants were found to cut significantly earlier than the single mutant (Fig. 2C). In these experiments, the level of cutting of the orp1-4 mutant cells was less than shown above (Fig. 2A), because in the present experiment the cells were synchronized in early G2 phase and therefore reached the arrest point later than the asynchronous cells described in Fig. 2A.
Chk1, but not Cds1, is involved in the checkpoint response
In the DNA replication or the G2/M damage pathways the targets of the checkpoint Rad proteins are the effector kinases Chk1 and Cds1(Walworth et al., 1993;Murakami and Okayama, 1995). Deletion of cds1 did not affect entry into aberrant mitosis (cutting)in orp1-4 (Fig. 3A),showing that Cds1 is not required for the checkpoint. Consistently, Cds1 kinase activity was not induced in orp1-4 upon temperature shift(Fig. 3B). This finding further supports the above conclusion that orp1-4 cells do not arrest in S phase, since Cds1 is known to be required for checkpoints activated during S phase (Murakami and Nurse,2001; Lindsay et al.,1998; Murakami and Okayama,1995). Surprisingly, deletion of chk1 abolished the checkpoint that ensures arrest of 70% of the orp1-4 cells(Fig. 3A). When orp1-4cells were shifted to the restrictive temperature, Chk1 became phosphorylated(Fig. 3C), further suggesting that Chk1 is required to prevent cutting. In contrast, in orp1-4 cdc18-K46, where the vast majority of cells enter mitosis in the absence of DNA replication, phosphorylation of Chk1 did not increase upon shift-up(Fig. 3C), strongly suggesting that the phosphorylated Chk1 is indeed responsible for arresting orp1-4 cells.
These results show that there is a checkpoint prior to DNA replication that is responsible for the arrest of 70% of orp1-4 cells. This checkpoint involves the checkpoint Rad proteins and Chk1 but not Cds1.
The checkpoint is independent of Rum1 and Ste9
The major target of the S/M and the G2/M checkpoints is the Cdc2 kinase,which also plays an important role in the initiation of DNA replication. Since inhibiting Cdc2 is a likely target of the checkpoint activated in orp1-4 cells, we investigated whether the known mechanisms of Cdc2 inhibition are required for the G1 arrest of 70% of orp1-4 cells. Rum1 can inhibit the mitotic CDK, Cdc2-Cdc13, in vitro and is required for efficient proteolysis of Cdc13 (Moreno and Nurse, 1994; Correa-Bordes and Nurse, 1995; Davis and Smith,2001; Correa-Bordes et al.,1997). Ste9 is required for efficient degradation of the mitotic cyclin Cdc13 during G1 arrest (Yamaguchi et al., 2000; Blanco et al.,2000; Guo and Dunphy,2000). We constructed double mutants of orp1-4 with rum1Δ and ste9Δ and compared cutting in the double mutants and the orp1-4 single mutant. Both double mutants cut with the same timing and to the same extent as the orp1-4 single mutant (data not shown). We conclude that Rum1 and Ste9 are not required for the arrest of orp1-4 cells prior to DNA replication.
Cdc2 is phosphorylated by Wee1 in arrested orp1-4 cells
In cycling cells, phosphorylation of Cdc2 is thought to start in late G1,close to the G1/S transition (Hayles and Nurse, 1995). The previously characterised S/M and DNA damage checkpoint pathways delay entry into mitosis by maintaining phosphorylation of Cdc2. When orp1-4 cells are incubated at the restrictive temperature,phosphorylated Cdc2 can be detected(Grallert and Nurse, 1996). This could result from the activation of the Chk1-dependent checkpoint pathway(above), or Cdc2 could have been phosphorylated before checkpoint activation. We shifted synchronous orp1-4 cells to the restrictive temperature and followed both the appearance of phosphorylated Cdc2 and the activation of the checkpoint, as marked by Chk1 phosphorylation. Chk1 and Cdc2 became phosphorylated 2.5 hours and 3.5 hours after shift-up, respectively(Fig. 4A,B), opening the possibility that Cdc2 phosphorylation in orp1-4 is a result of activation of the checkpoint and of Chk1.
cdc2 mutations conferring a wee phenotype, cdc2-1w and cdc2-3w, make Cdc2 insensitive to phosphorylation by Wee1 and independent of dephosphorylation by Cdc25,respectively. When we combined orp1 with the cdc2-1w and cdc2-3w mutations, both double mutants were found to cut more than the orp1-4 single mutant (Fig. 4C). This shows that phosphorylation of Cdc2 is important to keep the orp1-4 cell from entering aberrant mitosis.
Phosphorylation of Cdc2 can be carried out by either of two kinases, Wee1 or Mik1 (reviewed by Berry and Gould,1996). To investigate which kinase is responsible for phosphorylation of Cdc2 in arrested orp1-4 cells, we constructed orp1-4 wee1-50 and orp1-4 mik1Δ double mutants. Mutation of wee1, but not of mik1 exaggerated the cutting phenotype of orp1-4 (Fig. 4D). Expression of mik1 is cell cycle regulated and the protein is expressed in S phase. Activation of the intra-S phase and S/M checkpoints results in further induction of mik1 in a cds1-dependent manner (Christensen et al., 2000; Baber-Furnari et al., 2000; Borgne and Nurse,2000). Therefore, a low level of expression of Mik1 in orp1-4 cells at the restrictive temperature(Fig. 4E) is consistent with a lack of effect of a mik1Δ mutation and is further evidence that the cells have not entered S phase.
When the G2/M damage checkpoint is activated, the Wee1 protein accumulates and is phosphorylated in a Chk1-dependent manner(Raleigh and O'Connell, 2001). We investigated whether a similar process takes place in the pre-replication checkpoint arresting orp1-4 cells. When asynchronous orp1-4cells were arrested at the restrictive temperature an accumulation of Wee1 could be observed and at later times phosphorylated Wee1 was detected(Fig. 4F). We conclude that phosphorylation of Cdc2 in orp1-4 cells is due to Wee1 activity prior to DNA replication and occurs after and possibly in response to checkpoint activation.
Activation of a damage checkpoint pathway in G1 prevents cutting in germinating orp1Δ spores
The above results suggest that there is a checkpoint prior to DNA replication that requires the checkpoint Rad proteins and Chk1 and acts via inducing and/or maintaining phosphorylation of Cdc2. In the orp1-4mutant this checkpoint might be activated by aberrant origin structures recognized as DNA damage (see Discussion). One prediction of this model is that if we inflict DNA damage in the absence of Orp1 and thereby induce this G1 checkpoint, cutting should be prevented. To test this hypothesis, we irradiated germinating orp1Δ spores derived from an orp1-4/orp1Δ diploid. These orp1Δ spores contain the temperature-sensitive Orp1 from the parental diploid and, when germinated at the restrictive temperature, all of them enter mitosis in the absence of DNA replication (Grallert and Nurse,1996). The spores were irradiated with a UV dose (254 nm) that gave a 50% survival of wild-type cells. Irradiation of the germinating orp1Δ spores strongly reduced cutting(Fig. 5), suggesting that inflicting DNA damage in G1 in the absence of Orp1 can induce a checkpoint that inhibits the mitotic machinery.
orp1-4 cells become committed to cutting early in G1
Entry into aberrant mitosis (cutting) in orp1-4 cells can be observed already 4 hours after a shift to the restrictive temperature, but only about 30% of the cells cut even after 6 hours(Grallert and Nurse, 1996). We argue that these cells enter mitosis from G1 (i.e. before the occurrence of any DNA replication). First, if cells leaked into S phase before cutting, it would be detectable by 2D gel analysis(Fig. 1), although synthesis of very short pieces of DNA would not be detected by this technique. Second, we examined whether orp1-4 cells have to pass the cdc10 arrest point before they become committed to cutting. Cdc10 is a transcription factor that activates transcription of a number of genes required for initiation of DNA replication (Aves et al.,1985; Tanaka et al.,1992; Lowndes et al.,1992). Therefore, temperature-sensitive cdc10 mutants arrest at the restrictive temperature before any DNA replication has occurred and with a 1C DNA content. If the orp1-4 cells have to pass the cdc10 arrest point to become committed to cutting, we would expect an orp1-4 cdc10ts double mutant not to cut at the restrictive temperature. Even if the double mutant leaks through the cdc10 arrest point, it would cut only after a delay compared with orp1-4. When synchronous orp1-4 and orp1-4 cdc10-129 cells were selected by lactose gradient centrifugation and shifted to the restrictive temperature,the double mutant was not delayed at cutting compared with the single orp1-4 mutant (Fig. 6). On the contrary, the double mutant cut earlier than the single mutant. Asynchronous orp1-4 and orp1-4 cdc10-129 mutants cut to the same extent and with similar timing (data not shown). We conclude that commitment to cutting in orp1-4 occurs before entry into S phase and not later than the cdc10 arrest point.
Discussion
We have studied a G1 checkpoint by exploiting the orp1-4 mutant,which has two alternative fates at the restrictive temperature: cell cycle arrest and cutting. The arrest is dependent upon the checkpoint Rad proteins,which demonstrates that the arrest is due to a checkpoint. Several lines of evidence argue that the orp1-4 cells arrest in G1: they can mate from the arrest point, as shown previously(Grallert and Nurse, 1996),they arrest before expression of mik1, and they have not initiated DNA replication, as shown by 2D gel analysis of the early replication origin ars 3001. Furthermore, a leakage into S phase should elicit the cds1-dependent intra-S and/or the S/M checkpoint pathway. We have shown that Cds1 is not activated in orp1-4 cells(Fig. 3B).
It was shown earlier (Grallert and Nurse, 1996) that chromosomes prepared from arrested orp1-4 cells do not enter the gel upon PFGE. This result suggests the presence of aberrant DNA structures but is also consistent with an early S phase arrest. Two-dimensional gel analysis of ars3001 argues against the latter interpretation and leaves us to conclude that aberrant DNA structures are responsible for the arrest in orp1-4 cells.
In synchronous orp1-4 cells shifted to the restrictive temperature, Chk1 phosphorylation occurs before Cdc2 phosphorylation(Fig. 4A,B), leading us to speculate that activation of Chk1 brings about Cdc2 phosphorylation.
Of the two kinases that phosphorylate Cdc2, absence of Wee1 function abolishes the G1 arrest of orp1-4 cells, strongly suggesting that Wee1 phosphorylates Cdc2 during G1 in the present checkpoint(Fig. 6). The following observations suggest that Wee1 is activated by Chk1. Deleting chk1+ in orp1-4 cells has the same consequences as introducing a wee1-50 mutation (this work). Furthermore, wee1- cells are insensitive to the overexpression of Chk1,which can directly phosphorylate Wee1 in vitro(O'Connell et al., 1997) and activation of the G2/M damage checkpoint leads to accumulation and phosphorylation of the Wee1 protein(Raleigh and O'Connell, 2001). Accumulation and phosphorylation of Wee1 can also be observed in arrested orp1-4 cells. Therefore, it is tempting to speculate that phosphorylation and/or an increase of Wee1 activity are contributing mechanisms to bring about and maintain a G1 arrest of orp1-4 cells(Fig. 6). A direct way to examine this suggestion would be to measure Cdc2 phosphorylation in orp1-4 chk1Δ and orp1-4 wee1 double mutants. However, these mutants start cutting less than 2 hours after shift-up to the restrictive temperature. Since Cdc2 becomes dephosphorylated as the cells go into mitosis and cut (Fig. 4B), it is not possible to determine whether the deletion of chk1 or wee1results in lack of Cdc2 phosphorylation. In contrast to wee1,mutation of mik1 has no effect on cutting in orp1-4 cells,nor is mik1 expression induced at the restrictive temperature. Thus,Mik1 is not a major target of the checkpoint induced in orp1-4cells.
Most temperature-sensitive replication initiation mutants arrest in G1 or inside S phase, whereas several deletion mutants proceed to aberrant mitosis without completing S phase (cutting). In general, the tighter the mutants are,the more cutting occurs. These observations have led to the model that the preRC complex, in addition to initiating DNA replication, provides a signal that S phase is in preparation and mediates inhibition of mitosis through activation of a checkpoint pathway. An alternative explanation is that a preRC-independent checkpoint is activated in G1. A third alternative is that the cells attempt to replicate DNA in spite of compromised initiation, and that it is the S/M checkpoint pathway that prevents mitosis. The latter alternative has been excluded by the present experiments. We will discuss the two former alternatives below.
We suggest that the difference between the two populations of orp1-4 cells (cutting versus arresting) is that the residual function of the Orp1-4 protein allows changing the origin structure in 70% of the cells to a structure that activates a checkpoint pathway(Fig. 7). Although the preRC does not assemble properly in orp1-4 cells(Kearsey et al., 2000), some steps of preRC formation might be performed, at least on some origins. This incomplete preRC or the resulting origin structure might in turn activate the checkpoint (Fig. 7). The following observations support the idea that the arrested cells have proceeded further than cutting cells have, and that formation of a certain structure,possibly recognised as DNA damage, is responsible for the arrest. (1) The cutting cells become committed to cutting at a point before the cdc10arrest point (Fig. 6), while the arresting cells arrest at or later than the cdc10 arrest point(Grallert and Nurse, 1996).(2) orp1-4 cells rapidly lose viability upon shift to the restrictive temperature, before cells undergoing aberrant mitoses can be observed (data not shown). This argues for an irreversible formation of damage-like structures. (3) Chromosomes prepared from arrested orp1-4 cells do not enter the gel upon PFGE (Grallert and Nurse, 1996), consistent with the presence of aberrant DNA structures. (4) Complete loss of Orp1 function or a combination of orp1-4 with mutant alleles of genes encoding other preRC components leads to increased entry into mitosis in the absence of DNA replication relative to an orp1-4 single mutation(Fig. 2C)(Grallert and Nurse, 1996),strongly suggesting that the former mutants are unable to modify the origin structure enough to activate a checkpoint. Consistently, no Chk1 phosphorylation was detected in orp1-4 cdc18-K46 double mutant cells(Fig. 3C).
The checkpoint activated in orp1-4 cells is clearly different from the intra-S checkpoint, since the former requires Chk1 but does not require Cds1 (this work) while the latter has the opposite requirement(Walworth et al., 1993;Lindsay et al., 1998).
We argue that complete lack of replication does not activate any checkpoint and therefore results in entry into mitosis in the absence of DNA replication. Induction of the checkpoint depends on an attempt to form a preRC. Formation of an incomplete preRC results in checkpoint induction, but this does not imply that the checkpoint is specifically activated by incomplete preRCs. Our finding that irradiating germinating orp1Δ spores prevents entry into mitosis points to the existence of a damage checkpoint activated in G1 that can inhibit the mitotic machinery. We favour the interpretation that this checkpoint is identical to the checkpoint activated in arresting orp1-4 cells
Studying germinating spores carrying a deletion of the orp2 gene resulted in the conclusion that there is no checkpoint for a lack of DNA replication (Kiely et al.,2000), consistent with our interpretation. However, the orp2ts mutant was arrested in S phase and therefore those experiments did not allow the detection of a checkpoint prior to DNA replication.
Combination of the orp1-4 mutation with cdc10-129(Fig. 1B), cdc18-K46(Fig. 2C), checkpoint rad genes (Fig. 2A),or chk1Δ (Fig. 3A) makes the cells cut earlier and more extensively than the single orp1-4 mutant. We suggest that the first two cut earlier and more because the arrest in orp1 mutant cells is dependent on attempting preRC formation. To follow our line of reasoning above, more preRC formation will be achieved in the presence of Cdc18, which again will lead to more checkpoint arrest and less cutting. Consistently, it has been shown that Cdc18 overproduction can rescue the cutting phenotype of orp1-4(Grallert and Nurse, 1996). It has been shown (Greenwood et al.,1998) that overexpressing the C-terminus of Cdc18 in wild-type cells is sufficient for replication initiation and induction of a checkpoint inhibiting mitosis, but since these cells were shown to be arrested in S phase with between 1C and 2C DNA contents, the relevance for the present experiments is uncertain. The early-cut phenotype observed when orp1-4 is combined with checkpoint rad mutations suggests that the checkpoint proteins might be physically present and active at the origin complex in orp1-4 cells.
There are two contradictory reports on a G1 checkpoint in a cdc10ts mutant. The Chk1 protein was found to be required(Carr et al., 1995) and not required (Hayles and Nurse,1995) for arrest of cdc10ts cells. In the two papers different synchronisation methods were used, which might account for the discrepancy. Since Cdc2 is not phosphorylated in arrested cdc10ts cells (Hayles and Nurse, 1995), it is unlikely that the checkpoint pathways arresting cdc10ts and orp1-4 cells are one and the same. Consistently, Rum1, which is required only in pre-START G1, is necessary for a cdc10ts arrest(Moreno and Nurse, 1994), but not for arrest of orp1-4 cells (this work). However, we cannot exclude the possibility that cdc10ts cells leak into later parts of G1, although it was shown that they do not leak into S phase(Carr et al., 1995). If this is indeed the case, the Chk1-dependent checkpoint reported earlier(Carr et al., 1995) might be the same as the one arresting orp1-4 cells.
The checkpoint activated in orp1-4 cells appears to be similar to the G2/M damage checkpoint in that it requires Chk1 and the maintenance of Cdc2 phosphorylation. Also, failure of either checkpoint results in entry into mitosis. We have provided several pieces of evidence that orp1-4cells arrest in G1. Nevertheless, it is possible that the present G1/M checkpoint mechanism is using the same molecular signals and targets as the G2/M checkpoint. The phosphorylation state of Cdc2 and the levels and activities of the mitotic regulators Cdc13 and Cdc25 may be the same during a late G1 arrest as in a G2 arrest. In this context it is interesting to note that orp1-4 cdc2-3w cells are checkpoint deficient(Fig. 4C). This observation raises the possibility that, in addition to Wee 1, Cdc25 might also be targeted by Chk1 in orp1-4 cells, as in the G2/M checkpoint(Raleigh and O'Connell, 2001). In higher eukaryotes the biochemical differences during the G1 and G2 phases are more extensive, because of the divergent evolution of CDKs and their regulators. Therefore, failure of a G1 checkpoint cannot lead to premature entry into mitosis, but only to premature entry into S phase.
Checkpoint activation in G1 in higher eukaryotes depends upon phosphorylation and inhibition of CDKs, which is similar to the checkpoint activated in orp1-4 cells. In human cells, CDK2 activity is rapidly inhibited in response to activation of the G1/S damage pathway, by destruction of CDC25A (Mailand et al.,2000). It is noteworthy that hChk1 phosphorylates hCdc25A in vitro(Sanchez et al., 1997) and destruction of Cdc25A is Chk1 dependent(Mailand et al., 2000). Less is known about the possible involvement of Wee1 or Mik1 in the DNA damage checkpoints in higher eukaryotes. Xenopus Chk1 phosphorylates Wee1 upon checkpoint activation, which leads to increased Wee1 kinase activity(Lee et al., 2001). In Drosophila the rad3 homologue mei-41 and the chk1 homologue GRP are involved in the slowing of the early embryonic cycles, which indicates activation of a checkpoint once maternally provided replication functions become limiting(Fogarty et al., 1997;Sibon et al., 1999;Sibon et al., 1997;Zhou and Elledge, 2000;Rhind et al., 1997). Interestingly, mutation of Wee1 has similar phenotypic consequences to mutation of Grp (Price et al.,2000) suggesting that this Grp-dependent checkpoint might target Wee1. The relative contributions of inhibiting the phosphatases and inducing the kinases might vary in different organisms, but the strategy of maintaining CDK phosphorylation upon checkpoint activation in G1 appears to be conserved.
Acknowledgements
We thank Marit Osland Haugli and Joe Sandvik for technical assistance; Iain Hagan for his help with lactose gradient synchronisation; J. Sogo and his lab members for hospitality and instructions in 2D gel analysis; J. Huberman for communicating unpublished information; Thomas Caspari for helpful discussions;and T. Carr, T. Caspari, H. Lindsay, S. Moreno, P. Nurse, M. O'Connell, H. Okayama, N. Walworth and T. Wang for strains and antibodies. Work in our laboratory is funded by The Norwegian Cancer Society and the Norwegian Research Council.