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Mol Cell Biol. 2010 April; 30(7): 1570–1581.
Published online 2010 February 1. doi:  10.1128/MCB.00919-09
PMCID: PMC2838064

Roles of Hop1 and Mek1 in Meiotic Chromosome Pairing and Recombination Partner Choice in Schizosaccharomyces pombe[down-pointing small open triangle]

Abstract

Synaptonemal complex (SC) proteins Hop1 and Mek1 have been proposed to promote homologous recombination in meiosis of Saccharomyces cerevisiae by establishment of a barrier against sister chromatid recombination. Therefore, it is interesting to know whether the homologous proteins play a similar role in Schizosaccharomyces pombe. Unequal sister chromatid recombination (USCR) was found to be increased in hop1 and mek1 single and double deletion mutants in assays for intrachromosomal recombination (ICR). Meiotic intergenic (crossover) and intragenic (conversion) recombination between homologous chromosomes was reduced. Double-strand break (DSB) levels were also lowered. Notably, deletion of hop1 restored DSB repair in rad50S meiosis. This may indicate altered DSB repair kinetics in hop1 and mek1 deletion strains. A hypothesis is advanced proposing transient inhibition of DSB processing by Hop1 and Mek1 and thus providing more time for repair by interaction with the homologous chromosome. Loss of Hop1 and Mek1 would then result in faster repair and more interaction with the sister chromatid. Thus, in S. pombe meiosis, where an excess of sister Holliday junction over homologous Holliday junction formation has been demonstrated, Hop1 and Mek1 possibly enhance homolog interactions to ensure wild-type level of crossover formation rather than inhibiting sister chromatid interactions.

Sexual reproduction in eukaryotes involves formation of haploid gametes from diploid cells by one round of DNA replication, pairing of the homologous chromosomes, and recombination and then by the two meiotic divisions (53). In fungi the gametes differentiate into haploid spores, which germinate to form vegetative cells. Crossover (CO) formation between homologous chromosomes and DNA repair processes between sister chromatids are required for spore viability (10, 55, 58).

In vegetative cells homologous recombination (HR) is important for repair of DNA damage and stalled replication forks, with the sister chromatid as the preferred partner (28). Many of the enzymes involved in mitotic HR also contribute to meiotic recombination. In addition, meiosis-specific cytological structures and enzymes enhance recombination frequency (meiotic induction) and shift partner preference from sister chromatids to homologous chromosomes (3, 47, 64, 74). In detail the steps of HR vary between different types of sequence organization (allelic versus sister versus ectopic), between different types of DNA damage, between meiotic and mitotic cells, and between species (10, 55, 58).

Meiotic recombination, including CO formation, is initiated by DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae and other eukaryotes, DSBs are formed by Spo11. Many cofactors are required (29). The Schizosaccharomyces pombe homolog is Rec12, also requiring auxiliary factors whose elimination leads to loss of meiotic DSB formation (12). The 5′ single-strand ends at DSBs are processed by nucleases. In S. cerevisiae the MRX complex made up by the proteins Rad50, Mre11, and Xrs2 is required for this resection, as well as for DSB formation. The corresponding MRN complex of S. pombe (Rad50, Rad32, and Nbs1) is not required for DSB formation but is essential for DSB repair (43, 72). Deletion of rad50, rad32, or ctp1 (homologous to SAE2/COM1 in S. cerevisiae and CtIP in humans) leads to very low spore viability. These proteins are also essential for DSB processing (23, 24, 32, 43, 60, 62).

Free DNA 3′ ends at DSBs are recruited for invasion of a sister or homologous chromatid by the strand transfer proteins Rad51 and Dmc1, again involving many accessory proteins (16). This results in the central intermediates of HR: heteroduplex DNA consisting of single strands originating from different chromatids and Holliday junctions (HJs). In S. cerevisiae HJs form preferably between homologs with a two- to sixfold excess over intersister HJs (64). Surprisingly, meiotic HJs form with about a fourfold excess between sisters in S. pombe (11). Eventually the intermediates are resolved into crossover (CO) and noncrossover (NCO) events. COs show exchange of the flanking sequences of the two chromatids involved and usually carry a patch of conversion (unilateral transfer of DNA sequences from one chromatid to its interacting partner) near the DSB site. NCOs are conversion events without associated COs (22). In S. pombe loss of core HR functions leads to very low spore viability: deletion of rad51 but not of dmc1 (20), double mutation of rad54 and rdh54 (7), inactivation of the endonuclease activity encoded by mus81 and eme1 (5, 52), and combined deletion of rad22 and rti1 (homologs of RAD52 of S. cerevisiae). But, differently from the other core functions, Rad22 and Rti1 are not required for CO and NCO (50).

Early in meiotic prophase of many eukaryotes, axial elements (called lateral elements in later stages) form along sister chromatids, and pairing of homologous chromosomes is initiated, leading to juxtaposition of the homologous chromosomes along their whole length in the synaptonemal complex (SC) (54). In S. pombe no SC is formed, but linear elements (LEs), resembling axial elements of other eukaryotes, are formed. LEs do not form continuously along the chromosomes (1) but load the proteins Rec10, Hop1, and Mek1 (36, 44, 57), which are homologs of, or at least related, to the S. cerevisiae proteins Red1, Hop1, and Mek1, respectively, localizing to axial/lateral elements (2, 67). Hop1 carries a HORMA domain, also present in proteins associating with axial elements and regulating the progress of recombination in higher eukaryotes: Arabidopsis thaliana (61), Caenorhabditis elegans (9, 41), and mammals (18).

In S. cerevisiae localization of Hop1 and Mek1 (meiosis-specific protein kinase) to axial elements is dependent on Red1 (2, 67). Mutation of the three S. cerevisiae genes results in reduction of DSB formation, CO and conversion frequencies, and spore viability (26, 31, 59). Direct comparison of unequal sister chromatid recombination (USCR) frequencies in an assay excluding the scoring of intrachromatid recombination (ICR) revealed no increase in the hop1 null mutant but about fourfold increases in the red1 and mek1 null mutants (69). The S. cerevisiae Hop1, Red1, and Mek1 proteins are involved in biasing meiotic DSB repair to occur between homologous chromosomes rather than between sister chromatids (47). Activated Mek1 kinase is required for the inhibition of sister chromatid-mediated DSB repair by Rad51, when the DMC1 gene is deleted and the meiotic recombination checkpoint is activated (4, 27, 38, 47). For Mek1 activation, phosphorylation of Hop1 by the Mec1/Tel1 kinases is also required (6).

Less is known about the S. pombe proteins. Hop1 of S. pombe was identified as a nonsignificant hit by sequence comparison with full-length S. cerevisiae Hop1 and contains an N-terminal HORMA domain and a central zinc finger motif like Hop1 in S. cerevisiae. In addition they share a short homology block toward the C terminus (36). The Mek1 protein of S. pombe shares 34% identity and 54% similarity with its S. cerevisiae counterpart along the whole sequence. It contains an FHA domain in the N-terminal part like the other members of its family of checkpoint kinases and is involved in regulation of the meiotic cell cycle (57). Hop1 and Mek1 are strongly expressed in meiosis but not expressed or only slightly expressed in vegetative cells (42, 57). In prophase both proteins localize to LEs as defined by colocalization with the LE component Rec10 (36). Deletion of the distant RED1 homolog rec10 abolishes LE formation (36, 44) and strongly reduces meiotic recombination (17, 70). Rec10, but not Hop1 and Mek1, is required for localization of Rec7 (a distant homolog of S. cerevisiae Rec114) to meiotic chromosomes (34). Rec7 and Rec10 are required for Rec12 activity (12, 29).

Obtaining information on the functions of Hop1 and Mek1 in S. pombe was the aim of the work presented here, especially on their possible roles in homolog versus sister discrimination for DSB repair. Deletion mutants have been studied with respect to spore viability and the frequencies of CO and conversion. They have also been assessed for genetic recombination events between sister chromatids in the known PS1 assay (63) and the newly developed VL1 assay (for details, see Fig. Fig.3).3). Physical analysis of DSB formation and repair has been performed in meiotic time course experiments. It is proposed that S. pombe Hop1 and Mek1 are promoting interactions between homologous chromosomes rather than inhibiting interactions between sister chromatids.

FIG. 3.
PS1 and VL1 assay systems for intrachromosomal recombination. Strains with constructs carrying repeated DNA sequences have been assayed for prototroph formation either by intrachromatid recombination (ICR, yielding prototrophs only in PS1) or by unequal ...

MATERIALS AND METHODS

Strains, media, and standard genetic methods.

The genotypes of S. pombe strains used in this study are listed in Table S1 in the supplemental material. The standard media and general methods were as described previously (21, 45). Yeast extract agar (YEA) contained 0.5% Difco yeast extract, 3% glucose, and 1.8% agar; yeast extract liquid (YEL) contained 0.5% Difco yeast extract and 3% glucose; malt extract agar (MEA) contained 3% malt extract and 1.8% agar; minimal medium (MMA) contained 0.65% Difco nitrogen base without amino acids, 1% glucose, and 1.8% agar. When necessary, the media were supplemented with amino acids and bases at 100 mg/liter. YEA + 5 and MEA + 5 contained adenine, uracil, leucine, lysine, and histidine. Antibiotic resistance of strains was tested on YEA with 50 mg/liter Geneticin (G418 sulfate) or YEA with 100 mg/liter hygromycin. Standard crosses were carried out on MEA + 5 at 25°C. YEA + 5 was used as standard growth medium at 30°C. The synthetic EMM medium was prepared with 1% glucose and with or without a nitrogen source as described previously (49).

Construction of plasmids and strains by transformation.

For details of strain and plasmid construction, see the supplemental material. A strain where hop1 was replaced by the G418 cassette kanMX6 has been constructed, while the mek1 deletion strain, also marked with kanMX6, was a gift of the laboratory of P. San-Segundo (57). The preparation of the novel ade6-D20 deletion strain is described in the supplemental material, as well as the construction of the VL1 system for the analysis of USCR.

Spore viability and recombination between homologous chromosomes.

In general the classical methods have been used, including strain construction, for the different recombination assays (21, 45). Parental strains for crosses were grown in YEL, and cell material was mixed and plated on MEA, followed by incubation for 3 days at 25°C (unless indicated otherwise). The cross material was then suspended and treated with snail enzyme (1:1,000 [vol/vol] Helix pomatia juice; Biosepra) solution overnight at 30°C. The values obtained by the traditional method for spore viability determination have often varied considerably. An alternative, faster, and more reproducible method has been described recently (19). An 0.1-ml undiluted or 10-fold-diluted spore suspension was plated onto full medium, and the petri dishes were incubated at 30°C for 30 h. The plates were then inspected visually under the microscope of a Singer tetrad dissection apparatus, which allows systematic inspection of nonoverlapping fields of view. Three classes of spore fates were distinguished and quantified: one spore or cell (no division), 2 to 4 cells (not able to form colonies), and >4 cells (microcolonies). Earlier inspection revealed higher values for the first two classes, indicating that some spores germinate rather late. Spore viability was then calculated as a percentage of microcolonies of the total units counted.

The frequency of CO in a given genetic interval was estimated by calculation of the genetic distance between markers in centimorgans (cM). These intergenic recombination experiments based on random spore analysis of crosses involving auxotrophy mutations have been described before (20). Since there is no CO interference in S. pombe, the distances were calculated according to the Haldane formula as d = −50ln{1 − 2 [R/(R + P)]}, with R representing the number of progeny with recombinant genotypes and P representing that with parental genotypes (46). The determination of conversion frequencies in intragenic recombination experiments has been described in detail (20). Strains carrying heteroallelic auxotrophy mutations in a given gene were crossed, and random spores were analyzed for prototrophy. The number of prototrophs per million viable spores (ppm) was taken as a measure of conversion frequency.

Determination of intrachromosomal recombination frequency.

The formerly described PS1 system relies on red pigment formation of ade6 mutants on medium with limiting adenine concentration (63). It was used in crosses with strains carrying the ade6-D20 deletion to avoid prototroph formation by recombination with the homologous chromosome. The parental strains were grown on YEA without supplements to exclude colonies which had turned white due to a mitotic recombination event. After standard crossing and digestion of cells (see above), the spores were spread at a density of 3,000 per plate on YEA. The titer of germinating spores was determined by spreading the same volume of a 30-fold-higher dilution on YEA. Large white colonies (prototrophic due to recombination) were scored 3 to 4 days later (variable growth speed due to different genetic backgrounds) among the small red colonies (nonrecombinant parental auxotrophs). The determination of mitotic recombinant frequencies was done in the same way with cell suspensions of haploid strains carrying the PS1 repeat construct. Mitotic and meiotic USCR frequencies with the VL1 repeat construct were determined analogously, except that approximately 7,000 spores or cells were spread per plate.

Immunostaining of proteins and fluorescence in situ hybridization (FISH).

The preparation of meiotic cells and surface spreading for staining of proteins with antibodies were done exactly as described before (34). For the analysis of homologous chromosome pairing during meiotic prophase, the following cosmid clones were used as probes for FISH: SPAC132, SPAC1556, SPAC17D4, SPAC27D7, and SPAC922. They carry single-copy sequences from different regions on chromosome I and have been provided by the Wellcome Trust Sanger Institute (Hinxton, Cambridge, United Kingdom). The probes were labeled by nick translation with Cy3-dUTP (red) (Amersham Pharmacia Biotech UK Ltd., Little Chalfont, England) as described elsewhere (33). The hybridization to spread nuclei was performed as previously described for S. cerevisiae (35).

Meiotic time course experiments for DSB analysis.

Haploid or homozygous diploid strains carrying the pat1-114 mutation (temperature sensitive, induction of meiosis at 34°C) were used to obtain synchronous meiosis as described previously (8). The timing of meiotic S phase was determined by fluorescence-activated cell sorting (FACS) analysis (14) and found to be the same with all strains (see Fig. S4 in the supplemental material). The amount of cells undergoing the meiotic divisions was determined by DAPI (4′,6-diamidino-2-phenylindole) staining of the nuclei as described before (14). Only experimental mixtures with high yields of divisions (≥90%) were used for further experiments.

The diploid strains were constructed by protoplast fusion as described previously (14, 66). In brief, the parental h haploids were grown to late log phase (1 × 107 to 2 × 107) in YEL at 25°C. The cells were harvested, washed with 0.9 M sorbitol, and resuspended in 0.9 M sorbitol containing 5 to 10 mg/ml lysing enzymes (Sigma; L-1412). After 20 to 30 min of slow agitation at 25°C, the protoplasts were collected by centrifugation at 2,000 rpm for 5 min, washed, and mixed in 0.9 M sorbitol. Mixed protoplasts were pelleted for 5 min at 2,500 rpm, resuspended in 2 ml of polyethylene glycol (PEG)-CaCl2 (30% PEG 6000, 10 mM CaCl2), and kept for 20 to 40 min at room temperature. After centrifugation at 1,000 rpm for 2 min, the fused protoplasts were resuspended in 1 ml of YEL plus 0.6 M KCl, plated on selective medium (MMA without adenine), and incubated at 25°C. Diploid colonies are prototrophic on selective medium and white on YEA medium as a result of interallelic complementation between ade6-M216 and ade6-M210 strains (45).

Analysis of DNA breakage in meiotic prophase was carried out with minor modifications as described previously (8, 73). A preculture inoculated from a single colony of a given strain was incubated overnight in YEL at 25°C. It was used for inoculation into EMM medium. The cells were grown to a density of 8 × 106 per ml, centrifuged, washed once with water, resuspended in EMM without NH4Cl, and incubated at 34°C for induction of meiosis. At different times afterwards ~6 × 108 cells were retrieved from the culture, washed in cold 50 mM EDTA (pH 8.0), and resuspended in 200 μl spheroplasting buffer (50 mM EDTA [pH 8.0], 29 mM Na2HPO4, 8.4 mM citric acid, 0.9 M sorbitol, 0.3 mg/ml Zymolyase 100T [Seikagaku Inc.], 5 mg/ml lysing enzyme [L-1412]). Two hundred fifty microliters of 2% low-melting-point agarose in spheroplasting buffer was added, and the mixture was poured into 0.1-ml plug molds. After 10 min at 4°C, the plugs were transferred into 2 ml of spheroplasting buffer and incubated for 1 h at 37°C. Afterwards the buffer was replaced by 2 ml of TE/PK (0.5 M EDTA, pH 8.0, 10 mM Tris-HCI, pH 7.5, 1% N-laurylsarcosine, 1 mg/ml proteinase K). After 2 days of incubation at 50°C, the plugs were washed once with 10 ml water for 10 min, then with TE (10 mM Tris-HCl, pH 7.6, 50 mM EDTA, 10 mM phenylmethylsulfonyl fluoride [PMSF]) for 1 h, and finally twice with TE for 45 min at room temperature. The plugs were stored in TE at 4°C.

The chromosomal DNA in the plugs was analyzed by pulsed-field gel electrophoresis (PFGE) as described previously (8). For quantification of meiotic DSB formation at the mbs1 recombination hot spot, DNA was digested with the NotI restriction enzyme, followed by PFGE and Southern blot hybridization as reported previously (8, 73). The 32P-labeled probe (~1 kb) from the left end of the NotI fragment J was obtained by PCR of S. pombe genomic DNA using the following primers: c227L (5′-ACTTCTTACTACTGGAGCCG-3′) and c227R (5′-CGATTCTCTTCAGTGGGTTC-3′). PFGE was performed at 14°C with a CHEF-DR III system from Bio-Rad. The size of DNA fragments was estimated with the help of S. cerevisiae chromosomes as markers (CHEF DNA size standard; Bio-Rad). Quantification was done using a PhosphorImager and the Aida software. The amount of breakage at mbs1 was normalized to the unbroken NotI fragment J.

RESULTS

Hop1 and Mek1 proteins contribute to homologous pairing.

The hop1 and mek1 deletion strains showed no vegetative phenotypes, as reported before (34, 36, 57). Mating in homozygous crosses and the progression of azygotic meiosis in homozygous diploids were normal (data not shown). The temporal appearance and frequency of horsetail nuclei (elongated nuclei with telomeres clustered at the spindle pole body), as well as the timing of the meiotic divisions, were similar to those of the wild type (data not shown). Antibodies against Rec10 stain LEs in the hop1Δ and mek1Δ strains similarly to wild-type meiosis (34, 36). Homologous pairing as determined by the association of FISH-labeled chromosomal loci was reduced in the deletion strains at some locations on chromosome I (Fig. (Fig.1).1). The maximal reductions to 52 to 67% of the wild-type value were observed at two loci in the middle of the short arm. These reductions were statistically significant (P < 0.001, one-tailed Fisher's exact test). Slighter reductions (not statistically significant) were observed at the centromere. No notable reductions occurred at the right telomere. At a site in the middle of the short arm, a diploid strain homozygous for both the hop1 and mek1 deletions was also assayed and association was comparable to that in the single mutants (Fig. (Fig.11).

FIG. 1.
Intergenic recombination and chromosome pairing in meiosis with hop1Δ and mek1Δ mutants. A map of chromosome I is shown with genetic markers on the left (L) and right (R) arms and the centromere as a black oval. The positions of the mbs1 ...

Spore viability and meiotic recombination in the hop1Δ and mek1Δ mutants.

Microscopic inspection of crosses homozygous for hop1Δ showed a moderate increase of asci with fewer than four, and morphologically aberrant, spores compared to the wild type. This was not the case in crosses homozygous for mek1Δ. Quantitative evaluation (for details, see Materials and Methods and Table S2 in the supplemental material) yielded the following spore viability values in homozygous crosses: hop1+ mek1+, 93.6% ± 1.5%; hop1Δ, 83.7% ± 3.8%; mek1Δ, 92.9% ± 0.9%; hop1Δ mek1Δ, 84.3% ± 1.1%. Only the hop1 deletion leads to a significant decrease (χ2 test, P < 0.0001), which is identical in the double mutant (P > 0.1).

The CO frequency was assayed by determination of genetic distances in cM in intergenic recombination experiments for intervals on chromosome I (Fig. (Fig.1;1; see also Table S3 in the supplemental material). When hop1Δ was homozygous in the crosses, the genetic distances were moderately reduced (2.3- to 4.8-fold) in seven intervals located at the left end of the chromosome or adjacent to the centromere. Region-specific differences in reduction were not apparent. In mek1Δ crosses two intervals were assayed and showed 1.9- and 2.8-fold reductions. The same intervals were assayed in the hop1Δ mek1Δ strain and yielded 4.3- and 7.5-fold reductions, slightly higher than those in the hop1Δ strain (3.8- and 3.7-fold, respectively). Thus, single deletion of hop1 or mek1 and deletion of both led to moderate reduction of CO frequency.

Conversion frequencies were assayed in intragenic recombination experiments: determination of prototroph frequencies (prototrophs per million viable spores [ppm]) in crosses of heteroalleles in four genes (Fig. (Fig.2;2; see also Table S4 in the supplemental material). The patterns of the generally moderate ppm reductions in crosses homozygous for the single and double gene deletions differed. In general the reductions were stronger in the hop1 crosses (2.2- to 6.7-fold) than in the mek1 crosses (1.2- to 3.6-fold). The values for the double mutants (3.5- to 23.2-fold reductions) were all lower than those for the single mutants, ranging from epistasis at ura4A to synergism at ade6 with the M26 hot spot (reductions of 6.7-, 1.9-, and 23.3-fold). The crosses involving the ura4A hot spot showed moderate reductions (2- to 4-fold). The two “cold spot” genes ade7 and ade4 showed 1- to 6-fold reductions.

FIG. 2.
Intragenic recombination in meiosis with hop1Δ and mek1Δ mutants. The frequencies of prototrophs per million of viable spores (ppm) in crosses of pairs of heteroallelic auxotrophy mutants in four different genes are shown (means of at ...

A specific assay for quantification of USCR in S. pombe.

Since the DNA sequences of sister chromatids are identical, allelic HR events between them cannot be studied by genetic means. For this reason assays involving repeated DNA sequences have been developed for the assessment of intrachromatid recombination (ICR) and unequal sister chromatid recombination (USCR) (55). In S. cerevisiae ICR and USCR are increased in the absence of Red1 and Mek1 but not in the absence of Hop1 (69).

In S. pombe the known PS1 assay for quantification of intrachromosomal recombination (63) does not differentiate between ICR and USCR (Fig. (Fig.3A)3A) and also involves the ade6-M26 hot spot. Therefore, an assay avoiding scoring of ICR (VL1; see the supplemental material) was constructed. It consists of the 3′ part of the ade6 gene, followed by the marker gene for hygromycin resistance, and then by the promoter region and the 5′ part of the ade6 gene (Fig. (Fig.3B).3B). The two ade6 fragments share a 500-bp sequence of the central part of the gene. The construct was integrated into a large intergenic region on the left arm of chromosome I (Fig. (Fig.1).1). Proper insertion and the structure of resulting prototrophic recombinants were verified by PCR, Southern blot analysis, and DNA sequencing (see Fig. S2 in the supplemental material). The point mutations identified in the construct (see Fig. S2) obviously did not abolish the formation of prototrophic ade6 recombinants. All the recombinant frequency determinations with VL1 and PS1 were carried out in the background of the newly constructed ade6-D20 deletion strain (see the supplemental material) for exclusion of homologous recombination events other than those shown in Fig. Fig.3.3. The scoring of ade6+ recombinants (see Materials and Methods) was carried out on YEA as described previously (63). The results obtained with the two assays are given in Fig. Fig.44 and in Tables S5 and S6 in the supplemental material. The frequency of mitotic recombinants in haploid cell cultures (25-fold higher in the PS1 assay than in the VL1 assay) was clearly lower than that in the meiotic crosses: 20-fold meiotic stimulation in the PS1 assay and 6-fold in the VL1 assay (see Tables S4 and S5). The frequency data for PS1 are similar to those obtained with a similar repeat construct involving the non-hot spot ade6-M375 mutation instead of ade6-M26 (51). It is likely that the VL1 insertion on chromosome I did not result in recombination hot spot activity: the region of integration is devoid of a hot spot of Rec12 binding (37), and the prototroph frequencies in mitosis and meiosis are much lower than those in the PS1 assay. For discussion purposes, it should be kept in mind that the estimates of mitotic frequencies were based on small numbers of scored prototrophs.

FIG. 4.
Meiotic intrachromosomal recombination in DSB formation and repair mutants, including hop1Δ and mek1Δ. Ade+ recombinant frequencies (prototrophs per million viable spores [ppm]) have been assayed in the two systems PS1 and VL1, ...

For further characterization of the VL1 assay, several recombination mutants were assayed (Fig. (Fig.4A;4A; see also Table S6 in the supplemental material). Prototroph formation was reduced 10-fold in rec12Δ crosses and to a similar degree also in the PS1 assay (S. Mallela, personal communication). This indicates that the large majority of meiotic intrachromosomal events in the wild type derive from DSBs formed by Rec12. It had been shown before that deletion of the S. pombe RAD52 homologs rad22 and rti1 affected recombination between homologous chromosomes only slightly (maximal 2.5-fold reduction) but reduced prototroph formation in the PS1 assay 80-fold (50). In the VL1 assay the ppm reductions were 7-fold (rad22Δ), 1.9-fold (rti1Δ), and 14-fold (rad22Δ rti1Δ). Strong reductions were also observed for ctp1Δ (18-fold) and rad51Δ (13-fold) strains. Unexpected was the 10-fold reduction in the dmc1Δ strain. Crosses homozygous for the rad51Δ dmc1Δ double mutant did not yield sufficient viable spores to allow an estimate of the prototroph frequency, which is probably lower than that in the single mutants. Surprisingly, slight increases over the wild-type level have been found in crosses of strains carrying rad50 mutations: rad50Δ (1.4-fold), rad50S (1.4-fold), and rad50S with additional deletion of hop1 and/or mek1 (1.1- to 2-fold). These increases may be due to mitotic recombination before cell mating. As for the other mutants defective in functions essential for DSB repair, the scored numbers of prototrophs were in some cases rather low, especially in the VL1 assay.

The role of Hop1 and Mek1 in intrachromosomal recombination.

Strains with deletions of hop1, mek1, or both genes in ade6-D20, ade6-D20 PS1, and ade6-D20 VL1 backgrounds were constructed and assayed for mitotic and meiotic intrachromosomal recombination frequencies (see Table S5 in the supplemental material). The standard errors of the means of prototroph frequencies in the crosses homozygous for hop1Δ or mek1Δ overlapped with the error of the hop1+ mek1+ crosses in the PS1 assay. When both genes were deleted, a 1.5-fold increase with an error still overlapping with the wild type was observed (Fig. (Fig.4B;4B; see Table S5). Thus, active Hop1 and Mek1 may not affect recombination in the PS1 assay, but relative changes of ICR versus USCR frequencies in the mutants have not been excluded. In the VL1 system (Fig. (Fig.4C;4C; see Table S6) prototroph frequencies were slightly increased in crosses homozygous for hop1Δ (1.7-fold) and mek1Δ (1.2-fold) and more strongly when both genes were deleted (2.9-fold). In all cases the standard errors did not overlap with that of the wild type. Thus, active Hop1 and Mek1 seem to reduce USCR in the VL1 assay.

Global DSB formation and repair in hop1 and mek1 null mutants.

To assess formation and repair of meiotic DSBs, haploid or diploid strains carrying the pat1-114 mutation for optimally synchronizing meiosis were subjected to PFGE to visualize the fragmentation of chromosome-sized DNA molecules in time course experiments. Meiotic stages were determined by FACS analysis and microscopy. Meiotic DNA replication was complete at 3 h (see Fig. S4 in the supplemental material), and the second meiotic division was at 8 h after induction. The control experiments were performed with haploid (see Fig. S3) and diploid (Fig. (Fig.5A)5A) pat1-114 rad50+ and pat1-114 rad50S strains. In both rad50+ and rad50S strains a smear of shorter DNA fragments appeared 3 h after induction, besides the intact chromosomes. The smear reached maximal intensity at 4 h, when almost no intact chromosomal DNA remained. In the rad50+ strains this smear was weak and transient (Fig. (Fig.5A;5A; see also Fig. S3A), whereas in the rad50S strains the fragments accumulated and persisted without repair beyond 6 h after induction (Fig. (Fig.5A;5A; see also Fig. S3A), as had been reported before (8).

FIG. 5.
Global meiotic DSB formation and repair visualized by PFGE. Diploid (A, B, and C) or haploid (D) strains carrying pat1-114 were induced to undergo meiosis, cells were retrieved at the indicated time points and lysed, and the liberated DNA was subjected ...

In the rad50S hop1Δ strains, broken DNA appeared with similar timing, but the smear was weaker than that in the rad50S hop1+ strain. Intact chromosomes did not disappear completely, indicating that DSB formation is reduced in the absence of Hop1. Surprisingly, DSB repair seemed to take place at the later time points, as the amount of intact chromosomes increased again after 3.5 h (Fig. (Fig.5B;5B; see Fig. S3A in the supplemental material). In the strains carrying mek1Δ DSB fragments appeared with timing and amounts similar to those for the mek1+ strains but were visibly repaired and restored to intact chromosomes only in rad50+ strains and not in rad50S strains (Fig. (Fig.5C).5C). For the hop1Δ mek1Δ strains the timing was again similar to that for the other strains, but the amount of DSB fragments was small throughout the rad50+ and rad50S strain time courses (Fig. (Fig.5D).5D). Intact chromosomal DNA remained present at all time points. The phenotype of the double mutant strains resembled those of the hop1Δ single mutant strains (Fig. (Fig.5B5B).

Quantification of a DSB fragment at the mbs1 hot spot of recombination.

The above experiments provided a global view of DSB formation across the genome, but quantification was not possible. For this reason, the chromosomal DNA in the plugs was also digested with NotI, and the resulting fragments were separated by PFGE, transferred for Southern hybridization, and assayed for the specific DSB and recombination hot spot mbs1 on the NotI fragment J at the left end of chromosome I (Fig. (Fig.1),1), as described before (73) and in Materials and Methods. The 32P-labeled probe visualized the intact 500-kb NotI fragment J and shorter fragments resulting from DSB formation, among them the 250-kb DNA fragment resulting from breakage at mbs1. The amount of breakage was determined by normalization of the amount of radioactivity in the 250-kb fragment to that in the intact fragment at different time points of meiosis in various strains (Fig. (Fig.66).

FIG. 6.
Quantification of a DSB fragment at the mbs1 hot spot. DNA was isolated at the indicated time points from haploid pat1-114 strains undergoing meiosis, digested with NotI, subjected to PFGE, transferred to membranes, and hybridized with a probe that visualizes ...

In rad50S meiosis, broken DNA accumulated during the meiotic time course and reached 12% ± 0.7% at 6 h (Fig. (Fig.6A).6A). Deletion of the mek1 gene led to a mild reduction (9.2% ± 0.6%). When hop1 was deleted, the breakage level was low throughout the time course, maximally 2.2% ± 0.1% at 4 h, followed by a decline to 1% ± 0.05% at 6 h. In the hop1Δ mek1Δ strain the small amount of breakage (2%) was accumulated faster than in the hop1Δ strain, and no reduction in late prophase was detected. In rad50+ meiosis the maximal amount of broken DNA at 3.5 h reached only 2.3% ± 0.2%, followed by gradual reduction to 0.3% ± 0.1% at 6 h (Fig. (Fig.6B).6B). Only a slight reduction of DNA breakage was observed in the mek1Δ strain with a maximum of 1.5% ± 0.1% at 3.5 h. In the hop1Δ strain, the maximum of DNA breaks was observed earlier (1% ± 0.1% at 3 h), and smaller amounts of DSB were determined throughout the time course. In the hop1Δ mek1Δ strain marked reduction of DSB amounts was observed (maximally 0.22% ± 0.01%). Obviously DSB formation and repair did occur in all the investigated rad50+ strains.

DISCUSSION

The Hop1 and Mek1 proteins are moderately important for spore viability, pairing, and recombination between homologous chromosomes.

On average the three fission yeast chromosomes undergo 20, 15, and 10 crossovers per meiosis (46). Thus, even when crossovers are abolished, and the chromosomes segregate randomly, 12.5% of spores can be expected to be viable. In reality, it has been found that mutations abolishing meiotic DSB formation still show 20 to 30% spore viability (13, 15). Deletion of hop1, or of hop1 and mek1, reduced spore viability only to about 90% of the wild-type value, while mek1 deletion alone showed no reduction (see Table S2 in the supplemental material). In S. cerevisiae strong reduction of spore viability was observed after inactivation of HOP1 (1%) or MEK1 (13%) (26, 59), which can be attributed to the larger number of chromosomes and the lower CO-per-chromosome ratio. Also, mutation of HOP1 or MEK1 resulted in substantially stronger reduction of CO and conversion frequency (26, 31, 59) than that in S. pombe: in the hop1Δ and mek1Δ single mutants CO frequency was not reduced more than 5-fold (Fig. (Fig.1)1) and conversion frequency was reduced at most 7-fold (Fig. (Fig.2).2). Analysis of the double mutant revealed epistasis for some but mostly additivity for intergenic and intragenic intervals (maximally 27-fold reduction). In line with these results, pairing of homologous chromosomes was reduced maximally to 50% in the mutants at internal sites on a chromosome, but not at a telomere (Fig. (Fig.1).1). Genetic elimination of proteins that affect horsetail organization (bouquet) in fission yeast meiosis has led to similar reductions of pairing and to 5- to 10-fold reduction of CO frequency (25, 40, 48). In contrast, deletion of genes required for DSB formation and the core functions of DSB repair (except rad22Δ rti1Δ) typically leads to 100-fold or more reduction of CO and conversion (12) and spore viabilities of 1% or less (also rad22Δ rti1Δ) (50). In the case of rad51Δ spore viability is very low, but CO and conversion are drastically reduced only when dmc1 is deleted in addition (20).

Hop1 and Mek1 involvement in intrachromosomal and possibly also sister chromatid recombination.

The effect of single and double deletion of hop1 and mek1 was assayed in the PS1 assay (63) and the newly constructed VL1 assay (Fig. (Fig.3).3). The frequencies of prototrophs in the single mutants were similar to those of the wild type (PS1) or slightly increased (VL1). In both assays stimulation (1.5- and 2.9-fold, respectively) was observed in the double mutant (Fig. (Fig.4).4). These findings may be interpreted as an involvement of Hop1 and Mek1 in reduction of intrachromosomal recombination, and possibly also sister chromatid recombination, and as contributing to efficiency of recombination between the homologs based on the data presented above. The increases of intrachromosomal recombination in S. pombe (maximally 3-fold) are in line with those in S. cerevisiae mutants thought to be defective in barrier formation against SCR (47, 69). But the decreases of CO and conversion frequencies are much higher in the S. cerevisiae hop1 and mek1 mutants than in their S. pombe counterparts. As discussed further below, we propose that Hop1 and Mek1 may transiently inhibit DSB processing for promotion of interactions with the homolog, rather than setting up a barrier against SCR. The influence of Hop1 and Mek1 on the flow of intermediates into different S. pombe pathways remains to be determined by quantification of physical intermediates such as Holliday junctions.

Involvement of Hop1 and Mek1 in formation and processing of the covalent Rec12 DNA intermediate.

Substantial DSB formation occurred in rad50+ meiosis whether the hop1 or mek1 gene was deleted or not (transient disappearance of intact chromosomes), but not in the hop1Δ mek1Δ double mutant (Fig. (Fig.5).5). The maximal amounts of DSBs at the mbs1 hot spot were reduced in rad50+ and rad50S meiosis in the mutant strains in comparison with hop1+ mek1+ strains (Fig. (Fig.6).6). Additive reduction was obtained in the double mutant in rad50+ meiosis. In rad50S meiosis epistasis of hop1Δ over mek1Δ was observed, which can be attributed to restoration of DSB repair by deletion of hop1 (see below). The results indicate that Hop1 and Mek1 contribute to high-level DSB formation. This is in agreement with the recently reported quantification of the Rec12 oligonucleotide, the processing product resulting from removal of Rec12 bound covalently to DNA 5′ ends at DSBs. In rad50+ meiosis, mek1 deletion reduced the level to 70%, hop1 deletion reduced the level to 53%, and deletion of both reduced the level to 22% of the level in the hop1+ mek1+ strain (60). Since formation and repair of DSBs overlap in time and the rates of these processes may differ between mutants and the wild type, it is not possible to clearly distinguish between defects in DSB accumulation and increased efficiency of repair in the mutants. But a contribution of Hop1 and Mek1 to formation of DSBs is supported by the observation that the binding of Rec7 to the chromosomes (required for DSB formation) was reduced in hop1Δ and mek1Δ strains (34). In S. cerevisiae rad50S strains deletion of HOP1 reduced DSB levels at three hot spots 10-fold, while deletion of MEK1 reduced it as strongly at some hot spots but less or not at all at other hot spots (56, 71). Deletion of the S. pombe genes affects DSB formation less severely, especially in the case of mek1Δ.

In agreement with earlier observations (8), DSBs were found to accumulate in rad50S meiosis without repair. When in the rad50S strain the hop1 gene was deleted, repair did occur, but not when mek1 was deleted (Fig. (Fig.55 and and6;6; see also Fig. S3 in the supplemental material). Reappearance of intact chromosomes after fragmentation (best visible in Fig. S3) is the strongest evidence for restoration of repair. The similarly low levels of DSBs at mbs1 in rad50S hop1Δ and rad50S hop1Δ mek1Δ strains are in agreement with restoration of repair by hop1 deletion (Fig. (Fig.6A).6A). Further support for this interpretation has been published recently (60): while no Rec12 oligonucleotide processing product was formed in rad50S hop1+ meiosis, it appeared in rad50S hop1Δ meiosis (7% of the level found in the rad50+ hop1+ mek1+ strain). The processing product was also observed in the rad50S mek1Δ strain at a level of 3%, although no repair of DSBs has been observed in this case (Fig. (Fig.5C5C and and6A).6A). Thus, the processing occurring in the rad50S mek1Δ strain may not be sufficient for formation of intact chromosomes at a detectable level, unlike the situation in the rad50S hop1Δ strain. No Rec12 oligonucleotide was detected in the rad50S hop1Δ mek1Δ strain. Perhaps it occurs below the detection limit due to the additive reduction of DSB formation in the absence of Hop1 and Mek1.

The presented data are consistent with Hop1 and Mek1 transiently inhibiting DSB processing in wild-type meiosis. Retardation of processing may allow more time for collision with the homologous chromosome in the moving horsetail nucleus and thus may increase recombination versus repair from the sister chromatid. In rad50S meiosis this inhibition would then be permanent but could be released by deletion of hop1, and to a lesser extent also by deletion of mek1, for formation of the Rec12 oligonucleotide. While in the rad50S hop1Δ strain intact chromosomes can then be formed, Hop1 action in the rad50S mek1Δ strain may render repair ineffective, or active Mek1 may also be required at a later step of repair. Clearly, further analysis of the role of Hop1 and Mek1 in DSB repair is required. For example, a role of the two proteins, especially Hop1, in the inhibition of alternative DSB processing and repair pathways cannot be excluded. It has been shown that topoisomerase bound to DNA 5′ ends can be removed by a 5′-tyrosyl DNA phosphodiesterase (30).

Pathways for DSB formation and repair in S. pombe meiosis.

For visualization of the interpretations presented above and the discussion of further results, a hypothetical pathway scheme is presented in Fig. Fig.7.7. Rec10, and thus formation of LEs (36, 44), is essential for efficient recruitment of the Rec12 complex for DSB formation, but the MRN complex is not required, unlike in S. cerevisiae, where the MRX complex is essential for DSB formation (72). The meiotic proteins Hop1 and Mek1 are proposed to contribute to the efficiency of DSB formation as discussed above. At DSB sites, Hop1 and Mek1 are proposed to interact with the MRN complex, and perhaps also with Ctp1. At the same time in prophase, dynamic alignment and separation of homologous chromosomes occur due to the movement of the horsetail nucleus (25). It is proposed that Hop1 and Mek1 inhibit processing of the Rec12 DNA intermediate, until alignment with a chromatid of the homologous chromosome is achieved. This inhibition is either not very efficient or limited to early prophase, since there is a 4-fold excess of Holliday junctions forming with sister chromatids (11). Eventually, action of the MRN complex and Ctp1, and perhaps additional factors, leads to release of the Rec12 oligonucleotide and resection, followed by strand invasion, Holliday junction formation, and completion of repair.

FIG. 7.
Hypothesis on pathways of DSB formation and repair in S. pombe. Proteins involved in the different steps are shown next to the corresponding arrows. The fat arrows indicate the major DSB-processing pathway in meiosis. The formation of lesions leading ...

For further interpretations presented below, a caveat may be that different types of meiosis had to be studied for physical (pat1-114), for cytological (azygotic), and for spore viability and recombination analysis (zygotic). DSBs formed by Rec12 are intermediates for intrachromosomal recombination in the VL1 assay (Fig. (Fig.4A).4A). Repair of other types of DNA damage occurring in mitotic and meiotic cells (for instance, replication fork collapse) also involves the MRN complex. Thus, we did not expect to find wild-type levels of USCR among the very rare viable spores of rad50Δ and rad50S crosses (VL1 assay, Fig. Fig.4A;4A; see also Table S6 in the supplemental material). Additional work is required for verification of the observed prototroph frequencies and the exclusion of mitotic recombination events in the strains before mating. Also, a cryptic pathway in crosses homozygous for rad50Δ mutants has been described (23). Parallel increases of spore viability and intragenic recombination have been observed in homozygous rad50S crosses carrying in addition a hop1 or a mek1 deletion, as expected from the presence of Rec12 oligonucleotides in pat1-114 meiosis of these strains (60).

Very low spore viability is typical not only for rad50Δ crosses but also for other null mutants abolishing homologous repair pathway functions. Involvement of Ctp1 in DSB processing leading to USCR (and probably also to SCR) was indicated by 17-fold reduction of prototroph frequency in the VL1 assay (Fig. (Fig.4A).4A). In rad22Δ rti1Δ crosses prototroph frequency in the PS1 assay was synergistically reduced to 1% of the wild-type value, while CO and conversion were almost unaffected, but spore viability was very low (50). The same holds for the VL1 assay: rad22Δ rti1Δ crosses resulted in a 14-fold reduction (Fig. (Fig.4A).4A). S. pombe Rad51 and Dmc1 have overlapping functions in CO and conversion (20). The same seems to be the case for USCR in the VL1 assay: 15-fold reduction in the rad51Δ strain and 10-fold in the dmc1Δ strain (Fig. (Fig.4A).4A). This suggests involvement of Dmc1 in SCR, which is in line with the proposed absence of a barrier against SCR (12). No estimate of prototroph frequency could be obtained for the rad51Δ dmc1Δ crosses (extremely low spore viability). In accordance with this interpretation, no S. pombe homolog has been found for the S. cerevisiae Hed1 protein. Hed1 has been shown to inhibit participation of Dmc1 in SCR (65). Deletions of rad54 and rdh54 (homologs of S. cerevisiae RAD54 and RDH54, respectively) both showed increased meiotic intrachromosomal recombination frequency, but only the rdh54 deletion showed reduced CO and conversion frequencies (7). Spore viability was moderately reduced in the single mutants but very low in the double mutant, indicating that Rad54 and Rdh54 play an important role in meiotic sister chromatid repair (7). Since the structure-specific endonuclease Mus81 has been shown to be essential for repair of damaged DNA in vegetative cells, for spore viability, and for meiotic CO formation, but not for NCO formation (5, 52, 68), it is obvious that this enzyme is involved in sister chromatid repair and CO pathways.

The interactions of the core components of the mitotic homologous repair pathway with their meiotic paralogs and with other meiotic factors, including Hop1 and Mek1, obviously are complex. But the 4-fold excess of sister over homolog Holliday junctions (11) indicates that DNA repair involving sister chromatids is important. In S. pombe it contributes substantially to successful meiosis and spore viability. This may also apply to other eukaryotes.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Paul Russell, Scripps Research Institute, La Jolla, CA, for sending a ctp1 deletion strain prior to publication and John Woodward, the Wellcome Trust Institute, Hinxton, United Kingdom, for S. pombe cosmids. We thank also Benjamin Sakem and Katja Ludin from the Cell Biology Institute in Berne, Switzerland, for construction of strains and thorough commentary on the manuscript.

This work was supported by grants from the Swiss National Science Foundation to J.K., a short-term fellowship of the Swiss National Science Foundation to O.C., and a grant from the Austrian Science Fund (P18186) to J.L.

Footnotes

[down-pointing small open triangle]Published ahead of print on 1 February 2010.

Supplemental material for this article may be found at http://mcb.asm.org/.

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