INTRODUCTION

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Checkpoints maintain the integrity of the genome by ensuring the proper sequence of events during the cell division cycle (16). The dependency of later events in the cycle on the successful completion of earlier events prevents chromosome loss and missegregation leading to aneuploidy. Checkpoints also operate in meiosis, a specialized cell division that generates haploid gametes through two rounds of chromosome segregation.

Prior to the first meiotic division, homologous chromosomes pair, undergo high levels of genetic exchange, and become closely connected along their lengths by the synaptonemal complex (SC) (37). The SC consists of two lateral elements, corresponding to the individual chromosome cores, linked through a central region. Chromatin bridges, called chiasmata, form at the sites of recombination between homologs, and these connections persist after the SC has disassembled. Chiasmata ensure that chromosomes align on the meiosis I spindle such that homologs segregate to opposite poles at meiosis I. Correct reductional chromosome segregation also depends on synapsis (i.e., SC formation) between homologous chromosomes. The importance of recombination and synapsis to proper meiotic chromosome segregation is underscored by the existence of a checkpoint that monitors these processes and arrests cells at the pachytene stage of meiotic prophase in response to defects (4, 23, 26, 37, 51, 59).

Several yeast mutants delay or arrest at pachytene because of the checkpoint, including zip1, zip2, dmc1, and hop2 (4, 9, 23, 51). The zip1 mutant lacks a major component of the SC central region and arrests with homologously paired but unsynapsed chromosomes (51). zip1 mutant cells sustain wild-type levels of double-strand breaks (DSBs) (59), the initiators of meiotic recombination events; however, zip1 mutant cells are defective in processing double Holliday junctions into mature crossover products (49), and ~10% of DSBs remain unrepaired (49, 59). Like zip1, the zip2 mutant arrests in pachytene with paired but unsynapsed chromosomes (9). The Zip2 protein is thought to act at sites of synaptic initiation to promote Zip1 assembly (9). The dmc1 mutant lacks a meiosis-specific homolog of the Escherichia coli RecA protein (4); dmc1 mutant cells exhibit hyperresected 5' ends of DSBs (4) and defects in the progression from DSBs to double Holliday junctions (44). Also, chromosome synapsis is delayed in the dmc1 mutant (35). The hop2 mutant, like dmc1, is defective in DSB processing; in contrast to dmc1, however, hop2 mutant cells arrest with extensive synapsis between nonhomologous chromosomes (23). In the zip1, zip2, and dmc1 mutants, differences in yeast strain background determine whether cells arrest at pachytene or whether some cells complete sporulation after a delay in meiotic prophase progression (4, 9, 35, 49, 52).

Several observations indicate that the pachytene arrest of the zip1, zip2, dmc1, and hop2 mutants is due to the operation of a checkpoint rather than to a mechanical block in the meiotic cell cycle. First, the arrest of each of these mutants is alleviated by mutations that prevent the initiation of recombination and synapsis, and thereby prevent formation of the intermediates that trigger arrest (4, 8, 9, 23, 51). Second, pachytene arrest is bypassed by mutations in any one of several genes (RAD24, RAD17, MEC1) required to arrest the mitotic cell cycle in response to unrepaired DSBs and other types of DNA damage (26). Third, cells that arrest because of the pachytene checkpoint retain viability and can resume vegetative growth when returned to growth medium (4, 51).

Mutations in the meiosis-specific genes RED1, MEK1, and HOP1 allow zip1 to sporulate (59; K.-S. Tung and G. S. Roeder, unpublished data), although these mutations do not completely prevent the initiation of meiotic recombination (19, 31, 32). Red1 is a major component of SC lateral elements and the axial elements that serve as precursors to lateral elements (46). Hop1 colocalizes with Red1 to discrete sites on axial elements; however, Hop1 dissociates as these elements become incorporated into mature SCs (46). Mek1 is a meiosis-specific kinase that colocalizes with Red1 on meiotic chromosomes and phosphorylates Red1 (2, 11). Mek1-dependent phosphorylation of Red1 is required for wild-type levels of meiotic sister chromatid cohesion (2). Bypass of the zip1 arrest by deletion of RED1 or MEK1 led to the speculation that Red1 and Mek1 are required for the formation of a recombination structure that is monitored by the pachytene checkpoint machinery (59).

We have found that overproduction of either Red1 or Mek1, but not Hop1, suppresses checkpoint-induced arrest at pachytene. Red1 overproduction promotes nearly wild-type levels of sporulation in the zip1 mutant, whereas Mek1 overproduction promotes sporulation of a subset of zip1 mutant cells, after a delay. In each case, the checkpoint is inactivated without repairing all DSBs, suggesting that Red1 and Mek1 participate in checkpoint signaling. Cooverproduction of Red1 with Mek1 (or cooverproduction of Hop1 with either Red1 or Mek1) restores checkpoint function, indicating that the correct stoichiometry of these proteins is important for checkpoint function. In addition to inactivating the checkpoint, overproduction of Red1 decreases meiotic recombination in zip1 and in wild type and decreases homologous chromosome pairing in zip1. We speculate that Mek1 and Red1 function in checkpoint signaling and that alterations in Red1 phosphorylation allow defects in meiotic recombination and chromosome synapsis to escape detection by the checkpoint machinery.

	MATERIALS AND METHODS

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Plasmids. Plasmids were constructed by using standard protocols (39) and were propagated in E. coli XL1-Blue (Stratagene). pB133 contains the EcoRI-PvuII fragment containing MEK1 from pB124 (32) in YEp352 (18). pJ16 contains the EcoRI-SalI fragment containing MEK1 from pB124 in the EcoRI-SalI sites of the YEp351 polylinker (18). pJ17 contains the EcoRI-SalI fragment containing RED1 from pB86 (46) in the EcoRI-SalI sites of the YEp351 polylinker. The XbaI fragment containing MEK1 from pJ16 was inserted at the XbaI site of pB86 to generate pJ14, which is YEp352 containing both MEK1 and RED1. pJ66, which is YEp352 containing both RED1 and HOP1, was generated by cloning the NheI (filled in)-SalI fragment containing HOP1 from pNH83-2 (21) into the SphI (filled in)-SalI sites of pB86. The NheI (filled in)-SalI fragment containing HOP1 from pNH83-2 was inserted into the SphI (filled in)-SalI sites of pB133 (containing the EcoRI-PvuII fragment of MEK1 in YEp352) to generate pJ70, which is YEp352 containing both MEK1 and HOP1. pAV62 contains the XbaI-EcoRI fragment containing RED1 in YEplac112 (14). pB64 is the XbaI-EcoRI fragment containing RED1 from pB8 (31) in YCp50 (45). The following plasmids have been described: pL15 for hop2::LEU2 (23), pMB116 for zip1::LYS2 (52), pR976 for THR1 (42), pR978 for spo13::ADE2 (31), pJC303-4 for CENIII::URA3 (10), and pNKY422 for dmc1::URA3 (4).

Yeast strains. Table 1 lists the yeast strains used in this study. Strains were constructed and maintained by using standard procedures (45). Strains used for sporulation, nuclear division, gene conversion, and cytology are isogenic to BR2495 (31). Homozygous mutant strains were constructed by transforming the haploid parents of BR2495 (BR1919-8B and BR1373-6D [31]) with the appropriate plasmid(s) and then mating transformants. Disruptions were confirmed by Southern blot analysis (48). Wild-type and homozygous mutant diploids were then transformed with the appropriate multicopy or single-copy plasmids (see Table 1). BS223 and BS225 were constructed by first transforming haploids with pV180 (53) and then selecting for the red1::ura3-1 allele by plating haploids carrying the red1::URA3 and ura3-1 mutations on medium containing 5-fluoroorotic acid (7).

Sporulation and nuclear division. Three or four independent transformants from each strain were grown to saturation in 2 ml of 2× synthetic complete medium lacking either uracil or leucine. Cells (1.5 ml) were pelleted, resuspended in 2 ml of yeast extract-peptone-dextrose (45) supplemented with 60 µg of adenine per ml and 40 µg of uracil per ml, and grown for an additional 10 h. Samples (1.5 ml) of each culture were then collected, washed once with water, and resuspended in 10 ml of sporulation medium (2% potassium acetate) in 250-ml flasks. Cultures were incubated at 30°C with shaking. At the indicated time points, cells were analyzed for sporulation by light microscopy. Additionally, 180 µl of culture was removed at each time point and added to an Eppendorf tube containing 20 µl of 37% formaldehyde. After incubation at 4°C for 3 days, aliquots of each fixed culture were placed onto glass slides, allowed to air dry, washed in phosphate-buffered saline (39), and stained with 4'-6'-diamidino-2-phenylindole (DAPI). Nuclear division was assessed by fluorescence microscopy by using a Leica DMRB microscope. Sporulation and nuclear division were scored for 300 cells per culture per time point.

Recombination assays. Gene conversion was measured as the frequency of prototroph formation in heteroallelic diploids after 3 days of sporulation. For each strain, three or four independent transformants were grown and sporulated as described above. Recombinants were selected on solid medium lacking uracil and histidine, uracil and threonine, or uracil and tryptophan to select for meiotic gene convertants carrying the overexpression plasmid. The meiotic frequency of prototroph formation was calculated for each culture; average values for each strain were then calculated. Physical isolation of spores was performed as described (33). Reciprocal recombination was measured in dyads produced from spo13 mutants as described (30).

DSB assay. Strains were grown and sporulated as described above. After various time points in sporulation medium, cultures were harvested and analyzed by Southern blotting of pulsed-field gels (12). A DNA fragment containing THR4 (15) was labelled with ³²P with the Redi-Prime II kit (Amersham) and was used as probe. Signal intensity was calculated by using Multi-Analyst software for the Bio-Rad Imaging Densitometer (Bio-Rad). The intensity of meiotic DSBs (corresponding to fragments smaller than the intact chromosome III fragments) was analyzed and compared to the total intensity of chromosome III DNA (DSBs plus intact chromosome III). The discrete band that migrates faster than the intact chromosome III corresponds to the fragment from the end of the chromosome to the THR4 DSB hotspot; smaller fragments are generated by DSBs centromere distal to the THR4 hotspot.

Cytology. Meiotic nuclei were surface spread as described (9) and then incubated simultaneously with a chromosome III probe for fluorescent in situ hybridization (FISH) (9) and antibodies as described (23). Anti-Red1 antibodies (46) were used at a 1:100 dilution. For each strain, 50 spread nuclei that displayed anti-Red1 staining were scored for chromosome III pairing.

	RESULTS

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Overproduction of Red1 or Mek1 suppresses the zip1 sporulation defect. A screen for the Kluyveromyces lactis homolog of the ZIP1 gene identified the K. lactis RED1 gene as a high-copy-number suppressor of the zip1 sporulation defect (47). This observation raised the possibility that overexpression of the Saccharomyces cerevisiae RED1 gene might also allow the zip1 mutant to sporulate. To test this possibility, the RED1 gene was inserted into a plasmid containing the 2µm circle origin of DNA replication; such a plasmid is maintained in 20 to 50 copies per yeast cell (18). Overproduction of S. cerevisiae Red1 promotes wild-type levels of sporulation in the zip1 mutant in the BR2495 strain background (Fig. 1A; Table 2).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Sporulation of zip1 and dmc1 mutant strains overproducing Red1 or Mek1. (A) zip1 + Red1-OP, zip1 mutant overproducing Red1 (JM155); zip1 + Mek1-OP, zip1 mutant overproducing Mek1 (JM154); zip1, zip1 mutant carrying vector only (YEp352) (JM153); wild type, wild-type strain containing vector only (JM474). (B) dmc1 + Red1-OP, dmc1 mutant overproducing Red1 (JM161); dmc1 + Mek1-OP, dmc1 mutant overproducing Mek1 (JM160); dmc1, dmc1 mutant carrying vector only (YEp351) (JM159); wild type, wild-type strain containing vector only (JM152). Percent sporulation was calculated from triplicate cultures harvested at the times indicated; values shown are averages.

Overproduction of Red1 or Mek1 promotes sporulation of mutants that arrest at the pachytene checkpoint. To determine whether the overproduction phenotypes observed are specific to the zip1 mutant, the effect of Red1 or Mek1 overproduction was tested in other mutants that arrest at pachytene. Overproduction of either Red1 or Mek1 increases the sporulation frequency of the zip2 mutant (Table 3). In the dmc1 mutant, overproduction of either Red1 or Mek1 increases the rate of sporulation, but not the overall amount of sporulation (Fig. 1B; Table 3). The arrest of the hop2 mutant is partially suppressed by overproduction of Red1 or Mek1 (Table 3). In contrast, overproduction of Red1 or Mek1 does not promote sporulation of the sep1 mutant (data not shown), consistent with previous results indicating that sep1 arrest is triggered by signals different than those that activate the pachytene checkpoint (54). Mutants that arrest with meiotic recombination completed and chromosomes fully synapsed, such as top2 (38) and ndt80 (58), also do not sporulate when Red1 or Mek1 is overproduced (data not shown).

Overproduction of Red1, but not Mek1, decreases meiotic recombination. Most of the spores produced in zip1 mutant strains overproducing Red1 or Mek1 are inviable (Table 2), raising the possibility that recombination in these strains occurs at reduced levels. To test this possibility, both gene conversion and crossing over were measured in zip1 mutant strains overproducing Red1 or Mek1. Gene conversion was assayed by measuring prototroph formation in return-to-growth experiments, by using zip1 BR2495 strains. Crossing over was measured in the BR1919-8B strain background, in which the zip1 mutant sporulates, though sporulation is delayed and occurs with reduced efficiency relative to wild type. To improve the accuracy of recombination measurements, a spo13 mutation was introduced to improve spore viability. spo13 mutants undergo a single (predominantly equational) meiotic division to generate dyads containing diploid spores (22).

Overproduction of Red1 decreases DSBs. Red1 overproduction could decrease meiotic recombination either by reducing the number of recombination events initiated or by increasing the fraction of DSBs repaired by recombination between sister (rather than nonsister) chromatids. To distinguish these possibilities, DSB levels were assayed in strains carrying the rad50S mutation (1), which prevents DSB processing and results in the accumulation of broken DNA ends. DNA DSBs were analyzed by pulsed-field gel electrophoresis followed by Southern blotting and probing for DNA sequences from chromosome III; DSBs result in molecules that migrate faster than the intact chromosome (Fig. 2A). For each strain, the percent of total chromosome III DNA present in broken molecules was calculated; this frequency was then compared between strains containing either a multicopy RED1 plasmid or vector only (Fig. 2A).

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Effect of Red1 overproduction on DSB formation. (A) Mitotic (0 h) and meiotic (15 h) cells were analyzed for chromosome III DSBs by pulsed-field gel electrophoresis and Southern blotting. Mutant strains used were as follows: rad50S plus vector (JM446), red1 rad50S plus vector (JM442), zip1 rad50S plus vector (JM438), rad50S overproducing Red1 (Red1-OP) (JM447), red1 rad50S overproducing Red1 (JM443), and zip1 rad50S overproducing Red1 (JM439). DSBs migrate below the position of the full-length chromosome III. Both a discrete band (representing the fragment from the end of the chromosome to the THR4 hot spot) and fragments corresponding to the products of cleavage at other DSB sites are observed. (B) Quantitation of the results shown in panel A. % Meiotic DSBs, the amount of chromosome III DNA in broken molecules as a percent of the total amount of chromosome III DNA. Numbers in parentheses indicate the fold decrease in percent DSBs of strains overproducing Red1 compared to strains carrying vector only. (C) DSBs in rad50S strains carrying either vector (JM446) or overproducing Red1 (JM447) were analyzed at several time points during meiosis. Percent DSBs, the amount of chromosome III DNA in broken molecules as a percent of the total amount of chromosome III DNA.

Red1 overproduction alters homologous chromosome associations in zip1. In a wild-type strain, Red1 overproduction alters the Red1 localization pattern from semicontinuous to fully continuous along chromosome axes (Fig. 3A and B) (46). The Red1 protein localizes continuously along chromosomes in the zip1 mutant, even when Red1 is not overproduced (Fig. 3C) (46). To investigate the effects of Red1 overproduction on the morphology of zip1 chromosomes, meiotic chromosomes were surface spread and stained with anti-Red1 antibodies. In zip1 mutant strains, the Red1-stained chromosome cores (corresponding to homologous chromosomes) are closely apposed at a few sites, called axial associations (Fig. 3C); these are sites where chromosome synapsis is thought to initiate (9, 35). In contrast to zip1 mutant strains containing the normal amount of Red1 (Fig. 3C), the chromosomes in zip1 mutant strains overproducing Red1 appear disorganized and fragmented (Fig. 3D). Axial elements are not obviously paired in zip1 mutant cells overproducing Red1, and axial associations are less evident (Fig. 3D).

View larger version (92K): [in this window] [in a new window]   FIG. 3.   Red1 overproduction alters meiotic chromosome morphology. (A) Spread nucleus from a wild-type strain carrying the YEp352 vector (JM152). (B) Spread nucleus from a wild-type strain overproducing Red1 (BS354). (C) Spread nucleus from the zip1 mutant carrying the YEp352 vector only (JM153); arrows indicate examples of axial associations. (D) Spread nucleus from a zip1 mutant overproducing Red1 (JM155). Scale bar, 1 µm.

Some DSBs persist when the zip1 arrest is bypassed. If unrepaired DSBs persist in zip1 mutant cells that arrest because of the checkpoint, and if unrepaired DSBs can trigger checkpoint-mediated arrest, then overproduction of Red1 or Mek1 might promote zip1 sporulation either by masking the DSB signal or by allowing DSBs to be repaired. To investigate these possibilities, spread meiotic chromosomes were prepared from zip1 strains carrying vector or a multicopy plasmid containing RED1 or MEK1. By using a modification of the TUNEL assay (17), DSBs were labeled in situ and then detected by indirect immunofluorescence. DSBs in chromosome spreads were labeled with digoxigenin-tagged nucleotides by using Tdt, which specifically incorporates nucleotides onto free 3' hydroxyl ends of DNA (17). Incorporated digoxigenin-tagged nucleotides were detected by using antidigoxigenin antibody conjugated to rhodamine. The stage of the meiotic cell cycle was simultaneously monitored using antitubulin antibodies.

View larger version (39K): [in this window] [in a new window]   FIG. 4.   Most DSBs are repaired when the checkpoint is bypassed. Meiotic chromosomes were surface spread and then labeled in situ with Tdt (red) to detect DSBs. Antitubulin antibody (green) was used to visualize meiotic spindles (indicating that cells are no longer arrested at pachytene). Strains tested were as follows: (A) zip1 mutant carrying vector only (YEp352) (JM152), (B) zip1 mutant overproducing Red1 (JM155), (C) zip1 mutant overproducing Mek1 (JM154), (D) wild-type strain carrying vector only (YEp352) (JM152). Scale bar, 1 µm.

Cooverproduction of Red1 and Mek1 restores the checkpoint. Since Red1 and Mek1 interact with each other, overproduction of one of these proteins might impair checkpoint function by changing the stoichiometry of Red1 relative to Mek1. To test this possibility, zip1 mutant strains were transformed with a multicopy plasmid bearing both the RED1 and MEK1 genes. zip1 mutant strains overproducing both Red1 and Mek1 fail to sporulate, suggesting that the checkpoint is still active (Table 2). In principle, cooverproduction of Red1 and Mek1 might nonspecifically inhibit sporulation rather than restore checkpoint function. To address this possibility, sporulation was assayed in a wild-type strain in which Red1 and Mek1 are cooverproduced. In this control strain, sporulation occurs with wild-type kinetics and to wild-type levels, indicating that the multicopy plasmid carrying both MEK1 and RED1 is not deleterious to sporulation.

	DISCUSSION

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Imbalance of meiotic chromosomal proteins inactivates the pachytene checkpoint. Overproduction of Red1 or Mek1 specifically promotes sporulation of mutants that normally undergo checkpoint-mediated arrest at pachytene. The zip1, zip2, dmc1, and hop2 mutants all exhibit defects in both recombination and synapsis; however, the molecular signal that triggers arrest in these strains remains unknown.

Detection of meiotic DSBs. Meiotic DSBs are typically assayed by Southern blot analysis of restriction fragments or whole chromosomes (8, 12, 15, 50). Alternatively, the presence of meiotic DSBs has been assayed cytologically by using antibodies against the Rad51 protein (26). However, Southern blotting is not sensitive enough to detect very low levels of DSBs and cannot be used to assay DSBs in a subset of cells within a mixed population. Anti-Rad51 staining cannot be used to analyze rad51 mutant strains or other strains in which the Rad51 protein does not localize to chromosomes (13). In contrast, detection of meiotic DSBs by Tdt labeling is extremely sensitive and can be applied to all strain backgrounds.

Red1 overproduction antagonizes recombination in addition to suppressing pachytene arrest. Certain non-null alleles of RED1, such as red1-2 (30) and red1-DraI (B. Rockmill, A. V. Smith, and G. S. Roeder, unpublished data), decrease meiotic recombination below the level observed for the red1 null mutant. Our data indicate that excess Red1 also antagonizes meiotic recombination, both in the wild type and in the zip1 mutant. In addition, excess Red1 decreases recombination in a specific non-null mek1 mutant, mek1-974 (21). Thus, Red1 overproduction appears to antagonize recombination in strains of different genotypes. Our results suggest that the mechanism by which excess Red1 decreases recombination is different from the way in which deletion of RED1 decreases recombination. In the red1 null mutant, DSBs are decreased to ~10% of the wild-type level (44); however, in the red1 rad50S double mutant, DSBs are not decreased, indicating that rad50S is epistatic to red1 (59). In contrast, overproduction of Red1 decreases DSBs both in the wild type and in rad50S strains, suggesting that Red1 overproduction prevents DSB formation regardless of the status of RAD50.

Red1 and Mek1 as signals of meiotic prophase defects. Red1 overproduction might promote zip1 mutant sporulation by decreasing meiotic recombination to a level below that required to activate the checkpoint. However, the following observations argue against this interpretation. First, a fourfold reduction in DSBs results in ~60 DSBs per cell. In mitotic cells, a single unrepaired DSB is sufficient to trigger cell cycle arrest (40). Furthermore, wild-type meiotic cells do not sporulate until all DSBs have been repaired. Second, bypass of zip1 by Mek1 overproduction cannot be explained by a decrease in meiotic recombination.

View larger version (32K): [in this window] [in a new window]   FIG. 5.   Model for bypass of checkpoint-mediated arrest by overproduction of Red1 or Mek1. (A) In the zip1 mutant, continued Red1 phosphorylation serves as an inhibitory signal that prevents pachytene exit. (B) When Red1 is overproduced in the zip1 mutant, the excess Red1 protein may be inefficiently phosphorylated, resulting in little or no detectable signal to the checkpoint. (C) Overproduction of Mek1 in the zip1 mutant may result in excess or inappropriate phosphorylation of Red1, such that Red1 is not properly detected by the checkpoint machinery. (D) Cooverproduction of Mek1 and Red1 restores the appropriate phosphorylation of Red1, allowing proper checkpoint function. (E) Cooverproduction of Hop1 and Red1 may promote interaction between Red1 and Mek1, leading to Red1 phosphorylation. (F) Cooverproduction of Hop1 and Mek1 also may promote correct phosphorylation of Red1. P, phosphate group; R, Red1 overproduction; M, Mek1 overproduction; H, Hop1 overproduction.