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Vol. 16, Issue 4, 1651-1660, April 2005
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* Molecular & Cell Biology Laboratory, The Salk Institute, La Jolla, CA 92037;
Division of Biology, University of California, San Diego, La Jolla, CA 92093-0346; and
Molecular and Computational Biology Section, University of Southern California, Los Angeles, CA 90089-1340
Submitted October 27, 2004;
Accepted January 21, 2005
Monitoring Editor: Mark Solomon
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Recombination is initiated by DNA double strand breaks (DSBs) generated in replicated DNA by homologues of Spo11 (Keeney, 2001) and preferentially involve homologous chromosomes over sister chromatids (Schwacha and Kleckner, 1997). Thus, an unusual feature of meiosis is the deliberate generation and repair of DNA damage.
Conserved checkpoint responses recognize aberrant DNA structures and delay or arrest cell cycle progression until DNA breaks are repaired (Hartwell and Weinert, 1989; Weinert, 1998b; Nyberg et al., 2002). While even a single DNA break can activate checkpoint-mediated arrest (Sandell and Zakian, 1993), DNA breaks form normally during DNA replication and the threshold for checkpoint activation during S-phase is elevated relative to other stages of the cell cycle (Shimada et al., 2002a; Sogo et al., 2002). It is not clear whether meiotic DNA damage checkpoint thresholds are similarly elevated, either during meiotic S-phase or later in prophase when meiotic DSBs form.
Two DNA checkpoints have been identified in yeast meiosis. The first is a meiotic replication checkpoint (Murakami and Nurse, 1999), similar to the vegetative S-phase replication checkpoint, which stabilizes replication forks (Lopes et al., 2001; Tercero and Diffley, 2001) and restrains the firing of late replication origins (Santocanale and Diffley, 1998; Shirahige et al., 1998) in response to DNA damage (Paulovich and Hartwell, 1995) or nucleotide depletion by the ribonucleotide inhibitor hydroxyurea (HU) (Zhao et al., 1998) during S-phase. In the fission yeast Schizosaccharomyces pombe, both the vegetative and meiotic replication checkpoints are dependent on cds1+ (ScRAD53) (Murakami and Okayama, 1995; Boddy et al., 1998; Rhind and Russell, 1998) and the "checkpoint rad" genes rad1+, rad3+, rad9+, hus1+, rad17+, and rad26+ (al-Khodairy and Carr, 1992; Enoch et al., 1992; Lindsay et al., 1998; Murakami and Nurse, 1999).
Vegetative wild-type cells treated with HU arrest the cell cycle, and recover and complete replication when the drug is removed. In contrast, 
cds1 cells treated with HU undergo a lethal arrest in S-phase (Murakami and Okayama, 1995; Lindsay et al., 1998), while checkpoint rad mutants fail to arrest and enter an aberrant mitosis without completing DNA replication (Enoch et al., 1992). The failure of the replication checkpoint in vegetative cds1 cells exposed to HU causes DNA breaks as a consequence of replication fork collapse (Lopes et al., 2001), activating the chk1+ kinase (Lindsay et al., 1998). Chk1 is normally activated in a checkpoint rad-dependent manner in response to DNA damage in G2 phase of the cell cycle, preventing entry into mitosis (al-Khodairy et al., 1994; Walworth and Bernards, 1996; Martinho et al., 1998).
In contrast to the cell cycle, cds1 diploid cells blocked in meiosis with HU proceed with meiotic divisions after only a short delay, indistinguishable from diploid checkpoint rad mutants under similar conditions (Murakami and Nurse, 1999). This difference from the behavior of vegetative cells suggests that the chk1+-dependent response to replication fork collapse may be attenuated or inactive during meiosis (Murakami and Nurse, 1999). Curiously, haploid cds1 and rad3 cells induced to enter meiosis undergo prolonged arrest when treated with HU (Forsburg and Hodson, 2000). This raises the possibility that the absence of homologous chromosomes triggers an alternative rad3+- and cds1+-independent checkpoint, perhaps involving Chk1 or other kinases, in response to the DNA damage caused by the failure of the replication checkpoint.
The second characterized meiotic checkpoint is a recombination checkpoint that monitors defects in the processing of meiotic recombination intermediates in pachytene (Roeder and Bailis, 2000). Fission yeast defective in homolog pairing undergo a modest delay in prophase (Nabeshima et al., 2001). This delay, which can also be induced by ionizing radiation (IR) in prophase (Shimada et al., 2002b), is cds1+, mek1+, and checkpoint rad dependent (Shimada et al., 2002b; Perez-Hidalgo et al., 2003). However, fission yeast cells completely deficient for recombination repair can still proceed with meiotic divisions (Catlett and Forsburg, 2003), suggesting that the recombination checkpoint response in fission yeast does not permanently arrest meiotic divisions in the presence of DNA damage. This is in contrast to budding yeast recombination and homolog pairing mutants, which arrest permanently in prophase in a ScMEK1-dependent manner (Lydall et al., 1996; Xu et al., 1997), and suggests that fission yeast meiosis is particularly tolerant of DNA damage.
To investigate checkpoint responses to DNA damage during fission yeast meiosis in greater detail, we examined the effects of DNA damage from a variety of sources on meiotic progression in wild-type and checkpoint deficient fission yeast strains. We show that DNA damage during meiotic S-phase does not delay meiosis in a checkpoint-dependent manner. We also show that Cds1 and Chk1 proteins are not additionally phosphorylated in response to DNA damage early in meiosis. Further, we find that fission yeast can assemble MI spindles in the presence of Rhp51 foci, confirming that certain forms of ongoing repair do not cause arrest in meiotic prophase, in contrast to observations in budding yeast (Lydall et al., 1996). In addition, we observe that even unperturbed cds1 strains sustain elevated levels of spontaneous meiS-phase damage, which is adequately repaired by recombination before the completion of meiosis, suggesting that ongoing damage repair can occur during meiosis in the absence of checkpoint arrest. Taken together, these results show that damage thresholds for checkpoint activation in fission yeast meiosis are substantially elevated relative to thresholds during the cell cycle, such that cells do not delay or arrest their meiotic divisions in response to DNA damage.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Matings were performed on synthetic sporulation agar (SPAS) (Gutz et al., 1974). Rad3ts strains were derivatives of TC1669 and TC1670 (Martinho et al., 1998). Chk1-HA strains were derivatives of NW223 (Walworth and Bernards, 1996). rec12 strains were derivatives of GP1456 and GP1459 (Lin and Smith, 1994).
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Synchronous Meiosis
Synchronous haploid meiosis was performed in the pat1–114 genetic background essentially as described (Iino and Yamamoto, 1985b, a), except that starved cultures were refed at 34°C in the presence or absence of hydroxyurea (HU) (Sigma), methylmethane sulfonate (MMS) (Sigma) or camptothecin (CPT) (Calbiochem) as appropriate. FACS analysis and nuclei counts were performed as described (Forsburg and Hodson, 2000). Haploid strains used in meiotic timecourses were as follows: wild-type (FY2057), cds1 (FY2182), chk1 (FY2184), rad3 (FY2186), tel1 (FY2188), mek1 (FY2386), cds1 mek1 (FY2806), tel1 mek1 (FY2807), rad3ts (FY2808), rad3ts tel1 (FY2809).
For synchronous diploid meiosis, we generated diploid strains expressing mat1-Pc, mat1-Mc, and mat1-Mi, but not mat1-Pi, in the pat1–114 background using the mat2–102 allele (Kelly et al., 1988; Willer et al., 1995; Yamamoto and Hiraoka, 2003). The following haploid strains were cross-ed to generate fresh diploids for each timecourse: FY2004 x FY2057 (wild-type), FY2245 x FY2182 (cds1), FY2246 x FY2184 (chk1), FY2247 x FY2186 (rad3), FY2248 x FY2188 (tel1), and FY2322 x FY2386 (mek1). Strains were cross-ed on SPAS for 12–16 h at 25°C and selected for intragenic complementation at 25°C on EMM plates lacking supplemental adenine (EMM -ade). White ade+ colonies were streaked to YES + 5 µg/ml phloxin B and incubated 3–5 d at 25°C. Starter cultures in liquid EMM -ade were inoculated with large pink colonies and incubated 2–3 d at 22–25°C. 300 µl of cells were fixed in 70% ethanol and analyzed by FACS as described (Liang et al., 1999). Cultures with prominent 2C and 4C peaks and a minimum of sub-1C particles were used to inoculate 10–150 ml batch cultures in EMM -ade. Meiotic timecourses were performed as described for haploid meiosis (Forsburg and Hodson, 2000), except that EMM -ade is used for washes and overnight starvation.
Integration of Cds1-HA
A Cds1 expression construct C-terminally tagged with hemeagglutinin (3 x HA) (gift of G. Brown) was excised by XhoI and SalI digestion from pSLF272 (Forsburg and Sherman, 1997) and cloned into the XhoI and SalI sites of pJK210 (Keeney and Boeke, 1994). The construct was linearized in Cds1 by NheI digestion and transformed into FY527 (h-his3-D1 ura4-D18 leu1–32 ade6-M216) at the endogenous locus by electroporation (Kelly et al., 1993) to produce FY2244 (Table 1). Single copy insertion in ura+ transformants was confirmed by Southern blot and protein expression confirmed by western blot (not shown).
Protein Extraction and Western Blot Analysis
Diploid strains for analysis of Cds1 and Chk1 phosphorylation during mitosis and synchronous meiosis were generated from the following parental strains: FY2812 x FY3117 (Cds1-HA), FY2812 x FY2371 (Chk1-HA), FY2245 x FY2856 (cds1 Chk1-HA). Proteins were isolated by denaturing trichloroacetic acid extraction (Caspari et al., 2000) as described (Catlett and Forsburg, 2003). 7.5 µg (Cds1-HA strains) or 15 µg (Chk1-HA strains) total protein was separated on 10% SDS-PAGE gels run for 16 h at 4°C. Proteins were transferred to Immobilon-P membrane in 10 mM CAPS pH 11, 10% methanol for 1 h at 45V, blocked for 1 h at room temperature in Tris-buffered saline + 1% Tween 20 + 5% nonfat milk and HA-tagged proteins detected by overnight incubation with mouse anti-HA antibody HA.11 (Covance) at 1:2500 dilution. PCNA was detected using mouse anti-PCNA antibody PC10 (Santa Cruz Biotechnology) at 1:2000 dilution. Primary antibodies were detected by overnight incubation with HRP-conjugated anti-mouse secondary antibody (Jackson Immunoresearch) at 1:2000 dilution followed by chemiluminescent detection (ECL+, Amersham).
Immunofluorescence
Synchronous meiotic timecourses were sampled and meiotic chromosome spreads prepared as described (Hodson et al., 2003). Heat-fixed spread preparations on glass slides were blocked for 1 h in 100 µl phosphate-buffered saline (PBS)/10% calf serum/0.05% NaN3 (PCA) at room temperature under coverslips in a humidified chamber, followed by overnight incubation with either rabbit polyclonal antiphospho-H2A.X (Upstate) at 1:50 dilution or rabbit polyclonal anti-Rhp51 (Catlett and Forsburg, 2003) at 1:100 dilution in PCA. Slides were washed briefly in PBS followed by incubation for two hours with chicken anti-rabbit Alexa Fluor 488 (Molecular Probes) at 1:250 dilution in PCA. Slides were washed again briefly in PBS, air-dried vertically in the dark, and mounted in medium (1 mg/ml p-phenylenediamine, 1 mg/ml n-propyl gallate, 10% PBS, 90% glycerol, pH9.0) + DAPI (0.66 µl of 1 mg/ml DAPI in dimethyl sulfoxide per ml of mount medium) under coverslips sealed with nail polish. Immunofluorescence on spreads was visualized using a Leica DMR microscope with a Hamamatsu CCD camera and images were captured using Openlab (Improvision) software.
For MI spindle assembly assays, 5 x 106 diploid cells fixed in 70% ethanol were washed twice in 500 µl PBS with gentle spinning (1000g for 2 min) and resuspended by inversion in 0.5 mg/ml Zymolyase 20T in PBS. Cells were incubated for 10 min at room temperature, checked for spheroplast formation, washed twice in PBS and pipetted onto poly-L-lysine coated glass slides. Cells were incubated for 10 min, supernatant removed by pipetting and adherent spheroplasts heat fixed at low heat until remaining liquid evaporated (1–2 min). Slides were blocked for 1 h in PCA and incubated simultaneously overnight with mouse monoclonal antitubulin antibody 4A1 (a kind gift of Richard McIntosh) at 1:50 dilution and rabbit polyclonal anti-Rhp51 at 1:500 dilution in PCA. Slides were washed in PBS and incubated for 2 h with chicken anti-mouse Alexa Fluor 594 (Molecular Probes) and goat anti-rabbit Oregon Green 488 (Molecular Probes) at 1:250 dilution in PCA. Slides were washed in PBS, air-dried vertically in the dark, and mounted in mount medium + DAPI. Cells were visualized as described.
Recombination and Spore Viability Assays
Sister chromatid exchange (SCE) during meiosis was assayed at the ade6 locus using the ade6-L469/pUC8/his3+/ade6-M375 (Osman et al., 2000) and ade6-M375-M210 alleles as described (Catlett and Forsburg, 2003), except that spores were plated on YES to first assay viability and replica plated to EMM -ade to assay adenine prototrophy. Strains crossed to generate spores for the SCE assay were FY2314 and FY2321 (wild-type), FY2376 and FY2479 (cds1), FY2377 and FY2480 (chk1), FY2378 and FY2481 (rad3), and FY2379 and FY2482 (tel1). Spore viability assays were performed by tetrad dissection as described (Catlett and Forsburg, 2003), except spores were grown on YES for 5 d at 25°C following microdissection. Strains crossed for spore viability assays were FY12 and FY13 (wild-type), FY2189 and FY2190 (cds1), FY2191 and FY2192 (chk1), FY2193 and FY2194 (rad3), FY2195 and FY2196 (tel1), FY3073 and FY3074 (rec12), FY2925 and FY2926 (cds1 rec12), FY2928 and FY2929 (chk1 rec12), FY2931 and FY2932 (rad3 rec12), FY3077 and FY3078 (tel1 rec12). P values were calculated in Statview (SAS Institute) by unpaired t test; a p-value 
0.05 is considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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cds1 and rad3 mutants treated with HU during meiosis will proceed with abnormal meiotic divisions after a short delay (Murakami and Nurse, 1999). In contrast, haploid cds1 and rad3 mutants treated with HU during meiosis show a prolonged arrest of divisions (Forsburg and Hodson, 2000). We speculated that this difference in arrest phenotype was due to the difference in ploidy of the strains tested (Forsburg, 2002). To test this, we repeated the meiotic replication block experiments using isogenic diploid and haploid strains undergoing synchronous, pat1–114 driven meiosis (Iino and Yamamoto, 1985b, a) (Figure S1A). In agreement with the published work (Murakami and Nurse, 1999; Forsburg and Hodson, 2000), we observe that diploid cds1 and rad3 strains released into meiosis in the presence of HU showed a short, 
1 h delay before proceeding into abnormal meiotic divisions while cds1 and rad3 haploids under similar conditions reproducibly displayed a substantially prolonged delay of divisions (Figure S1B). To determine whether other known checkpoint proteins play a role in restraining meiotic divisions when homologues are absent, we examined meiotic progression in mek1 and tel1 mutants as well. We observed no substantial alterations in meiotic progression in mek1 and tel1 single mutant haploids (Figure S1C) or diploids (data not shown) relative to wild-type, in the presence or absence of HU. Furthermore, meiotic divisions in cds1 mek1, rad3ts tel1 (Figure S1C), or tel1 mek1 double mutant haploids (data not shown) exposed to HU are not accelerated relative to cds1 and rad3ts single mutants, suggesting that Mek1 and Tel1 do not play a role in restraining meiotic progression in haploid fission yeast under conditions of replication checkpoint failure. Nevertheless, these results confirm our hypothesis that homologous chromosomes influence meiotic progression by an unknown mechanism under conditions of irreversible replication fork collapse. To further our understanding of the normal meiotic response to DNA damage, we next examined the response of meiotic diploids to DNA damage from a variety of sources, including damage induced by exogenous chemical agents, the failure of the replication checkpoint, and spontaneous damage arising during meiotic S-phase.
DNA Damage Does Not Induce a Checkpoint Dependent Delay of Meiotic Divisions
Vegetative cds1 cells treated with HU arrest cell cycle progression in a chk1+ -dependent manner (Lindsay et al., 1998), which contrasts with the lack of arrest observed in cds1 diploids treated with HU during meiosis (Murakami and Nurse, 1999) (Figure S1). This suggests that the chk1+-dependent response to DNA damage caused by the collapse of replication forks (Lopes et al., 2001) may be attenuated during fission yeast meiosis. To examine the meiotic response to other forms of DNA damage in S-phase, we examined meiotic progression in diploid cells exposed to exogenous, S-phase specific DNA damaging agents. The DNA topoisomerase I inhibitor camptothecin (CPT) causes double strand break (DSB) formation in the presence of active replication forks (Wan et al., 1999). Wild-type cells released into synchronous meiosis in the presence of 10 µM CPT, a concentration sufficient to activate Chk1 and block mitosis in cycling cells (Wan et al., 1999), show only a slightly delayed entry into MI relative to untreated cells (45% mononucleates in CPT-treated cells at 5 h vs. 10% mononucleate cells in untreated wild-type, Figure 1A). However, a substantial increase in aberrant chromosome segregation is observed under these conditions (Figure 1B), suggesting that treatment with CPT early in fission yeast meiosis causes persistent DNA damage to chromosomes. A similar delay of meiotic divisions occurred in chk1 mutants treated with CPT as well as cds1, rad3, tel1 and mek1 cells, relative to wild-type (Figure 1A and data not shown). These data suggest that the minor CPT-induced meiotic delay we observe is not checkpoint-dependent, and that this level of CPT causes damage to chromosomes which results in aberrant segregation later in meiosis. Thus, DSB formation in S-phase induced by CPT does not restrain meiotic divisions in a checkpoint-dependent manner, suggesting that the DNA damage response to DSBs is attenuated during fission yeast meiosis.
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The DNA alkylating agent methylmethane sulfonate (MMS) slows progression through vegetative S-phase in a checkpoint-independent manner and causes S-phase dependent lethality in replication checkpoint mutants (Tercero and Diffley, 2001). Checkpoint mutants released into synchronous meiosis in the presence of 0.01% MMS undergo delayed, abnormal meiotic divisions at a similar rate (chk1 and tel1) or slightly accelerated rate (cds1 and rad3) relative to wild-type (Figure 1A and data not shown). Accelerated divisions in cds1 or rad3 cells is accompanied by accelerated DNA synthesis in MMS in these mutants, as assayed by FACS analysis (data not shown); this may be due to the inappropriate firing of late origins in these mutants (Tercero and Diffley, 2001) and makes the accelerated divisions we observe under these conditions (Figure 1A) difficult to interpret. Nevertheless, these data suggest that some forms of DNA damage can cause a delay of meiotic divisions in fission yeast. However, the extent of the delay appears dependent on the nature and extent of the damage and occurs independently of a classic checkpoint arrest response.
Chromosome Missegregation Induced by DNA Damage Is not Elevated in Checkpoint Mutants
In addition to preventing cell division, checkpoints also activate repair (Weinert, 1998a; Nyberg et al., 2002). If a meiotic checkpoint response normally activates the repair of damage without delaying meiotic divisions, then a repair defect in checkpoint mutants might result in increased aberrant chromosome segregation as mutant cells enter divisions with unrepaired lesions. To test this possibility, we examined the relative frequencies of aberrant nuclear morphology as compared with normal morphology ten hours after meiotic induction in checkpoint mutants and wild-type. In untreated cells, a modest increase in aberrant chromosome segregation is observed in checkpoint mutants relative to wild-type (Figure 1B), however checkpoint mutant spore viability is very similar to wild-type in untreated cells (Figure 4C), suggesting that this modest increase in aberrant chromosome segregation does not lead to a substantial loss of meiotic viability. Cells treated with CPT show elevated levels of aberrant segregation which occurs to a similar extent in checkpoint mutants and wild-type (Figure 1B), suggesting that checkpoint proteins are not important for promoting the repair of DSBs induced during meiotic S-phase by this drug. In contrast, MMS treated checkpoint mutants show a subtle increase in abnormal chromosome segregation relative to similarly treated wild-type cells (Figure 1B), suggesting that these checkpoint proteins might be important for either the prevention or repair of damage induced by MMS. While these data raise the possibility that checkpoint proteins may play a role in the activation of meiotic damage repair, the effect is subtle and occurs independent of a canonical checkpoint arrest response.
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Cds1 and Chk1 Are not Activated by DNA Damage during Meiosis
Cds1 is activated in vegetative cells exposed to ionizing radiation in S-phase (Lindsay et al., 1998) and is required for S-phase delay in response to UV (Rhind and Russell, 1998) or MMS (Marchetti et al., 2002). We examined Cds1 phosphorylation, a marker of Cds1 activation, in response to DNA damage during meiotic S-phase in wild-type diploids (Figure 1C). Cds1 protein levels increase and show a modest electrophoretic shift early in meiosis (Figure 1C), as previously reported (Shimada et al., 2002b). We observe no additional shift in meiotic Cds1 mobility in response to levels of the radiomimetic drug bleomycin sufficient to arrest vegetative cells (Rhind and Russell, 2001) or levels of MMS sufficient to induce a 1-h delay of meiotic S-phase (Figure 1C and data not shown) and aberrant chromosome segregation in wild-type meiotic cells (66% aberrant asci in 0.005% MMS vs. 12% in untreated cells). We observe substantially increased expression and phosphorylation of Cds1 in HU-treated meiotic cells relative to untreated meiotic cells (Figure 1C), consistent with a Cds1-dependent meiotic replication checkpoint response (Murakami and Nurse, 1999) (Figure S1). These results show that DNA damaging agents do not result in an increased expression or upregulation of Cds1 protein during meiosis analogous to that observed in response to HU.
We next monitored the electrophoretic mobility of Chk1 protein, which is activated by DNA damage in G2 phase (al-Khodairy et al., 1994; Walworth and Bernards, 1996; Martinho et al., 1998) and in cds1 cells under replication block in S-phase (Lindsay et al., 1998). There was very little shifted Chk1 visible in meiotic wild-type cells treated with bleomycin or MMS or in meiotic cds1 cells treated with HU (Figure 1C, right panel). In contrast, vegetative wild-type cells treated with MMS or vegetative cds1 cells treated with HU (Figure 1C; asyn. +MMS, cds1 asyn. +HU lanes) show a dramatic alteration in the mobility of a subset of Chk1 protein. Thus, Chk1 phosphorylation in response to DNA damage during meiosis appears severely attenuated relative to mitotic responses. These results are consistent with the absence of checkpoint-dependent delay of meiosis in response to damage (Figure 1A), and suggests that Cds1- and Chk1-dependent damage responses during fission yeast meiosis are insensitive to levels of damage sufficient to activate vegetative checkpoints.
Meiotic Divisions Can Occur in the Presence of Rhp51 Foci
If DNA damage during meiotic S-phase in S. pombe does not activate checkpoint delay, repair may not initiated or completed before cells proceed with meiotic divisions. To test whether DNA damage induced in meiotic S-phase is repaired by recombination before chromosome segregation or persists through the first meiotic division (MI), we examined whether MI spindles assemble in the presence of Rhp51 foci during diploid meiosis. rhp51+ is the fission yeast homolog of ScRAD51, which is a marker of single-stranded DNA intermediates in the repair of damage by homologous recombination (HR) (Lydall et al., 1996; Caspari et al., 2002). In S. cerevisiae, checkpoint mutants assemble MI spindles in the presence of Rad51 foci and undergo meiotic divisions in the presence of incomplete recombination while wild-type cells do not (Lydall et al., 1996).
In marked contrast to observations in budding yeast, we observe that greater than 25% of wild-type S. pombe cells assemble MI spindles in the presence of Rhp51 foci, while 34% of untreated cds1 and 39% of untreated chk1 cells show Rhp51 foci on assembled MI spindles. More strikingly, >86% of wild-type cells undergoing meiosis in the presence of 0.01% MMS show Rhp51 foci in cells with assembled MI spindles, as well as the majority of cds1 cells in HU (Figure 2), indicating that the population of cells with Rhp51 foci on assembled spindles increases under conditions of DNA damage. We propose that fission yeast cells undergo at least the first meiotic division in the presence of DNA breaks or incomplete recombination structures as marked by Rhp51 foci. Our observation that the majority of cells are viable, despite the presence of Rhp51 foci at MI, suggests that damage is repaired subsequent to meiosis I or that Rhp51 foci do not represent actual DNA breaks. Nevertheless, these results strongly suggest that S-phase damage does not activate a checkpoint arrest either during S-phase or later, during meiotic prophase, and demonstrates that fission yeast meiosis is particularly tolerant of DNA damage.
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cds1 and rad3 Mutants Sustain Elevated Levels of Spontaneous Damage during Meiotic S-phase
Spontaneous DNA damage can occur upon passage through a normal S-phase (Zou and Rothstein, 1997). In S. cerevisiae, the cds1+ homolog RAD53 and the rad3+ homolog MEC1 are implicated in replication fork stability (Lopes et al., 2001; Cha and Kleckner, 2002). Increased levels of Rad52 foci, a marker for DNA repair, are observed in mec1 mutants during S and G2 phase (Lisby et al., 2001), raising the possibility that endogenous levels of spontaneous S-phase DNA breaks might be elevated in replication checkpoint mutants. Therefore, we examined cds1 and rad3 cells during meiotic S-phase and prophase for DSB formation by indirect immunofluorescence against phosphorylated histone H2A, a marker of DNA breaks (reviewed in Pilch et al., 2003). In S. pombe, the two H2A homologues Hta1 and Hta2 are phosphorylated in a rad3+- and tel1+-dependent manner in response to ionizing radiation, and are required for the maintenance of checkpoint arrest (Nakamura et al., 2004). Phospho-H2AX formation in mammalian (Mahadevaiah et al., 2001) and yeast meiosis (Perera et al., 2004) has been correlated with Spo11 activity (Keeney et al., 1997), suggesting meiosis-specific DSB formation corresponds to the formation of phospho-H2A domains.
We compared the fraction of cells with phospho-H2A immunoreactivity during normal meiotic S-phase and prophase in cds1, rad3, and wild-type strains (Figure 3). We found that 73% of chromosome spreads from cds1 cells in meiotic S-phase show phospho-H2A immunoreactivity compared with 44.6% in wild-type (Figure 3A and B, 2 h). Significantly, immunoreactive cds1 spreads tend to show larger domains of phospho-H2A phosphorylation than wild-type (17.7% ± 4% of spreads with <50% area immunoreactive in cds1 vs. 0.3% ± 0.6% in wild-type) (Figure 3B). These domains decrease in signal intensity after S phase is complete in both untreated wild-type and cds1 cells (Figure 3B, 3 h), suggesting repair during meiotic prophase. Similarly large domains of phospho-H2A immunoreactivity are also observed in a subset of spreads from untreated rad3 cells (Figure 3B). Because progression through meiosis is similar in wild-type and mutant strains (Figure 1 and data not shown), this suggests that an increased rate of DNA damage occurs in checkpoint mutants even during an unperturbed meiosis.
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Under replication block in HU, cds1 and rad3 cells accumulate larger and more persistent phospho-H2A domains than wild-type (Figure 3A). This correlates with the irreversible replication fork collapse and DNA damage thought to occur in replication checkpoint mutants in HU (Lindsay et al., 1998; Lopes et al., 2001) and suggests that extensive Tel1-dependent H2A phosphorylation occurs during diploid meiosis. Thus, cds1 and rad3 cells accumulate increased levels of spontaneous DNA damage during a normal meiotic S-phase, as well as under replication block in HU.
Checkpoint Mutants Show Increased Meiotic Recombination
Recombination intermediates increase during S-phase, suggesting that S-phase DNA lesions can be repaired by recombination (Zou and Rothstein, 1997). Meiotic recombination, which is initiated by Spo11-dependent DSB formation (Keeney et al., 1997), occurs preferentially between homologous chromosomes and is required for chromosome disjunction at MI (Schwacha and Kleckner, 1997). Radiation-induced DNA damage can partially rescue the viability defect in spo11 mutants (Thorne and Byers, 1993; Dernburg et al., 1998), suggesting that some forms of DNA damage in meiosis can become a substrate for meiotic recombination. Therefore, we reasoned that the elevated spontaneous S-phase breaks we observe in checkpoint mutants might be repaired in part by homologous recombination during meiosis.
To investigate this, we examined whether checkpoint mutants rescue spore viability in cells lacking the S. pombe SPO11 homolog rec12+ (Sharif et al., 2002). By tetrad dissection we observed a statistically significant elevation in spore viability in the progeny of cds1 rec12 (52.5% ± 16.2%), chk1 rec12 (48.6% ± 9.9%), and rad3 rec12 (45.0% ± 5.9%) double mutants relative to rec12 single mutant parentals (25% ± 6.8%); spore viability in tel1 rec12 double mutants (30.3% ± 14.4%) is not statistically elevated relative to rec12 (Figure 4A). These data suggest that the spontaneous S-phase DNA breaks we observe in cds1 and rad3 checkpoint mutants can become a substrate of meiotic recombination, and that spontaneous DNA damage is also increased in chk1, but not tel1 mutants.
While meiotic recombination occurs preferentially between homologous chromosomes, an increase in ectopic sister chromatid exchange events is observed in some recombination as well as checkpoint mutants during meiosis (Grushcow et al., 1999; Catlett and Forsburg, 2003), suggesting either damage repair or a direct involvement of checkpoint proteins in regulating recombination partner choice (Grushcow et al., 1999). We examined sister chromatid exchange (SCE) events at the ade6 locus using a nontandem heteroallelic duplication (ade6-M375int::pUC8/his3+/ade6-L469) as an intra- and interchromosomal recombination substrate (Osman et al., 2000; Catlett and Forsburg, 2003). SCE is significantly elevated in tel1 and cds1 and mildly elevated in chk1 and rad3 mutants (Figure 4B), suggesting that some of the DNA damage incurred in cds1 mutants (and perhaps chk1 and rad3 as well) is repaired by sister chromatid recombination. As these increases in recombination occur without a substantial alteration in spore viability (Figure 4C) (Shimada et al., 2002b), this result suggests that the levels of spontaneous damage we observe in checkpoint mutants are adequately repaired during the course of meiosis. Therefore, we suggest that loss of DNA checkpoint proteins during meiosis leads to an increase in spontaneous DNA damage which is adequately repaired by recombination by the time meiosis is complete. This repair occurs without slowing meiotic progression, further evidence that DNA damage does not trigger checkpoint-dependent arrest during fission yeast meiosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), raising the possibility that fission yeast cells do not arrest meiotic divisions in response to unrepaired or unrepairable DNA damage. Consistent with this possibility, we show that extensive DNA breaks, as marked by phospho-H2A domains, accumulate in cds1 and rad3 cells blocked in meiosis with HU (Figure 3), conditions under which these cells proceed with abnormal divisions (Murakami and Nurse, 1999) (Figure S1). Similarly, we show that treatments with the DNA damaging agents camptothecin and MMS, which result in increased abnormal chromosome segregation (Figure 1B), also fail to activate a checkpoint-dependent arrest of meiosis, as monitored by meiotic divisions and checkpoint protein mobility (Figure 1). We also show that fission yeast assemble MI spindles in the presence of Rhp51 foci during a normal meiosis and with increasing frequency in the presence of elevated DNA damage (Figure 2), suggesting that fission yeast can proceed with at least the first meiotic division in the presence of recombination structures. Therefore, DNA damage from a variety of sources does not trigger checkpoint-mediated meiotic arrest in fission yeast, either in S-phase or later in meiotic prophase. This is in marked contrast to observations in budding yeast of checkpoint-dependent meiotic arrest in prophase (Lydall et al., 1996) in response to incomplete recombination (Xu et al., 1997), and may explain why some replication mutants in fission yeast do not cause meiotic arrest (Forsburg and Hodson, 2000).
It is not clear why DNA damage evades checkpoint activation during fission yeast meiosis. However, the absence of cross-over interference (Munz, 1994) and a classic synaptonemal complex (SC) (Bähler et al., 1993) in fission yeast meiosis raises the possibility that checkpoint monitoring of damage repair and recombination may be dispensable during a simplified eukaryotic meiosis. Importantly, linear elements containing homologues of several of the proteins found in the SC do form during fission yeast meiosis (Lorenz et al., 2004), indicating that conserved structural components are nevertheless required for efficient meiotic recombination (Molnar et al., 2003).
Studies in budding yeast suggest that checkpoint proteins promote recombination in the context of the SC and chromosome cores, where cross-over events are favored and recombination can be monitored (Xu et al., 1997; Grushcow et al., 1999). As the SC and chromosome cores form during meiotic prophase (Roeder, 1997), damage early in S-phase might occur before the formation of meiotic chromosomal structures. Evidence of increased DSB formation in the HIS4-LEU2 recombination hotspot in Scrad24 and Scrad17 mutants (Shinohara et al., 2003), and chromosome fragmentation (Cha and Kleckner, 2002) and increased Rad52 focus formation (Lisby et al., 2001) during S-phase in Scmec1 mutants raises the possibility that loss of checkpoint function in S. cerevisiae could lead to increased DSB formation early in meiosis. We observe increased spontaneous DNA breaks during meiotic S-phase in fission yeast cds1 (and to a lesser extent, rad3) checkpoint mutants by phospho-H2A staining (Figure 3), demonstrating that loss of checkpoint proteins can lead to elevated spontaneous DNA damage early in meiosis even in the absence of extraneous damage. This result suggests a possible nonessential role for checkpoint proteins during a normal meiotic S-phase.
If damage occurs before the formation of meiosis-specific chromosome structures, damage repair might show altered repair template and checkpoint surveillance characteristics. The increase in spontaneous DNA damage we observe in checkpoint mutants is repaired using both homologous chromosome and sister chromatid repair templates, as suggested by the partial rescue of rec12 spore viability, increased meiotic SCE, and high meiotic viability we observe in checkpoint mutants (Figure 4). Because S-phase damage from a variety of sources does not prevent chromosome segregation in MI, it is likely that the processing of spontaneous S-phase damage is not monitored by the recombination checkpoint; indeed, we see no evidence of meiotic delay in checkpoint mutants (Figure 1 and data not shown). Taken together, these data suggest that the partial rec12 rescue and elevated SCE we observe in some checkpoint mutants may be secondary to the repair of increased spontaneous S-phase damage.
Our data do not rule out a second, direct role for checkpoint proteins in the regulation of meiotic recombination, distinct from their role in preventing DNA damage in meiotic S-phase. Overall levels of meiotic gene conversion are modestly decreased in cds1+ and rad3+ checkpoint mutants (Shimada et al., 2002b), suggestive of an additional, conserved role for checkpoint proteins in the regulation of meiotic recombination. The mammalian rad3+ homolog ATR localizes to meiotic chromosome cores (Moens et al., 1999; Tarsounas and Moens, 2001) while the C. elegans cds1+ homologues Ce-CDS-1 and Ce-CDS-2 are required for meiotic crossing over (Oishi et al., 2001), suggesting that roles in regulating meiotic recombination may be evolutionarily conserved. Our initial analysis of tel1 strains show an increase in SCE without a concomitant increase in rec12 rescue (Figure 4). This could mean that Tel1 is directly involved in the regulation of meiotic recombination without influencing levels of spontaneous damage in meiotic S-phase, in contrast to our results for Cds1 and Rad3. The prophase I defects (Barlow et al., 1998) and infertility (Barlow et al., 1996) observed in ATM deficient mice also reinforces the conserved importance of the Tel1 homolog in meiotic progression. Our observation of phospho-H2A domains in rad3 cells in meiotic S-phase suggests a role for Tel1 signaling early in meiosis; however a recent study in mammals showed that DNA-PK can also phosphorylate H2AX after IR (Stiff et al., 2004). Future studies will determine the full extent of Tel1 functions during fission yeast meiosis.
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ACKNOWLEDGMENTS |
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rec12 strains GP1456 and GP1459, and A. Carr for the rad3ts strains TC1669 and TC1670. We thank J. Bailis for protocols, technical advice and extensive comments, M. Catlett for technical advice and J. Marlett and J. Hodson for technical assistance. This work was supported by the NIH (R01-GM59321 to S.L.F.) and the San Diego Foundation (to D.G.P.). |
Footnotes |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Present address: Oregon Health & Science University, NRC3, 3181 SW Sam Jackson Park Road, Portland, OR 97239.
Address correspondence to: Susan L. Forsburg (forsburg{at}usc.edu).
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-tubulin and Rhp51 was performed on ethanol fixed diploid cells released into synchronous meiosis in the presence or absence of 15 mM hydroxyurea (+HU) or 0.01% methylmethane sulfonate (+MMS). Cells with assembled MI spindles (tubulin, red) were examined for the presence of Rhp51 foci (green) on DNA (DAPI, blue). Representative MI cells with (wild-type +MMS, 
H2AX, green) was performed on chromosome spreads prepared hourly from diploid cultures released into synchronous meiosis in the presence or absence of 15 mM hydroxyurea (HU). DAPI staining for DNA is shown in blue; phospho-H2A localizes to DNA (merge). Representative spreads with less (wild-type, 2 h) or more (