The Fission Yeast Crb2/Chk1 Pathway Coordinates the DNA Damage and Spindle Checkpoint in Response to Replication Stress Induced by Topoisomerase I Inhibitor

Abstract

Living organisms experience constant threats that challenge their genome stability. The DNA damage checkpoint pathway coordinates cell cycle progression with DNA repair when DNA is damaged, thus ensuring faithful transmission of the genome. The spindle assembly checkpoint inhibits chromosome segregation until all chromosomes are properly attached to the spindle, ensuring accurate partition of the genetic material. Both the DNA damage and spindle checkpoint pathways participate in genome integrity. However, no clear connection between these two pathways has been described. Here, we analyze mutants in the BRCT domains of fission yeast Crb2, which mediates Chk1 activation, and provide evidence for a novel function of the Chk1 pathway. When the Crb2 mutants experience damaged replication forks upon inhibition of the religation activity of topoisomerase I, the Chk1 DNA damage pathway induces sustained activation of the spindle checkpoint, which in turn delays metaphase-to-anaphase transition in a Mad2-dependent fashion. This new pathway enhances cell survival and genome stability when cells undergo replicative stress in the absence of a proficient G₂/M DNA damage checkpoint.

Accurate transmission of genetic information to daughter cells requires checkpoint pathways monitoring completion of DNA replication and DNA damage and correct attachment of replicated chromosomes to the mitotic spindle. These pathways are highly conserved through evolution.

Double-strand breaks (DSBs) are the most dangerous threat to the integrity of the genome. They can be repaired either by nonhomologous end joining or by homology-dependent repair mechanisms such as homologous recombination, break-induced replication, single-strand annealing, and synthesis-dependent strand annealing (22, 31, 42). DSBs can arise spontaneously during DNA replication, or they can be induced by exogenous treatments such as ionizing radiation (IR).

Treatment of cells with inhibitors of the topoisomerase I enzyme (Top1), such as the anticancer drug camptothecin (CPT), leads to single-strand breaks by trapping Top1-DNA intermediates and inhibiting the enzyme's religation activity. Such protein-DNA complexes are converted into DSBs upon DNA replication (32). In fission yeast as well as in vertebrates, exposure to both IR and CPT results in activation of the DNA damage checkpoint pathway in which the Chk1 kinase acts as a downstream effector (18, 43, 46-48, 50).

Fission yeast Chk1 kinase is activated in response to damaged DNA in late S and G₂ phases of the cell cycle and delays mitotic entry by maintaining the Cdc2-cyclin B complex as inactive. Upregulation of Chk1 activity occurs through phosphorylation at S345 by the Rad3 kinase (6, 19), a member of the phosphatidylinositol 3-kinase-like family and a homologue to vertebrate ATR (1). Rad3-dependent activation of Chk1 requires the checkpoint mediator Crb2, a protein sharing sequence and functional similarity with budding yeast Rad9 and human proteins 53BP1 and BRCA1 (34, 49). The sequence similarity concerns the C-terminal BRCT domains, which are protein-protein interaction domains (5), and the two tandem Tudor folds in the central part of the proteins, which are protein-protein and protein-DNA interaction domains (8, 15). It has been shown that 53BP1 recruitment to DSBs depends on the interaction between its Tudor domains and the methylated K 79 of histone H3, which becomes accessible for the interaction at the sites of DSBs (15). Recent work has demonstrated that the Crb2 BRCT domains, similarly to Rad9 BRCT domains, are required for homo-oligomerization of the protein. In fission yeast, Crb2 homo-oligomerization is needed for Rad3-dependent Chk1 activation. Crb2 is recruited to DNA repair foci induced by DSBs in an apparently BRCT domain-dependent fashion. Moreover, Crb2 recruitment to foci depends on histone H2A phosphorylation by the Rad3 or Tel1 kinases (9, 29) and on histone H4-K20 residue methylation by Set9 (36).

Crb2 is also involved in regulation of homologous recombination in the G₂ phase by modulating the activity of Rqh1 helicase. This function is mediated by the Cdc2-cyclin B-dependent phosphorylation of Crb2 at residue T215, an event occurring at mid-mitosis in an unperturbed cell cycle. T215 phosphorylation allows further phosphorylation of Crb2 by the Rad3 kinase in response to DNA damage (7, 10). Furthermore, deletion of crb2 renders cells sensitive to chronic hydroxyurea (HU) treatment, a drug that inhibits the ribonucleotide reductase and induces stalling of DNA replication forks (39, 49). This phenotype is not due to checkpoint failure, since in fission yeast, stalled replication forks activate the Cds1 rather than the Chk1 pathway (44). Sensitivity to HU may result from a role of Crb2 in processing DNA structures that result from damaged replication forks, a process sometimes termed “recovery.”

The spindle assembly checkpoint blocks chromosome segregation until proper attachment of chromosomes to the mitotic spindle is achieved. This checkpoint acts by inhibiting the anaphase-promoting complex (APC), a multisubunit E3 ubiquitin ligase required to promote degradation of both cyclin B and cohesin. Inhibition occurs by preventing APC association with Slp1/Cdc20, a task performed by the checkpoint protein Mad2 (2). In fission yeast, as well as in others organisms, Mad2 inhibition of APC requires Mad3 and the upstream checkpoint kinase Mps1 (14, 24).

To better understand the function of Crb2, we undertook a functional analysis of crb2 mutant alleles which are defective in Chk1 activation when DNA is damaged. This analysis provides evidence that the Crb2/Chk1 pathway causes the spindle checkpoint to delay metaphase-to-anaphase transition when cells enter mitosis with abnormal DNA structures resulting from damaged replication forks. This function enhances cell survival and ensures genome stability in the absence of a proficient G₂/M damage checkpoint.

MATERIALS AND METHODS

Fission yeast physiological and genetic methods.

Strains used in this study are listed in Table 1. Additional strains were obtained by standard genetic crosses followed by tetrad analysis using the listed strains. Genetic procedures and media were as described previously (13, 26). Overexpression of proteins from the nmt1 promoter was induced by first growing cells in selective medium with 5 μg/ml of thiamine (promoter off) and then washing and inoculating cells in thiamine-free medium (promoter on) (21). Mutagenesis of the pRSP HISCrb2 plasmid was carried out in vitro with 1 M hydroxylamine-clorohydrate (Sigma) according to a method described previously (38). Mutagenized plasmids were transformed in the SP808 strain, and transformants were selected on medium containing thiamine and then replica plated on thiamine-free medium with phloxine B to select for viable clones.

TABLE 1.

S. pombe strains used in this study

Strain	Genotype	Source or reference
SP808	h+ura4-D18 leu1-32 ade6-M216	D. Beach
crb2+	h−Myc-crb2+chk1+-HA ura4-D18 leu1-32	39
Δcrb2	h−crb2::ura4+chk1+-HA ura4-D18 leu1-32 ade6-M216	39
Δchk1	h+chk1::ura4+ura4-D18 leu1-32 ade6-M216	45
crb2PH	h−Myc-crb2PH chk1+-HA ura4-D18 leu1-32 ade6-M216	This study
crb2(1-669)	h+crb2(1-669) chk1+-HA ura4-D18 leu1-32 ade6-M216	This study
crb2(1-533)	h+crb2(1-533) chk1+-HA ura4-D18 leu1-32 ade6-M216	This study
cdc25 crb2+	h+cdc25-M22 Myc-crb2+chk1+-HA ura4-D18 leu1-32	39
cdc25 crb2PH	h+cdc25-M22 Myc-crb2PH chk1+-HA ura4-D18 leu1-32	This study
Δcds1	h−cds1::ura4+ura4-D18 leu1-32	27
Δrad3	h−rad3::ura4+ura4-D18 leu1-32	16
Δtel1	h−tel1::kanR ura4-D18 leu1-32 ade6-704	A. M. Carr
Δmad2	h−mad2::ura4+ura4-D18 leu1-32 ade6-M210	A. M. Carr
chk1S345A	h+chk1S345A-HA ade6-M216 leu1-32	6
cut12GFP	h−cut12NEGFP::ura4+ura4-D18 leu1-32	4
MKY7A-4	h+his7::lacO lys1::lacIGFP	28
wt(Ch16)	h−leu1-32 ura4-D18 ade6-M210 + Ch16 ade6-M216	J. P. Javerzat

crb2(1-669) and crb2(1-533) alleles were generated by mutating G to T at position 2008 or at position 1600, respectively, using the Altered Sites II in vitro mutagenesis system (Promega). These mutations result in the creation of a stop codon at positions 670 and 534, respectively. Mutant alleles were integrated at the genomic locus using the pop-in/pop-out method.

Synchronization in G₂ phase of cells harboring the cdc25-22 mutation was performed by shifting cultures in early log phase grown at 25°C for 3.5 h to 36.5°C. Cells were then released into cell cycle at 25°C with or without 20 μM CPT (Sigma). To synchronize in early S phase, cells were treated with 12 mM HU (Sigma) for 4 h. Cells were then washed twice in medium lacking HU and released into cell cycle without or with 20 μM CPT. Two to four independent experiments were performed for each strain.

Genotoxic sensitivity of strains was tested by drop assay. Cells were grown in yeast extract-adenine medium and diluted to 1.3 × 10⁶ cells/ml, and 7.5 μl of sequential fourfold dilutions were spotted onto the appropriate plates. To determine UV and IR sensitivities, plates were irradiated with a 254-nm light source in a Stratalinker (Stratagene) or with a Cs¹³⁷ gamma source at a dose rate of 0.16 Gy/s, respectively. Plates were incubated at 30°C for 3 to 4 days and then photographed.

Survival curves for CPT were performed by exposing asynchronous cultures grown to 4 × 10⁶ cells/ml to 20 μM CPT at 30°C. Starting at time zero, cells were counted, diluted, and plated for survival estimation. Survival curves for IR were performed by cumulative irradiation of 10⁸ cells in 1 ml of distilled water collected from an asynchronous culture in log phase. After each irradiation, an aliquot of cells was diluted and plated for survival estimation. Three to five independent experiments have been performed for each strain.

Determination of chromosome loss rates after CPT treatment was done as described previously (40). Three to five independent experiments have been performed for each strain.

Microscopy.

Plates were photographed under a phase-contrast microscope with plan objective using a SONY 3 charge-coupled-device camera. All microphotographs were taken using a Leica Microsystems DMRD microscope with a ×100 oil immersion objective and a Princeton CoolSnap fx cooled charge-coupled-device camera. The light source for fluorescence excitation was an HBO 100 W Hg arc lamp. The percentage of cells passing mitosis was scored after DAPI (4′,6′-diamino-2-phenylindole; Sigma) staining of cells fixed in 70% ethanol. Pictures of cells stained with DAPI were taken when the binning was set at 1. For observation of living cells expressing green fluorescent protein (GFP)-tagged proteins, the exposure time was 5 s and binning was set at 2. Image capture software used was MetaView (Universal Imaging), whereas image processing software used was Metamorph Offline (Universal Imaging). The latter was used to count cell types on pictures. About 200 cells were counted for each time point.

Protein methods.

Proteins were extracted after harvesting cells with ice-cold stop buffer as previously described (39). Protein concentration was determined by Bradford assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting were standard. Monoclonal anti-His antibodies (QIAGEN) or anti-hemagglutinin (HA) antibodies (clone 12CA5; Roche) were used to probe membranes. Chemiluminescent detection of horseradish peroxidase (HRP)-conjugated secondary antibodies was carried out using Renaissance reagents (DuPont NEN).

RESULTS

Isolation of crb2 alleles that do not arrest cell cycle progression when overexpressed.

We used strain SP808 (Table 1) transformed with plasmid pRSP HISCrb2 to overexpress the Crb2 protein tagged at the N terminus with 10 His residues. Induction of the nmt1 promoter occurs at about 16 h of growth without thiamine. At 20 h, cell survival is about 30% (Fig. 1A), and cells are elongated with a single medial nucleus (Fig. 1B, left) and have a DNA content of 2C (not shown). The tagged Crb2 protein is clearly detected in Western analysis using anti-His-tag antibodies (Fig. 1C). Similar phenotypes were observed when untagged wild-type Crb2 protein was overexpressed (not shown). Thus, similar to Chk1, overexpression of Crb2 blocks cell cycle progression and results in cell death associated with a cdc terminal phenotype (11).

FIG. 1.

Mutations in the Crb2 BRCT domains abolish cell cycle arrest upon Crb2 overexpression. (A) Survival of cells overexpressing HIS-Crb2PH protein (pRSP HISCrb2PH − thiamine) compared to cells overexpressing wild-type HIS-Crb2 (pRSP HISCrb2 − thiamine). (B) DAPI staining of cells overexpressing HIS-Crb2 (left panel) or HIS-Crb2PH (right panel) protein. (C) HIS-Crb2 and HIS-Crb2PH proteins are overexpressed at similar levels. (D) Overexpression (− thiamine) of HIS-Crb2PH, HIS-Crb2(1-669), and HIS-Crb2(1-533) proteins does not arrest cell growth. The Crb2(1-669) protein lacks the last BRCT domain, while Crb2(1-533) lacks both BRCT domains.

To isolate crb2 mutants that are unable to block cell cycle progression when overexpressed, we transformed the SP808 strain with random mutagenized plasmid pRSP HISCrb2 and selected for transformants on plates containing thiamine. After replica plating on selective medium lacking thiamine (Crb2 induced), we isolated several clones which did not exhibit the cdc terminal phenotype associated with Crb2 overexpression. We focused on one clone harboring a plasmid overexpressing full-length HISCrb2 protein (pRSP HISCrb2PH). The plasmid was recovered and retransformed in SP808 cells to confirm the absence of cell cycle arrest (Fig. 1A). Cells overexpressing the mutant protein Crb2PH have a wild-type phenotype (Fig. 1B, right). Protein analysis confirmed that the mutated plasmid overexpresses the HISCrb2PH protein at a level comparable with that of the wild-type pRSP HISCrb2 plasmid (Fig. 1C).

Sequence analysis revealed that the mutated plasmid has two point mutations in the crb2 gene changing P629 to S and H632 to Y (allele crb2PH). The mutations are located in a conserved region of the first BRCT domain and concern two residues conserved in the budding yeast protein Rad9 (P1098 and H1101). Residue P629 is also conserved in the first BRCT domain of the human protein 53BP1 (P1824) (5).

Next, we constructed plasmids expressing the truncated Crb2 proteins Crb2(1-669) and Crb2(1-533), which lack the second or both BRCT domains, respectively. Neither of these two alleles was able to induce cell cycle arrest when overexpressed (Fig. 1D). Thus, either both BRCT domains or the second BRCT alone was required for the overexpression phenotype. However, considering that mutations P629S and H632Y in the first BRCT domain also abolish the overexpression phenotype, the former possibility seems more likely.

Analysis of crb2 mutants integrated at the genomic locus.

We produced three mutant strains carrying the mutant alleles crb2PH, crb2(1-669), and crb2(1-533) and analyzed their phenotypes in response to genotoxic treatments. All strains behave like the crb2 null strain when exposed to UV and IR. However, they are significantly more resistant than the Δcrb2 strain when treated with CPT, methyl methanesulfonate (MMS), and HU (Fig. 2A).

FIG. 2.

Drop test analysis of DNA damage sensitivity of crb2 mutants integrated at the genomic locus. (A) crb2PH, crb2(1-669), and crb2(1-533) are as sensitive as Δcrb2 cells to IR and UV irradiations but are significantly more resistant to CPT and to chronic exposure to low concentrations of HU and MMS. (B) Survival curves of Δcrb2 and crb2PH strains to IR. (C) Chk1-HA phosphorylation after IR in wild-type (crb2⁺) and crb2PH cells.

Figure 2B shows that strain crb2PH is as sensitive as Δcrb2 when exponentially growing cultures are IR irradiated. Analysis of Chk1 phosphorylation indicates that crb2PH cells, like Δcrb2 cells (not shown), do not activate Chk1 upon irradiation (Fig. 2C). Crb2 mutants treated with the different genotoxic reagents in asynchronous culture also did not activate Chk1 (not shown). Thus, when integrated at the genomic locus, the crb2PH allele behaves like allele crb2(1-533), indicating that it has nonfunctional BRCT domains. These observations are consistent with the recent published finding that the BRCT domains are required for efficient G₂/M DNA damage checkpoint signaling to Chk1 (9).

CPT resistance of crb2PH cells is independent of the functional G₂/M DNA damage checkpoint.

CPT treatment induces DNA damage when cells pass through S phase which activates the Chk1 pathway (47). CPT-induced DNA damage occurs during DNA replication when the trapped Top1 cleavage complexes collide with the replication forks, leading to the formation of DSBs (32). Because of better understanding of CPT-induced DNA damage compared to HU and MMS, we focused on the CPT resistance of the crb2PH allele.

To ensure that CPT resistance of crb2PH cells is not due to Chk1 activation, we analyzed cultures synchronized in the G₂ phase using the cdc25-22 allele. Synchronized cells were released into cell cycle in the presence or absence of CPT. Synchronization and cell cycle progression were followed by fluorescence-activated cell sorter analysis (not shown) and by DAPI staining of cells every 30 min starting at release (Fig. 3A). Aliquots of cultures were also processed every 30 min from time zero to analyze expression and phosphorylation of Chk1, which is HA tagged in the used strains (Fig. 3A). While the cdc25 crb2⁺ strain delays the G₂ phase, cdc25 crb2PH cultures treated with CPT only show a slight delay in the appearance of cells with two nuclei in the second cell cycle after release (compare time point 210 between untreated and treated synchronized cultures in Fig. 3A). However, phosphorylation of Chk1 upon CPT treatment, which, as expected, was easily detectable in the cdc25 crb2⁺ strain, was completely absent in cdc25 crb2PH cells (Fig. 3A). The Δcrb2 mutant did not exhibit any cell cycle delay or Chk1 phosphorylation upon CPT treatment (not shown).

FIG. 3.

Analysis of CPT response in synchronized wild-type (crb2⁺) and crb2PH cells. (A) Cell cycle progression and Chk1 phosphorylation of cdc25 crb2⁺ and cdc25 crb2PH strains expressing Chk1HA synchronized in G₂ and released into cell cycle without (left panels) or with (right panels) CPT. (B) Cell cycle progression and Chk1 phosphorylation of crb2⁺ and crb2PH strains expressing Chk1HA synchronized in early S phase and released into the cell cycle with (black circles) or without (black squares) CPT.

We confirmed these results using a different synchronization protocol that allows for analysis of the effects of the treatment in the first cell cycle following release. Cells were synchronized in early S phase by HU treatment for 4 h and then released into cell cycle in the absence or presence of CPT (time zero). Cell cycle progression was monitored by fluorescence-activated cell sorter analysis (not shown) and DAPI staining of cells every 15 min starting at time zero. Chk1 protein was analyzed every 30 min starting at time zero. As expected, wild-type (crb2⁺) cells delay cell cycle progression when treated with CPT and have phosphorylated Chk1 kinase (Fig. 3B). In contrast, no Chk1 phosphorylation is detected in the crb2PH strain exposed to CPT. However, crb2PH cells have a small delay in cell cycle progression after CPT treatment (Fig. 3B). This small delay, which was also observed after synchronization with the cdc25 allele (Fig. 3A), is reproducible and occurs at the metaphase-to-anaphase transition (see below and see Fig. 5 and 6). Note that as a result of the HU block-and-release protocol, low levels of Chk1 phosphorylation are detected in the untreated wild-type strain but not in the crb2PH mutant (Fig. 3B). This difference could explain why the crb2PH cells cycle slightly faster than wild-type cells even in the absence of CPT.

FIG. 5.

crb2PH cells delay metaphase-to-anaphase transition after CPT treatment. (A) Survival of wild-type (crb2⁺), Δcrb2, and crb2PH strains following synchronization in early S phase by HU treatment in the absence (black squares) or presence (black circles) of CPT. (B) Strains expressing the fluorescent protein Cut12GFP were synchronized in early S phase and released into the cell cycle with (black circles) or without (black squares) CPT. Upper panels indicate the percentage of cells with two nuclei as judged by DAPI staining. Lower panels indicate the percentage of cells in prometaphase and metaphase as judged by the presence of two adjacent Cut12GFP spots. (C) Representative microphotographs of the indicated strains expressing Cut12GFP protein at 75 min after release into the cell cycle in the presence of CPT. (D) Separation of centromere 1 in crb2PH cells is delayed by the CPT treatment. The left panel shows the percentage of cells with two nuclei as judged by DAPI staining, while the right panel indicates the percentage of cells with unsplit centromere 1.

FIG. 6.

Analysis of the delay in metaphase-to-anaphase transition of the indicated strains expressing the fluorescent protein Cut12GFP. Cells were synchronized in early S phase and released into the cell cycle in the presence (black circles) or absence (black squares) of CPT. Left panels indicate the percentage of cells with two nuclei as judged by DAPI staining. Right panels indicate the percentage of cells in prometaphase and metaphase as judged by the presence of two adjacent Cut12GFP spots.

In conclusion, the crb2PH allele is unable to activate the G₂/M DNA damage checkpoint by promoting Chk1 phosphorylation when DNA is damaged. However, at the same time, the Crb2PH protein retains a function required to survive CPT treatment.

CPT resistance of crb2PH requires Chk1, Mad2, and Cds1 proteins.

Having established that CPT resistance of crb2PH is independent of Chk1 phosphorylation, we asked if resistance requires the presence of Chk1 protein. As shown in Fig. 4A, deletion of chk1 in the crb2PH background renders cells sensitive to CPT. Importantly, strain Δchk1 crb2PH is as sensitive as Δcrb2 to CPT. This indicates that the function retained by the Crb2PH protein mediating survival under CPT treatment requires the presence but not the phosphorylation of Chk1 kinase. Furthermore, the chk1S345A allele, in which residue 345 cannot be phosphorylated by the Rad3 kinase in response to damaged DNA (6), is more resistant to CPT than the chk1 null allele (Fig. 4A). Furthermore, double mutant strain chk1S345A crb2PH is not more sensitive than each single mutant to the treatment (Fig. 4A). All of these results are consistent with the observation that crb2PH resistance to CPT requires the Chk1 protein (Fig. 4A) but does not implicate G₂/M delay and Chk1 phosphorylation by Rad3 (Fig. 3A and B).

FIG. 4.

Cell-killing experiments to analyze the genetic interaction between the crb2PH mutant and other checkpoint genes. (A) crb2PH, chk1S345A, and double mutant crb2PH chk1S345A are equally sensitive to CPT. (B) Double mutant Δmad2 crb2PH is as sensitive as Δcrb2 to CPT. (C) Double mutant Δmad2 chk1S345A is as sensitive as Δchk1 to CPT. (D) Triple mutant Δmad2 crb2PH Δchk1 is not significantly more sensitive to CPT than control double mutants. (E) Δcds1 interacts synergistically with crb2PH. Double mutant Δcds1 crb2PH is as sensitive as Δrad3 to CPT. Error bars indicate standard errors of the means.

Next, we asked whether the spindle checkpoint is required for the CPT resistance of crb2PH cells. Deletion of the spindle checkpoint protein Mad2 renders wild-type cells only slightly sensitive to CPT (Fig. 4B). Moreover, mad2 deletion does not affect the CPT sensitivity of Δcrb2 cells (Fig. 4B). In contrast, mad2 deletion abolishes the CPT resistance of mutant crb2PH (Fig. 4B), rendering the crb2PH Δmad2 double mutant effectively as sensitive as Δcrb2. This suggests that the function retained by the Crb2PH protein requires Mad2. Similarly, mad2 interacts genetically with the chk1S345A mutant. Thus, mad2 deletion abolishes the CPT resistance of mutant chk1S345A but does not affect the CPT sensitivity of Δchk1 cells (Fig. 4C). Furthermore, we show that triple mutant crb2PH Δchk1 Δmad2 is not more sensitive to CPT than the control double mutants (Fig. 4D), indicating that Mad2 and Chk1 act in the same pathway that allows CPT survival of crb2PH cells.

Combining the crb2PH allele with additional mutants provided further evidence for a connection between the DNA damage and spindle checkpoint pathways. A genetic interaction resembling that with Mad2 was observed between the crb2 alleles and mph1 deletion (not shown), which encodes the upstream kinase of the spindle checkpoint pathway (14).

Given that Cds1 kinase can act in response to S-phase DNA damage, we asked whether it is required for CPT survival of the crb2PH strain. As shown in Fig. 4E, Δcds1 is only slightly sensitive to CPT but shows a synergistic effect with the crb2PH mutation. This genetic interaction is likely due to a greater “requirement” for an intra-S DNA damage checkpoint in CPT-treated cells which lack the G₂/M checkpoint pathway and thus enter the next S phase with damaged chromosomes. This interpretation is consistent with our observation that the crb2PH strain, similarly to Δcrb2, passes mitosis with assembled Rad22 foci (not shown). Moreover, Δcds1 crb2PH cells are as sensitive as Δrad3 to CPT, corroborating further the idea that survival of crb2PH cells depends on the ability to perform a Rad3-dependent activation of Cds1 in the following S phase (Fig. 4E). In contrast to rad3, deletion of the tel1 gene did not affect the CPT response in any of the analyzed strains (Fig. 4E).

Mad2-dependent delay of metaphase-to-anaphase transition in crb2PH cells treated with CPT.

The genetic interaction between crb2PH and Δmad2 prompted us to investigate the possibility that crb2PH cells have a metaphase-to-anaphase delay following exposure to CPT. We constructed crb2 mutants expressing the Cut12GFP fusion protein that localizes at the spindle pole body. This protein allowed us to distinguish cells that have entered mitosis (two adjacent Cut12 spots) from cells in interphase (one spot) and from cells that have passed metaphase (two distant spots) (4).

Cells were synchronized in early S phase by HU treatment for 4 h and then released into cell cycle in the absence or presence of CPT. First of all, survival of the strains was monitored by plating the cells at the beginning of the experiment (time −4), at the moment of release (time zero), and every hour after release (Fig. 5A). Note that in this experiment, both crb2 mutants are more sensitive than in the survival curves shown in Fig. 4. This is likely due to the fact that in this experiment, differently from that shown in Fig. 4, cells are passing into the S phase in a synchronized fashion when exposed to CPT. However, even in this experiment, the crb2PH mutant is significantly more resistant to CPT than Δcrb2.

Every 15 min from time zero, the percentage of cells with two nuclei and the percentage of cells with two adjacent Cut12 spots were monitored (Fig. 5B and C). As expected, the wild-type strain (crb2⁺) treated with CPT delays cell cycle progression as judged by the delay in the appearance of cells with two nuclei and with two adjacent Cut12GFP spots (Fig. 5B, left). In contrast, cell cycle progression did not differ between CPT-treated and untreated Δcrb2 cultures (Fig. 5B, center). As predicted, CPT treatment altered cell cycle progression of the crb2PH mutant (Fig. 5B, right). The number of cells in prometaphase or metaphase (two adjacent Cut12GFP spots) at 75 min in the treated culture is about 23%, while it drops to 13% in the untreated culture. We conclude that CPT treatment has no effect on the kinetics of mitosis entry of crb2PH cells but delays the metaphase-to-anaphase transition by 15 min in the cells. In fission yeast, mitosis takes about 10% of mitotic cell cycle. In rich medium at 30°C, the generation time is 2.5 h, and thus, mitosis last about 15 min. Thus, in the crb2PH strain treated with CPT, the 15-min delay at mitosis is highly significant.

We confirmed the mitotic delay in crb2PH cells exposed to CPT by monitoring the separation of centromere 1 targeted by a fluorescent marker (28). As shown in Fig. 5D, separation of centromere 1 is delayed in crb2PH cells treated with CPT. Indeed, the percentage of cells with one fluorescent spot, which indicates the percentage of cells with unsplit centromere 1, is higher between 30 and 60 min in the treated culture. In all experiments presented in Fig. 5, the delay in the metaphase-to-anaphase transition of the crb2PH strain correlates with the delay observed when the appearance of binucleated cells is monitored by DAPI staining.

Importantly, we show that the chk1S345A mutant (Fig. 6A) as well as the chk1S345A crb2PH double mutant (Fig. 6B) have a mitotic delay similarly to crb2PH after CPT exposure. In contrast, Δchk1 cells behave like Δcrb2 cells and do not show mitotic delay (Fig. 6C); moreover, chk1 deletion abolishes the mitotic delay of crb2PH cells (Fig. 6D). The mitotic delay is also abolished in the Δmad2 crb2PH double mutant, indicating that cells expressing the Crb2PH protein delay metaphase-to-anaphase transition through the Mad2 pathway (Fig. 6E). Finally, Mad2 is required for the mitotic delay observed in chk1S345A cells treated with CPT (Fig. 6F). All of these results are in accordance with the genetic analysis presented in Fig. 4 and indicate that the mitotic delay observed in crb2PH cells treated with CPT requires Mad2 and the Chk1 protein but not Chk1 phosphorylation at S345.

The function retained by the crb2PH cells allows accurate chromosome segregation following CPT treatment in the absence of a G₂/M DNA damage checkpoint.

The above-described experiments established that the crb2PH allele enhances survival to CPT treatment by delaying the metaphase-to-anaphase transition and that this delay is Mad2 dependent. To determine whether this function contributes to genome stability, we measured the chromosome loss rate in wild-type, Δcrb2, crb2PH, Δmad2, and crb2PH Δmad2 cells after CPT treatment. For this experiment, mutant strains containing the ade6-M210 allele at the genomic locus and the minichromosome Ch16 with the ade6-M216 allele were constructed (30). These ade6 alleles complement each other, and thus, cells are ade⁺ and colonies are white on YE medium. Upon loss of Ch16, cells become ade⁻ and colonies are colored red. We measured the rate of chromosome loss of the different strains under normal growth conditions and after 4 h of treatment with 20 μM CPT. As shown in Fig. 7, all mutants are similar to the wild type in the absence of CPT exposure. As expected, strain Δmad2, which has a functional G₂/M DNA damage checkpoint, is similar to the wild type both in the absence and presence of CPT treatment. Interestingly, while Δcrb2 cells exposed to CPT are more likely to lose the Ch16 than wild-type cells, exposed crb2PH cells behaved like wild-type cells. In contrast, crb2PH Δmad2 cells behaved like Δcrb2. Therefore, the function retained by allele crb2PH, which requires the spindle checkpoint Mad2 protein, participates in genome stability. The observation that the rate of chromosome loss in crb2PH cells is the same as that in the wild type indicates that this mutant is defective in neither microtubule-kinetochore attachment nor sister chromatid dynamics. This is consistent with our findings showing that crb2PH cells are not CBZ sensitive and that they have normal silencing at centromere 1 (data not shown).

FIG. 7.

Chromosome stability in wild-type, Δcrb2, crb2PH, Δmad2, and Δmad2 crb2PH mutants under normal growth conditions (white bars) and after 4 h of treatment with 20 μM of CPT (black bars). Error bars are the standard errors of the means.

DISCUSSION

Checkpoint pathways are surveillance systems that ensure genome integrity through ordered execution of cell cycle events when the cell cycle is subject to perturbations. Among checkpoints, the DNA damage surveillance system transiently halts cell cycle progression in response to damaged DNA while the spindle checkpoint prevents metaphase-to-anaphase transition until all chromosomes are correctly attached to the spindle. Here, we provide evidence of a link between these two checkpoints that becomes apparent when cells experience damaged replication forks.

In fission yeast, mitotic entry is inhibited by the activation of the conserved Chk1 kinase in response to damaged DNA in late S and G₂ phases of the cell cycle (20, 46). Furthermore, the Chk1 pathway is activated in response to damaged replication forks, a situation occurring either following treatment with CPT or by exposing cells to HU in the absence of the replication checkpoint kinase Cds1 (3, 17, 47). Crb2 is a BRCT domain-containing protein that functions as a mediator for Chk1 activation (34, 49).

The principal findings in this paper derive from studying mutant crb2PH cells. We demonstrate that this mutant expresses a Crb2 protein with nonfunctional BRCT domains that is deficient in the G₂/M damage checkpoint. This is consistent with the reported requirement of Crb2 BRCT domains for Rad3-dependent Chk1 phosphorylation and subsequent G₂/M checkpoint activation (9, 25).

By comparing the CPT resistance of the crb2PH mutant to that of crb2 null cells, we show that the Chk1 pathway, beside its major role in the G₂/M damage checkpoint, acts on the spindle checkpoint in response to DNA structures arising from damaged replication forks. This function allows cells to delay the metaphase-to-anaphase transition in a Mad2-dependent mechanism. Importantly, we show that this function enhances not only cell survival but also genome stability in the presence of this type of DNA damage.

The effect of CPT treatment is well understood, while the effects of chronic exposure to low doses of HU or MMS are not. The DSBs induced by CPT are generated when replication forks collapse at the 5′ end of the Top1-cleaved DNA present on the leading strand (32). It is possible that the replication stress induced by chronic exposure to HU and MMS also results in damaged DNA arising from collapsed replication forks. In agreement with our results, a recently published paper suggested that Mad2 is required for the DNA replication checkpoint (HU response) when the Cds1 kinase is compromised (41). This is consistent with our model, since compromising Cds1 kinase leads to activation of the Chk1 pathway when cells are exposed to HU (17). This most likely occurs because HU-arrested replication forks cannot be stabilized by Cds1 and thus collapse.

A similar role in response to collapsed replication forks has been recently proposed for the Saccharomyces cerevisiae Chk1 pathway (37). In budding yeast, differently from Schizosaccharomyces pombe, the major role of the Chk1 pathway in response to damaged DNA detected in late S/G₂ is to block the transition from metaphase to anaphase by stabilizing the Pds1 protein (securin). In fission yeast, as well as in vertebrates, the major execution point of the Chk1 pathway is the transition from G₂ to mitosis. However, our work with S. pombe demonstrates that this is not the sole role of the Chk1 pathway and that, similarly to budding yeast, it can delay the metaphase-to-anaphase transition when cells accumulate abnormal DNA structures resulting from damaged replication forks. Thus, this function of the Chk1 pathway seems to be conserved. It is interesting that an interaction between the spindle pole body protein Sad1 and Crb2 has been observed in a two-hybrid assay (23).

Our results indicate that the ability of the Chk1 pathway to sustain the spindle checkpoint in response to a damaged replication fork implicates the N-terminal part of the Crb2 protein but not the BRCT domains. It has been shown that the N-terminal part of Crb2 contains the moieties for binding to Cut5 and Chk1 (9). Therefore, the Crb2PH protein likely is able to bind both Cut5 and Chk1 in vivo. This would explain why Chk1 is required for the observed resistance to CPT of crb2PH cells and for delaying the metaphase-to-anaphase transition after treatment. Cut5 is an essential checkpoint protein that shares sequence and functional similarities with mammalian TopBP1 protein (34, 35). It has been recently shown that Cut5 forms a complex with the PCNA-like checkpoint protein Rad9 during normal S phase and that this complex allows Crb2 and Chk1 recruitment and activation in response to replication fork collapse or IR treatment. Formation of this complex requires Rad9 C-terminal phosphorylation at T412 and S423 by the Rad3 or Tel1 kinase. Activation of the checkpoint in both cases leads to additional phosphorylation of Rad9 at T225 by Rad3. However, differently from IR, Rad9 phosphorylation at T225 is dependent on previous modification of T412 and S423 when cells accumulate DSBs induced by damaged replication forks (12). Because all these events occur upstream of Crb2 and Chk1, it is possible that Crb2PH and Chk1 can still be recruited to this complex upon CPT exposure but that Rad3-dependent activation of Chk1 is prevented because of a mutation in Crb2, resulting in entry into mitosis with damaged DNA. However, the formation of the complex might still function to delay metaphase-to-anaphase transition in a Mad2-dependent fashion. Consistent with this interpretation, the role of Mad2 in the response to HU of Cds1-compromised cells is Rad3 dependent (41).

Our model implies that the basal kinase activity of Chk1 might be sufficient to directly or indirectly target the spindle checkpoint when cells accumulate damaged DNA arising from replicative stress. Note that following DNA damage, at least 40% of the total Chk1 kinase does not undergo Rad3-dependent phosphorylation and upregulation. Thus, it is possible that in response to replication fork collapse, Chk1 is recruited by Crb2 in two different complexes, one that delays mitotic entry through Chk1 upregulation and another that acts on the spindle checkpoint pathway and involves a nonphosphorylated form of Chk1. This model is consistent with our finding that the relative CPT resistance of chk1S345A compared to the Δchk1 allele is abolished by deleting the mad2 gene.

Similarly to fission yeast, vertebrate cells respond to CPT treatment by activating the Chk1 pathway. The common use of this drug in cancer therapy underlines the interest in a detailed understanding of its actions. Tumor cells are often checkpoint defective and thus highly vulnerable to DNA-damaging agents. However, certain tumors are resistant to CPT treatment. The mechanisms underlying this resistance are different, ranging from insufficient accumulation of the drug over alterations in Top1 to alterations in the cellular response to the Top1-CPT complexes (33). The work presented here opens the possibility that cells lacking the G₂/M DNA damage checkpoint may survive CPT treatment because of sustained activation of the spindle checkpoint.