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1 The G0 Cell Unit, Initial Research Project (IRP), Okinawa Institute of Science and Technology (OIST) Corporation, 12-22 Suzaki, Uruma, Okinawa 904-2234, Japan
2 Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan
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Abstract |
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-ray than in VE cells. The sensitivity like in VE cells is restored after replication in the nitrogen-replenished medium, suggesting that the damage hyper-sensitive nature of G0 cells is due to the error-prone repair for single DNA duplex chromosome. The double-strand break (DSB) repair in G0 cells required Pku80, one of non-homologous end joining (NHEJ) proteins. S. pombe G0 cells upon DNA damages thus respond distinctively from VE cells in regard with regulation of checkpoint proteins and the mode of repair that is dependent upon the use of NHEJ. |
Introduction |
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-ray, methyl methane sulfonate (MMS) etc., have been isolated in these organisms, and utilized for the analyses how cells respond to and repair the damages (e.g. Fishman-Lobell & Haber 1992; al-Khodairy & Carr 1992; Navas et al. 1995; Furnari et al. 1997; Saka et al. 1997). A classic example of the analyses was the identification of the checkpoint gene RAD9 that appeared to monitor the damages and could restrain the progression of cell division cycle before the completion of repair processes (Hartwell & Weinert 1989). If a checkpoint gene is absent, the cell cycle undergoes without repairing the damaged lesions, resulting into lethal mitosis. The restraining of cell cycle progression occurs through inhibiting the regulators, Cdc25 and Wee1 of cyclin-dependent kinase (CDK) in fission yeast (Rowley et al. 1992; Furnari et al. 1997; O’Connell et al. 1997), and APC/C (anaphase promoting complex/cyclosome) that promotes anaphase proteolysis in budding yeast (Sanchez et al. 1999; Agarwal et al. 2003). In mammalian cells, kinases (ATM, ATR, Chk1) that restrain CDKs have been identified (Savitsky et al. 1995; Cimprich et al. 1996; Sanchez et al. 1997). In multicellular organisms, the DNA damages occur often on the body surface, the tissue where a large number of cells are resting and non-dividing. In the term of cell cycle, they are in the state of quiescence or in G0. G0 is different from G1, as G1 cells progress into the cell cycle, whilst G0 cells are arrested. In the mammalian cell cultures, quiescent cells experimentally produced by removing calf serum from the culture medium are thought to be in the G0 state (Zetterberg & Larsson 1985), but there is no general agreement yet whether they are basically identical to the G0 cells in vivo. In fungi, unicellular eukaryotic organisms, most studies on the DNA damage repair have been done using complete or rich media that allow cells to rapidly divide. If the cellular regulatory systems for the DNA damage repair may differ considerably between proliferating and quiescent states, rapidly dividing fungal systems may not be ideal for studying the cellular responses to DNA damages in the quiescent state. A model system that is suitable for the analyses of damage responses in quiescent cells is needed. One possible physiological state that could be analyzed is cells in the stationary phase, which is somewhat vague in definition but generally considered to be the arrested stage due to declined nutritional sources, over populated cells, production of toxic substances in the culture, etc. Stationary phase cells often lose cell viability and may not stay stably in one physiological state because of the quantitative changes in the above parameters in the culture conditions.
The life cycle of S. pombe depends on nutrition and sexuality. In the synthetic medium EMM2 containing glucose, inorganic ions and vitamins (Mitchison 1970), rod-shaped cells vegetatively divide every 2–2.5 h (Fig. 1, upper panel). The elongation of rods occurs in G2, the post-replicative stage. The short rod (7.5 µm long) is in early G2, while the long rod (15 µm long) is in late G2. The length of rod is constant during the period of mitosis, septation, the brief G1 and S phases. Cells that become into a half length after cytokinesis are in early G2, and grow again. Cell growth (cell mass increase) of wild-type S. pombe in the vegetative phase of the life cycle is thus under the G2-control. The majority cell population (
75%) of exponentially growing S. pombe cells are in G2.
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4 µm; Fig. 1, lower panel). The resulting arrested cells mostly (90%) contain 1C DNA, thus arrested in the uncommitted G1-like stage. If, in the culture, h– cells are mixed with h+ cells (they have the opposite mating types), mating and meiosis will take place (Nurse & Bissett 1981). If h– cells have no h+ sexual partner cells in the nitrogen-starved medium, they shift to the committed G0 stage after prolonged culture (Su et al. 1996). The G0 cells in medium lacking a nitrogen source are perfectly viable for many weeks and months, presumably utilizing a nitrogen source recycled within cell. The strategy of G2-driven S. pombe vegetative cells toward meiosis or the dormant G0 state must include cell division in order to enter the G1 stage. In contrast, the stationary cells of S. pombe keep the rod shape and contain 2C DNA (Costello et al. 1986). Upon the replenishment of nitrogen source, the G0 cells begin to elongate, synthesize DNA and enter the first mitosis after 10 h, followed by the rapid vegetative divisions with 2–2.5 h intervals. We aimed to study how the G0 cells of S. pombe are maintained. To this end, transcriptome and proteome analyses in combination with genetic approaches have been employed (M. Shimanuki, Y. Chikashige, S. Y. Chung, Y. Kawasaki, M. Hatanaka, K. Nagao, C. Tsutsumi, Y. Hiraoka & M. Yanagida, in preparation). As an attempt to investigate DNA metabolism, the cellular responses after DNA damages have been investigated in G0 cells. We here report that the DNA damage responses in the S. pombe G0 cells are dramatically different from those of vegetatively growing cells.
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Results |
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To examine the actual repair process in the G0 cell state, restoring the DNA damage was directly monitored in the UV-irradiated G0 cells. The culture of S. pombe wild-type h– cells exponentially grown in the synthetic EMM2 medium was shifted to the same medium that lacked the nitrogen source NH4Cl (designated EMM2-N) for 24 h. Resulting G0 arrested cells were irradiated by UV (50 J/m2) on the EMM2-N plates. Immunoblot against the genomic DNA isolated from G0 and VE cells before and after 50 J/m2 UV irradiation was done (Fig. 2A, top), using antibodies against thymine dimer (designated anti-TD, McCready & Cox 1993). The level of thymine dimer detected by antibodies was high in both G0 and VE cells immediately after irradiation (0 h), but rapidly decreased at the rates slightly slower in G0 cells (see the pattern of long exposure), indicating that G0 cells were efficient in repair for the removal of UV-damaged lesions.
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-ray irradiation (200Gy), the pulsed field gel electrophoresis (PFGE) of S. pombe chromosome DNA was performed (Fig. 2A, bottom). In both VE and G0 cells immediately after -ray irradiation (0 h), the three bands (I, II and III) of chromosomal DNA disappeared and only broken DNA was seen. Chromosome DNA bands were clearly restored in VE after 2–4 h, but more slowly in G0 cells. The apparent rate of rejoining broken chromosomal DNA was roughly 2–3 fold slower in G0 than VE cells.
G0 cells are hypersensitive to UV and -ray
The above experiment did not examine the actual physiological consequence in G0 cells after the damage so that cell viability was determined. To test the sensitivities of G0 cells for UV, G0 arrested cells were irradiated by different doses of UV (0–200 J/m2) on the EMM2-N plates. To count the number of viable cells after irradiation, each of the plates was supplemented by an aliquot of NH4Cl solution to adjust the concentration of NH4Cl in EMM2 to allow the cells to form colonies (Fig. 2B, top left panel). The viability of vegetative wild type (indicated by VE) is also shown as control. We found that G0 cells were much more sensitive to UV irradiation than VE cells: the viability of G0 cells was below 0.1% after 200 J/m2, whilst 10% of VE cells were still viable under the same dose. Difference in the sensitivities to this dose was the magnitude of 102. The FACS analysis (shown at top right) indicated that most (90%) of the arrested G0 cells contained 1C DNA, whereas most of the growing VE cells contained 2C DNA: not only the G2 phase in VE cells, postanaphase cells before cytokinesis are bi-nucleated so that they also contain 2C DNA. Basically the same hypersensitivity was obtained for diploid G0 cells to the above haploid result (data not shown). The ploidy showed no influence over the loss of viability upon UV damage. Considering the facts that G0 cells could eliminate thymine-dimer efficiently and that the loss of cell viability by UV was much severer in G0 than VE cells, we concluded that damage repair processes other than the elimination of thymine-dimer was very inefficient in G0.
The -ray sensitivities of these G0 and VE cells were then tested at doses of 100–1000 Gy (Fig. 2B, bottom left). In the dose range 100–200 Gy, G0 cells were considerably more sensitive to -ray than VE. The VE cells were almost completely viable even after the irradiation of 200 Gy -ray, whilst 80% of G0 cells were dead after the same dose of -ray. To the high dose -ray (> 400 Gy), however, the rates of loss of cell viability were similar between G0 and VE cells, indicating that the loss of cell viability for G0 was bi-phasic. We presumed that the minor population of G0 cells containing 2C DNA (Fig. 2B, bottom right) might have a resistance to the -ray, the degree of which was comparable to that of VE cells, because homologous recombination was possible. This result was consistent with the slower rejoining of DSBs in G0 than in VE cells.
Low glucose and sulfur-starvation do not produce 1C DNA G0 cells
To compare the UV sensitivity in EMM2-N with other culture media, VE cells were transferred to the media containing low glucose (0.1%) or no sulfur for 24 h at 26 °C. These starved conditions did not affect cell potencies of colony formation on a complete medium. Their UV sensitivities under these conditions were examined. As shown in Fig. 2C (top), only nitrogen-deficient G0 cells were significantly more sensitive to UV than vegetative cells. FACS analysis was done to examine the contents of intracellular DNA in these arrested cells (Fig. 2C, bottom). Most cells under sulfur-deficient or low glucose conditions displayed the 2C DNA contents in sharp contrast to G0 cells under nitrogen starvation. In addition, they were not small, and their cell shape was regular rod (right bottom), suggesting that the cell properties (2C DNA, rod, not small) were like VE, and strikingly different from those of G0 cells under nitrogen starvation.
The UV hypersensitivity of G0 cells was restored after DNA replication
To determine when the VE-like UV sensitivity was restored after nitrogen source replenishment, cells were taken at time intervals after the shift to EMM2 and irradiated by UV. Resulting survival curves obtained by plating are shown in Fig. 2D (left panel). The cellular DNA contents (determined by FACS) and cell shape (under a microscope) at different time points after nitrogen replenishment are shown in the middle and right panels, respectively. Cells obtained from 2 to 4 h after nitrogen replenishment were still hypersensitive to UV like G0, while cells from 8 h were like VE. The UV sensitivities from 5 to 7 h were intermediate between G0 and VE. As FACS showed that DNA replication occurred around 5–7 h Fig. 2D (middle panel), the UV sensitivity became like VE in nitrogen-replenished cells after DNA replication.
Crb2-YFP signal intensities in G0 cells depend on DNA contents
S. pombe Crb2 (similar to human 53BP1) is required for the activation of a checkpoint kinase Chk1 and the repair of damaged DNA (Esashi & Yanagida 1999; Mochida et al. 2004). Crb2 is localized at the site of DSB in a histone H2A phosphorylation- and H4 methylation-dependent manner (Du et al. 2003; Nakamura et al. 2004; Sanders et al. 2004). To examine the foci formation of Crb2-YFP (Du et al. 2003) in G0 arrested cells, we employed the strain that expressed Crb2-YFP by the chromosomally integrated gene under the native promoter. Unexpectedly, there were two classes of cells displaying the distinct intensities of Crb2-YFP (Fig. 3A, top left). Approximately 15–20% of G0 cells revealed the strong Crb2-YFP signals in the nucleus (indicated by the arrows). Crb2 might be abundant only in the G0 arrested cells containing 2C DNA content. This speculation was verified by measuring the DNA contents in a number of cells (141 cells), using the fluorescent dye, propidium iodide (PI) after RNase digestion (Fig. 3A, right panel). The DNA contents in the strong Crb2-YFP signals were nearly twice higher than those in cells showing the weak nuclear signals.
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Response to nitrogen starvation differs in cell cycle stages of VE cells
To determine the origin of G0 cells containing 2C DNA, we examined whether the response of VE cells to nitrogen starvation varied depending on the cell cycle stages. For this end, exponentially growing wild-type S. pombe cells were elutriated, and the synchronous culture by selecting the early G2 cells was performed (time 0 in Fig. 3B). The mitotic and septation indices (SI) peaked at 120 and 160 min, respectively (Fig. 3B, left panel). Synchronous cells collected at different cell cycle stages were then nitrogen starved, followed by FACS that analyzed the DNA contents of resulting arrested cells (Fig. 3B, right panel). The frequencies of cells containing 2C DNA increased to 22–34% during mitosis, septation and cell separation, but interphase cells produced only 1C cells (Fig. 3C). Cells in the period for the constant cell length might have different susceptibility to nitrogen starvation, leading to more frequent post-replicative state (see Discussion).
The G0 cells containing 2C DNA skip replication before mitosis
The question was addressed whether G0 cells containing 2C DNA (designated 2C G0 cells) might skip replication before entering mitosis after nitrogen replenishment in order to maintain the same ploidy. To this end, a strain that expresses both Crb2-CFP and Ams2-GFP was used. Ams2 shows an intense periodic fluorescence in the nucleus only during replication (Chen et al. 2003) so that the Ams2-GFP signal is a marker of replicative cells. The intense Crb2-CFP signal is the marker of 2C G0 cells. Movies of G0 cells were taken after nitrogen replenishment (Fig. 3D, Supplementary Fig. S1). We found that those 2C G0 cells displaying the intense Crb2-CFP (cells 3 and 5 indicated by the arrowheads, see figure legend) did not show the periodic Ams2-GFP signals before the first cell division after nitrogen replenishment, whereas the majority of cells with the weak Crb2-CFP signals (cells 1, 2, 4 and 6) all showed the intense Ams2-GFP signals before the first cell division. The cells in the replication period with the intense Ams2-GFP are marked with S in Supplementary Fig. S1. We hence concluded that the arrested 2C G0 cells entered the first mitosis by skipping DNA replication for keeping ploidy.
Weak Crb2-foci form in 1C G0 cells, but Rad22-foci do not
DNA-damaging drug phleomycin (20 µg/mL) was used to induce the foci formation in wild-type G0 cells expressing chromosomally integrated Crb2-YFP and Rad22-CFP under the native promoter. As shown in Fig. 4A (left panel), the small, relatively weak foci (indicated by arrowheads) were observed in the G0 cells 3 h after the addition of phleomycin to EMM2-N, while a small population of the G0 cells displaying the intense Crb2 signals showed the strong foci (indicated by asterisks). Control G0 cells not treated with phleomycin did not show the foci (upper left photo).
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Hyper-phosphorylation of Crb2 occurred in 2C G0 cells
To examine whether a small population of G0 cells containing 2C DNA might contribute to producing the upper bands upon irradiation, we employed two different G0 cells consisting of those containing nearly entirely (97%) 1C DNA (G01C in Fig, 4D; isolated by elutriation) and of those 20% of which contained 2C DNA (G01C2C). Upon UV irradiation, the upper bands of Crb2 were virtually absent for G01C cells, whilst the weak upper bands were observed for G01C2C. The control VE cells produced the intense upper bands upon irradiation. These results strongly suggested that the hyper upper bands of Crb2 were specific for cells containing 2C DNA regardless of growth or arrest.
To confirm that the arrested G0 cells containing 2C DNA could produce the hyper upper bands upon irradiation, we used nuc2-663 mutant that was defective in APC (anaphase promoting complex)/cyclosome and reported to be arrested with the 2C DNA contents under nitrogen starvation (Kumada et al. 1995; also see the bottom FACS panel of Fig. 4E). Immunoblot of Crb2 in nuc2-663 cells arrested by nitrogen source deficiency (24 h, 26 °C) produced the hyper upper bands upon UV irradiation like in wild-type VE cells. We therefore concluded that the hyper upper band formation of Crb2 by UV irradiation was dependent on the presence of post-replicative sister chromatids.
Chk1 in G0 does not show the upper band after UV irradiation
In VE cells, the hyper-phosphorylated form of Crb2 physically interacts with Chk1 kinase after DNA damage (Mochida et al. 2004). To test whether the checkpoint kinase was activated in G0 cells after UV irradiation, immunoblot was done for monitoring the hyper upper band of activated Chk1 (Walworth & Bernards 1996). The activated Chk1 upper band (Chk1P) was seen in VE cells 1–2 h after UV irradiation (50 J/m2) for checkpoint arrest (Fig. 5A upper panel, in VE). In G0 cells, however, the level of Chk1 was found to be rather low, and the upper band did not appear after UV irradiation (the band indicated by asterisk was a G0-specific contaminating band). These results may be explained because the checkpoint arrest was not required for repairing the damaged DNA in nitrogen-starved G0 cells, so that Chk1 might not have to be activated. Hyper-phosphorylation of Crb2 was also unnecessary in G0 cells. Control Cds1/Chk2 required for intra-S phase checkpoint (Murakami & Okayama 1995) showed the bands with similar intensities in both G0 and VE cells before and after UV irradiation. Other control Cut5/Rad4 required for replication checkpoint (Saka & Yanagida 1993) showed the slight increase in the band intensities after irradiation in VE cells. Whilst the upper band of Cut5 was seen in irradiated VE cells, such change did not occur in G0 cells.
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To identify the regulatory genes for repair, we attempted to examine effect of various checkpoint mutations in the arrested G0 cells after UV irradiation. These mutants showed almost similar profile of DNA content to that of the wild-type strain in G0. As shown in Fig. 5B, G0 cells deleting Rad3 (
rad3) and Crb2 (crb2) showed significant enhancement in the hypersensitivity to UV: (rad3 was most sensitive). The sensitivity enhancement in chk1 and cds1 cells was less than rad3 and crb2. We then tested whether the elimination of thymine dimer in G0 cells required Rad3 and Crb2. Immunoblot against thymine dimer using anti-TD antibodies showed that the rate of thymine dimer repair was slightly slower in rad3 than in wild type (Fig. 5C). Otherwise the repair was indistinguishable to that in wild-type cells. The above results were unexpected, suggesting an additional role for Rad3 in G0, and are discussed below.
UV irradiation causes DNA breakages in a Uvde endonuclease-dependent manner both in G0 and VE cells
An intriguing question was why G0 cells were much more sensitive to UV than VE cells, whilst the elimination of thymine dimer in G0 cells was as efficient as in VE cells. PFGE analysis was used to separate chromosomes after UV irradiation. We unexpectedly discovered that highly efficient apparent formation of DSB occurred by UV irradiation (100 J/m2) in both VE and G0 cells (result of VE cells shown in Fig. 6A). It was unclear how the apparent DSBs were made after UV irradiation, as UV irradiation is known to produce various kinds of DNA damages. We suspected that UV damage-directed endonuclease(s) might be implicated in this DSB formation. We therefore examined effect of Rad13 and Uvde deletions on DSB formation (Fig. 6B). G0 and VE cells of rad13 and uvde deletions were run in PFGE before or immediately after UV irradiation. We indeed found that VE and G0 cells did not produce broken chromosome DNA after irradiation in the absence of UVDE. In the absence of Rad13, DSB was formed in both G0 and VE. These results indicated that UVDE was essential for the UV-induced double strand DNA breakages in both G0 and VE. In G0 cells, both rad13 and uvde deletions were further more hypersensitive to UV than in wild-type G0 (data not shown).
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The repair of UV-induced DNA breakages seemed to be somewhat slow in G0 cells in comparison with that in VE cells (Fig. 6C). Three chromosome bands were seen after 8 h in VE cells, while they were seen only after 24 h in G0 cells. Note that the time course patterns of the repair process were quite different between G0 and VE cells (compare G0 and VE in Fig. 6C). In VE cells, the bulk of chromosomal DNA did not enter the gel during the repair process (1–2 h after irradiation). This was probably due to the homologous recombination complex formed during the repair. In G0 cells, non-homologous end joining (NHEJ) of broken chromosomes might be necessary for the repair of broken DNA (Ferreira & Cooper 2004 and see below), and DNA shorter than the full length was observed in the PFGE patterns.
The above interpretation regarding the involvement of NHEJ was substantiated by examining the cell viability of G0 cells that lacked Pku80 (pku80) after UV and -ray. Cell viability of the strain pku80 was more sharply decreased in G0 than in VE (data not shown). NHEJ requires Pku80 (Taccioli et al. 1994; Miyoshi et al. 2003). In G0 cells after UV (100 J/m2) or -ray irradiation (200 Gy), the repair of broken DNA took 24 h (Fig. 6D, upper panel WT). In the absence of Pku80 (pku80), the bulk of broken chromosome DNA still remained in G0 cells after 24 h (Fig. 6D, upper panel pku80). In control VE cells, the effect of pku80 was not significant for the broken DNA repair 8 h after UV or -ray irradiation (Fig. 6D, lower panels). These results showed that the repair of broken DNA formed by UV or -ray in G0 cells was dependent on error-prone NHEJ (Wilson et al. 1999) so that the cell viability of G0 cells was low after irradiation whilst broken DNA was apparently rejoined to form the three chromosome DNA bands.
The damage repair in G0 cells requires Rad3/ATR
We supposed that UV-induced DNA breakages might cause the UV hypersensitivity of checkpoint mutants as shown in Fig. 5B. We hence examined the repair of breakage in G0 and VE cells in the absence of Crb2, Rad3, Chk1 or Cds1 (Fig. 7A,B). In rad3 cells, the breakage was hardly repaired in both G0 and VE cells. In crb2 and chk1 cells, the breakage was mostly repaired in G0 cells, but not at all in VE cells. We concluded that Rad3 was essential for UV-induced DNA breakage repair in G0 cells. However, we could not explain why crb2 cells were hypersensitive to UV irradiation in G0, whilst the breakage in crb2 was mostly rejoined. The highly error-prone NHEJ repair might occur in G0 cells in the absence of Crb2. Survivor in a high dose range (100–150 J/m2) of UV might come mainly from 2C G0 cells. Crb2 has been proposed to be implicated in homologous recombination repair (Caspari et al. 2002; Du et al. 2003; Sanders et al. 2004) so that the cell viability of crb2 after UV irradiation was exceedingly lower than that of chk1 and cds1 cells.
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Discussion |
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ray-induced damage were quite distinct between G0 and VE cells. Ferreira & Cooper (2004) showed that the general levels of NHEJ and HR were reciprocally regulated thorough the cell cycle, so that NHEJ was higher in G1 (including nitrogen-starved cell state) than in other cell cycle stages; the reverse was true for HR. Our results are consistent in this regard, and the G0 cells containing 2C DNA (10% population) appear to respond to the damage by HR through the foci containing Rad22.
To understand the cell cycle aspect of non-dividing G0 cells, knowledge of the state of checkpoint proteins upon DNA damage is crucial. We provided evidence that Chk1 kinase (Walworth & Bernards 1996) was not activated in the nitrogen-starved S. pombe G0 cells after UV irradiation. Firstly, the hyper-phosphorylated band, which was presumably an active form upon damage (Capasso et al. 2002), was not produced in G0 cells on UV irradiation similar to that in cdc10-arrested G1 cells (Martinho et al. 1998). In our previous study (Mochida et al. 2004) we showed that Chk1 binds to the phosphorylated form of Crb2, which was shown in this study to be absent in 1C G0 cells. Hence Chk1 might not be activated. Secondly, the protein level of Chk1 in undamaged G0 cells was much lower than VE cells, and did not change level on damage. Thirdly, the G0 cells of chk1 deletion mutant showed the UV sensitivities similar to the wild type compared with rad3 and crb2 mutants, perhaps because Chk1 was not required in the arrested G0. Fourthly, PFGE showed that UV-induced DNA breakage was repaired in chk1 deletion mutant with the rate similar to that in wild-type cells. In VE cells, however, the repair was greatly defective in chk1 deletion mutant cells. Although the basal activity of Chk1 per se might be needed for repair, restraining Cdc2 kinase through the hyperactive Chk1 was unlikely to be necessary in G0 cells on damage, as these G0 cells were already completely arrested. This was consistent with the results of crb2 T215A Cdc2 phosphorylation site mutant; the modification of Crb2 by Cdc2 was required for the repair of the UV-induced DSB in VE cells, but not in G0 cells. We thus concluded that neither restraining nor activating Cdc2 kinase was necessary during the damage response and repair in G0 cells.
We showed that, in G0 cells, Rad3, an ATR-like checkpoint kinase (Bentley et al. 1996), was absolutely needed for the damage repair after UV irradiation. G0 cells of rad3 deletion were extremely sensitive to UV and failed to repair UV-induced breakage. One should be cautious, however, when considering cell viability of G0 cell, because the viability in this case means a potential of colony formation, which includes a recovery process from G0 to proliferation. The recovery process is not well understood. The targets of Rad3 in G0 cells remained to be clarified, as neither of Chk1 nor Cds1, the targets of Rad3 in VE cells, did not show the hyper upper bands upon damage. A potential target of Rad3 may be Crb2. Crb2 seemed to respond to damage in G0 cells, as the weak and strong foci were formed in G0 cells containing 1C and 2C DNA, respectively; however, no hyper upper band was produced for Crb2 in G0 cells (Fig. 4D). Rad3 activity and Cdc2 phosphorylation site of Crb2 (Saka et al. 1997; Esashi & Yanagida 1999) were required for the hyper-phosphorylation of Crb2 in VE cells. Modification of Crb2 protein was thus very different between VE and 1C G0 cells, and between 1C and 2C G0 cells. Crb2 might be differently modified according to pre- or post-replicative cell states.
Another candidate for Rad3 downstream targets is Rad21 cohesion protein. Nagao et al. (2004) showed that cleavage of cohesin in interphase is required for efficient DNA repair and phosphorylation of Rad21 after UV irradiation was dependent on Rad3, but not on Chk1 or Cds1. Furthermore one mutant of Cut2/securin EA2, which was responsible for Rad21 cleavage, showed UV hypersensitivity and its sensitivity was synergistic with that of rad13 but not with uvde (Nagao et al. 2004). These observations suggest that Rad3 may facilitate UVDE pathway.
The present study established that certain repair process such as the elimination of a thymine dimer was highly effective in G0 cells, being comparable to vegetative cells. In spite of such efficient repair, however, G0 cells were far more (100-fold by 200 J/m2) sensitive to UV than VE cells. This UV hypersensitivity in G0 was restored to the VE level after DNA replication in the nitrogen-replenished medium, strongly suggesting that the low DNA (1C) content in G0 cells might be implicated in the unknown cause of such hypersensitivity. The sensitivity of G0 cells to -ray, on the other hand, was only moderately (5-fold by 100 Gy) more sensitive than VE cells. Consistently, the rejoining rate of broken DNA for -ray induced DSB was slower (several fold) in G0 than in VE cells (Fig. 2A). For UV-induced DSB, the rate of repair seen in PFGE was also slower in G0 than VE cells and the modes of repair were distinct (Fig. 6C). Because homologous recombination was not possible in 1C G0 cells, error-prone NHEJ should be largely responsible for this slow repair. Indeed, we showed that the DNA breakage repair in G0 cells was dependent on the presence of Pku80 (Fig. 6D). Ferreira & Cooper (2004) showed that the activity of homologous recombination was high in VE but low in nitrogen starved G1 (their terminology) cells. In contrast, the activity of NHEJ was high in G1. Our results were consistent with their finding.
We showed that G0 cells consisted of two kinds of cells containing 1C or 2C DNA. Their modes of repair and re-entry into cell proliferation were distinct. In the minor population (10%) of 2C G0 cells, the Crb2 nuclear signal was intense in the absence of damage, and responded to form the strong foci upon DNA damage, as in VE cells (Du et al. 2003). We showed that hyper-phosphorylation of Crb2 occurred only in postreplicative cells, regardless of the state of the cells: either arrested or dividing. Crb2 has been proposed to be implicated in homologous recombination repair (Caspari et al. 2002; Du et al. 2003; Sanders et al. 2004). Consistently, the Rad22 signals specific for homologous recombination formed the foci only in the minor post-replicative G0 cells upon damage, and the foci produced by Crb2 and Rad22 upon damage were identically located. This might also be true in diploid cells because UV sensitivity of diploid G0 cells was similar to that of haploid G0 cells. It should be stressed that post-replicative G0 cells showed no anomaly except for skipping the DNA replication stage upon the replenishment of a nitrogen source to maintain the haploid state. It is quite possible that higher eukaryotic somatic tissues may contain minor populations of post-replicative G0 cells.
The cause for generating the post-replicative G0 cells under nitrogen starvation is unclear. The previous report that nuc2 mutant cells defective in APC/cyclosome were arrested in the post-replicative G0 state at the permissive temperature (Kumada et al. 1995) suggested a hypothesis that the normal nitrogen starvation signal might be transmitted through APC/cyclosome for arresting at a pre-replicative stage in G0. It is unclear whether this speculation is consistent with the present result that the highest frequencies of post-replicative G0 cells were produced when cells in the period of constant length (including mitosis, septation, G1, S) were exposed to nitrogen starvation, whilst post-replicative G0 cells were negligible when G2 cells were exposed to nitrogen starvation.
An unexpected result in the present report was that DNA breakage formed highly efficiently in G0 and VE cells upon UV-irradiation. To our knowledge, UV-induced DSB formation has been little investigated in fungi. In addition, the modes of repair for this DSB were distinct between G0 and VE, and the rate of repair was slow in G0 cells. We interpreted our data that homologous recombination intermediates were abundant in VE cells during repair and did not enter PFGE gel. In prereplicative G0 cells, however, the mobility of broken DNA generally decreased during the recovery process, indicating that the broken chromosome DNA fragments were directly and non-homologously joined through the NHEJ mechanism (Pastwa & Blasiak 2003).
We found that the UV damage endonuclease UVDE (Yasui & McCready 1998) was essential for DSB formation in both G0 and VE cells. UVDE has been reported to have a nicking activity to cut only a damaged DNA strand at the sites immediately 5' to the UV-induced cyclobutane pyrimidine dimers and (6-4) photoproduct (Bowman et al. 1994; Takao et al. 1996). It is possible that two independent nicks on two different strands locating close enough to separate DNA in vivo may cause a DNA breakage, which might be repaired by HR or NHEJ. Actually we observed that pku80 mutant showed higher sensitivity to UV than wild-type in G0 but not in VE (our unpublished observation), and a significant portion of UV-induced broken DNA in pku80 remained even after 24 h incubation (Fig. 6D, upper left). So we assume that UV induces DNA strand breaks in vivo in fission yeast that must be repaired in NHEJ-dependent manner in G0. The UVDE-like nucleases were found mainly in fungi (but not in S. cerevisiae), archae and bacterial cells, but authentic homologs were not found in higher eukaryotes. Since bacterial and various fungal species have definitive homologs of UVDE, its implication in the repair of UV-induced DNA breakage appears to be evolutionarily conserved.
In short, we showed in this study that S. pombe G0 cells upon DNA damages responded distinctively from VE cells in regard to regulation of checkpoint proteins and the mode of repair that was dependent upon the use of NHEJ. Nevertheless, a possibility remained that these S. pombe G0 cells might be similar to G1 cells in the mechanistic aspects for repair of DNA damages. As the G1 phase is very brief in the regular cell cycle of S. pombe, it is not plausible for mechanistic analysis of repair in the G1 cells. The use of ts mutant cdc10 (Simanis & Nurse 1989), which causes the accumulation of rod-shaped G1-arrested cells at the restrictive temperature in the nutrient medium, may provide a useful system to compare the modes of repair in G1 with those in G2 and G0 cells.
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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S. pombe cells of the heterothallic haploids 972 h–, 975 h+ (Gutz et al. 1974) and the derivatives were used. Mutant strains, crb2 (Saka et al. 1997), crb2T215 A (Caspari et al. 2002), rad3 (Bentley et al. 1996), chk1 (Walworth & Bernards 1996), cds1 (Murakami & Okayama 1995), nuc2-663 (Hirano et al. 1988), pku80 (Miyoshi et al. 2003), rad13 (Carr et al. 1993) and uvde (Yonemasu et al. 1997), were previously described. The EMM2 medium for vegetative growth and EMM2-N (EMM2 lacking NH4Cl) for nitrogen starvation were employed (Mitchison 1970). All the irradiation experiments except -ray, which was set at 30 °C, were done at 26 °C. The -ray irradiation was performed at the Research Reactor Institute of Kyoto University in Kumatori, Osaka. The nitrogen-starved cells designated G0 were prepared as follows. The cells were grown in EMM2 to the concentration of 8 x 106/mL. They were then filtrated and washed in EMM2-N twice on the Biodyne PLUS membrane (PALL, #60406), followed by re-suspension into EMM2-N with the cell concentration of 2 x 107/mL and incubation at 26 °C for 22–24 h. The G0 cells after 2 divisions during the first 4–5 h under nitrogen starvation became small and spherical (Su et al. 1996). Cell viability should be 100%, and the DNA content measured by FACS (Costello et al. 1986) was 1C for 90% of cell populations. Nitrogen replenishment was done by adding the 4 volumes of EMM2 to the G0 culture. FACScalibur (Beckton Dickinson) was employed for FACS analysis.
Synchronous culture
One to two liters of the culture containing exponentially growing S. pombe cells (1–2 x 1010) were separated by a size distribution using the HITACHI R5E centrifugal elutriation system. Small early-G2 cells (1–2 x 109) were collected and re-suspended in the fresh EMM2 to start the selection synchrony.
-ray & UV irradiations
-ray irradiation was performed, using a Cobalt 60 source at the dose rate of 17 Gy/min in the Institute of Kyoto University. To assay the number of survivals, cells were irradiated for the fixed time (30 min) with varying distances from the -ray source. For UV irradiation (254 nm, UV STRATALINKER 2400, Stratagene), cells were plated on EMM2 or EMM2-N agar plates (2 x 108 cells/10 x 14 cm2 plate). For PFGE analysis, -ray or UV irradiated cells were re-suspended into the EMM2-N or EMM2 liquid medium. For colony formation of G0 irradiated cells, an appropriate amount of NH4Cl solution was replenished directly to the EMM2-N plate.
PFGE
For PFGE, 1 x 108 vegetative cells and 2 x 108 G0 cells were collected and suspended in the ice-cold STOP-PBS buffer (291 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.5 mM KH2PO4, 10 mM EDTA, 50 mM NaF and 0.1% NaN3) and kept at 4 °C to stop DNA repair. DNA plug preparation was performed as described in the protocol provided by BIORAD (CHEF Genomic DNA plug Kits #170-3593) with modifications. The addition of Lyticase was found to be insufficient for preparing the DNA plug of G0 cells, so that zymolyase 100T (SEIKAGAKU, #120493) was instead used at the concentration of 0.5 mg/mL and a prolonged digestion was performed for 12 h at 37 °C. The CHEF Mapper system (BIORAD) was employed for electrophoresis with 0.8% Megabase agarose gel (BIORAD #161-3109) and 1X TAE buffer. Electrophoresis was done at 14 °C with the voltage of 2 V/cm using 3 blocks: 1st block was for 8 h at a 96° angle with 20 min of a switching time: 2nd block was for 8 h at a 102° angle with 25 min of a switching time: 3rd block was for 8 h at a 108° angle with 30 min of a switching time (Naito et al. 1998). Ethidium bromide stained gels were applied to obtain the images by the LAS-3000 (Fuji film).
Fluorescence microscopy
Light microscopy of S. pombe cells that expressed Crb2-YFP, Rad22-CFP or Ams2-GFP localization was done using a DeltaVision optical sectioning microscope model 283 (Applied Precision LLC) with a YFP/CFP filter set and a CH350L CCD camera (Photometrics). Images were taken with a x 60, 1.4 NA. objective lens (PlanApo, Olympus). Twelve Z-sections at 0.4 µm intervals were photographed, deconvoluted and projected into one image using soft-WoRx software. Time course observation of Ams2-GFP was done in 30 min intervals at 25–26 °C, while this integrant strain showed slightly longer cell length in G0 compared with wild type. The DNA content of single cells was measured by staining DNA with PI after methanol fixation and RNase A treatment.
Protein extraction
Total protein extracts were prepared by the TCA precipitation method (Nagao et al. 2004). Cells (1–2 x 108) were washed twice in 10% ice-cold TCA and kept at –30 °C freezer until all the samples were prepared. Cells were disrupted in 200 µL of 10% TCA containing 1 mM PMSF by glass beads using Multi Beads-Shocker (2700 r.p.m., 4 x 60 s disruption with 30 s intervals at 4 °C using the apparatus YASUI KIKAI MB601U(S)). After centrifugation (5800 g at 4 °C for 5 min), precipitates were dissolved in 1 x NuPAGE LDS sample buffer (Invitrogen). The pHs of each sample were adjusted to neutral by adding 2 M Tris-Cl (pH 9.5). After boiling them for 5 min at 95 °C, supernatants were retrieved as the extracts after centrifugation at 5800 g at room temperature for 5 min.
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Acknowledgements |
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-ray experiments in Kumatori. Tagged strains chk1-HA, cds1-HA and ams2-GFP are gifts from Drs N. Walworth, P. Russell and K. Takahashi. We thank all the members of the G0 Cell Unit for discussion and encouragement. The present study was supported by the JST (Japan Science and Technology Corporation) fund for the Initial Research Project (IRP) of Okinawa Institute of Science and Technology (OIST) that is set up by the Japanese Government. |
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* Correspondence: E-mail: yanagida{at}kozo.lif.kyoto-u.ac.jp
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Received: 1 September 2005
Accepted: 6 October 2005
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