Meiotic DNA Breaks Associated with Recombination in S. pombe

Molecular Cell, Vol. 5, 883–888, May, 2000, Copyright 2000 by Cell Press

Meiotic DNA Breaks Associated with Recombination in S. pombe Marcella D. Cervantes, Joseph A. Farah, and Gerald R. Smith* Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North Seattle, Washington 98109

Summary In the fission yeast Schizosaccharomyces pombe, we have detected prominent DNA breaks that appeared shortly after premeiotic DNA replication. These breaks, like meiotic recombination, required the products of the six rec genes tested. Prominent breaks were detected at widely separated sites, about 100–300 kb apart, equivalent to about 50–150 sites per genome or approximately the number of meiotic recombination events. Certain features of these breaks are similar to those in the distantly related yeast Saccharomyces cerevisiae, the only other organism in which meiotic DNA breaks have been reported. Other features, however, appear to be different. These results suggest that, although DNA breaks may be a general feature of meiotic recombination, the breaks in S. pombe may play a role different from those in S. cerevisiae. Introduction Homologous genetic recombination serves at least three important functions in living cells: to repair DNA double-strand (ds) breaks, to aid meiotic chromosome segregation, and to generate genetic diversity. During meiosis of the budding yeast Saccharomyces cerevisiae, these three functions appear to be combined. DNA ds breaks are actively made at high frequency, and their repair produces connections between homologous chromosomes that allow their faithful segregation at the first meiotic division (reviewed by Roeder, 1997). Concomitantly, these recombinational exchanges generate diverse combinations of alleles, with respect to which the homologs differ. In the fission yeast Schizosaccharomyces pombe, studied here, DNA ds breaks also appear to provoke meiotic recombination, but these breaks seem to be much farther apart than in S. cerevisiae, the only other organism in which meiotic DNA ds breaks have been reported. In S. cerevisiae, as in various organisms, homologous recombination occurs at high frequency near special sites called hotspots (reviewed by Smith, 1994; Lichten and Goldman, 1995). These sites were first identified genetically as sites at and near which gene conversion, or nonreciprocal recombination, occurs at high frequency. They were postulated by Szostak et al. (1983) and shown by Sun et al. (1989) and Cao et al. (1990) to be sites of DNA ds break formation in S. cerevisiae. In S. pombe, the M26 hotspot behaves genetically much * To whom correspondence should be addressed (e-mail: gsmith@ fhcrc.org).

like the hotspots described in S. cerevisiae. M26 was created by the ade6-M26 mutation and was noted because of its high frequency of intragenic recombination and gene conversion (Gutz, 1971). The hotspot is meiosis specific and is active when in one or both homologs (Ponticelli et al., 1988; Schuchert and Kohli, 1988). The sequence 5⬘-ATGACGT-3⬘ or a closely related sequence is essential for its activity (Schuchert et al., 1991; M. Fox, T. Yamada, K. Ohta, and G. R. S., submitted for publication), as is the Atf1·Pcr1 transcription factor, which binds to this sequence (Kon et al., 1997). In addition to converting at high frequency, M26 stimulates the coconversion of adjacent markers; the frequency of coconversion decreases approximately 2-fold for each 500 bp from M26 (Gutz, 1971; Grimm et al., 1994). This distance dependence is similar to that of S. cerevisiae hotspots. Notwithstanding these similarities, no DNA ds breaks at or near M26 have been reported despite repeated searches in our lab and others (Ponticelli, 1988; Ba¨hler et al., 1991), although the methods used may not have been adequate to detect low-level breaks. Formation of ds breaks in S. cerevisiae requires multiple gene products that are also required for meiotic recombination and proper homolog segregation (Roeder, 1997). One of these gene products, Spo11, is covalently attached to the 5⬘ end of the DNA at the ds break (Keeney et al., 1997). Spo11 shares amino acid sequence homology with a class of DNA topoisomerases, which form DNA-tyrosine phosphodiester intermediates during their action. A particular tyrosine (Tyr-135) of Spo11 is essential for its action, suggesting that the DNA-Spo11 covalent linkage is via this tyrosine (Bergerat et al., 1997). The S. pombe Rec12 protein is also essential for meiotic recombination (DeVeaux et al., 1992) and shares regions of amino acid sequence homology with Spo11, including the region containing Tyr-135 (Bergerat et al., 1997; Keeney et al., 1997). As shown in this paper, the corresponding tyrosine (Tyr-98) of Rec12 is also essential for meiotic recombination. These results suggested that meiotic DNA ds breaks might occur in S. pombe. To widen the search for DNA ds breaks beyond the M26 hotspot, we took advantage of pulsed-field gel electrophoresis, which can resolve the three intact chromosomes of S. pombe (Fan et al., 1989) and have been used to analyze meiotic DNA breaks in S. cerevisiae (Game, 1992; Zenvirth et al., 1992). A break anywhere on a chromosome would result in disappearance of the intact DNA. Since S. pombe has about 20, 15, and 10 meiotic crossovers on chromosomes I, II, and III, respectively (Munz et al., 1989), and one crossover might result from one break, intact DNA molecules might disappear even if the breaks were transient and the cells in the culture were not completely synchronous. We report here the detection of S. pombe meiotic DNA ds breaks and their dependence on multiple rec gene products. Certain features of these breaks appear to differ from those in S. cerevisiae, suggesting differences in the mechanism or regulation of meiotic recombination in these two distantly related yeasts.

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Table 1. Tyr-98 of Rec12 Is Essential for Meiotic Recombination Plasmid

rec12 Allele

Ade⫹ Recombinants/106 Viable Sporesa

pJF05 pJF03 pJF04 None

⫹ 164 (Y98F) 165 (Y97F) ⫺

4700, 6400 ⬍66, ⬍70, ⬍34, 6 5200, 4100 20, ⬍34, 10

a

Strains with the indicated plasmids and bearing the chromosomal ade6-M26 rec12-152::LEU2 alleles were mated with a strain bearing the ade6-52 rec12-152::LEU2 alleles, and Ade⫹ recombinant frequencies were measured among the resulting spores. Figure 1. Synchrony of Meiotic DNA Replication in an S. pombe pat1 Mutant

Results Tyr-98 of Rec12 Is Essential for Meiotic Recombination To determine whether the tyrosine of Rec12 corresponding to the crucial tyrosine (Tyr-135) of Spo11 is also required for meiotic recombination, we mutated the codon for Tyr-98 to encode Phe. A plasmid-borne copy of the mutation, rec12–164 (Y98F), was tested for complementation of the chromosomal rec12–152::LEU2 deletion mutation by measuring ade6 intragenic recombination (Table 1). This mutation reduced the Ade⫹ recombinant frequency by at least a factor of 100. In contrast, mutation of the adjacent codon to Y97F (rec12–165) had no significant effect, in keeping with the functional Spo11 protein having Phe at this position (Keeney et al., 1997). These data show that Tyr-98 of Rec12 is critical for its function, although the adjacent Tyr-97 can tolerate changes, and imply that Rec12 and Spo11 act similarly. This finding, plus the meiosis specificity of the expression of rec12⫹ and the phenotype of rec12 mutations (Lin and Smith, 1994), encouraged the search for DNA breaks during S. pombe meiosis.

Detection of DNA Breaks during Meiosis We set out to search for meiotic DNA breaks anywhere along the chromosomes rather than specifically at the M26 hotspot as in previous searches (Ponticelli, 1988; Ba¨hler et al., 1991). To this end, we induced cells for meiosis and examined their chromosomal DNA at multiple times after induction by pulsed-field gel electrophoresis. Total DNA was detected by staining the gel with ethidium bromide. Maximal synchrony was sought by using pat1–114 (ts) mutants, which initiate meiosis when the mutant Pat1 protein kinase homolog is inactivated at high temperature (Iino and Yamamoto, 1985; Nurse, 1985; McLeod and Beach, 1986). Synchrony was further enhanced by inducing cultures arrested in the G1 phase of the cell cycle (after nitrogen starvation). In such cultures, the bulk of premeiotic DNA synthesis occurs during an ⵑ1 hr interval at about 2 hr after induction (Figure 1; additional data not shown; Li and Smith, 1997); the first and second meiotic divisions occur at about 5 hr, and spore formation begins at about 6 hr (Li and Smith, 1997; data not shown). Using cultures induced for meiosis in this way, we observed transient breakage of the chromosomal DNA. Before induction and for the first hour after induction, the chromosomal DNA was intact (Figure 2A). At the time of DNA replication (ⵑ2 hr), the total amount of

Cells of the rec⫹ diploid strain GP338 were induced for meiosis as described in the Experimental Procedures. At the indicated times after induction, samples of cells were analyzed for DNA content by flow cytometry. DNA replication occurred between 1 and 2 hr. Additional samples from this induction were analyzed for DNA integrity (see Figures 2A and 3).

DNA observed was diminished, presumably because replicating DNA, with its “bubble” forms, fails to migrate out of the agarose wells. After this time of replication, intact DNA molecules of the two largest chromosomes largely disappeared, and those of the smallest diminished in intensity. After 4.5 hr, intact DNA molecules

Figure 2. DNA Breakage during Meiosis Requires rec Gene Products S. pombe cells were harvested at the indicated times (hr) after the induction of meiosis, and their DNA was analyzed by pulsed-field gel electrophoresis and staining with ethidium bromide as described in the Experimental Procedures. (A) Diploid rec⫹ strain GP338; (B) haploid rec⫹ strain GP535; (C) diploid rec12-117 strain GP2740; (D) haploid rec6-103 strain GP701. The bands in the mitotic (0 hr) samples are, from top to bottom, the wells into which the DNA was loaded, chromosome I (5.7 Mb), chromosome II (4.6 Mb), and chromosome III (3.5 Mb). The smear (*) is broken DNA apparent during meiosis in the rec⫹ strains but not in the rec mutant strains. The arrow at the left indicates occasionally observed mechanically broken DNA due to premature lysis of the spheroplasts during preparation.

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Figure 3. Meiotic DNA Breakage Occurs at a Limited Number of Prominent Sites Cells of strain GP338 (rec⫹ diploid) were induced for meiosis, and their DNA was analyzed as in Figure 2, using altered electrophoresis conditions. (A) Ethidium bromide–stained gel. S. cerevisiae chromosomal DNA markers are in lane M. The arrow indicates mechanically broken DNA (see Figure 2). Intact S. pombe chromosomal DNA is unresolved, in the region from the top of the wells to the 1.6 Mb marker. (B) Southern blot hybridization with a radioactive probe containing the ura1 gene located about 0.75 Mb from the left end of chromosome I. The band marked with an asterisk (*) is deduced to extend from the left telomere, through the ura1 gene, to the meiotic break site. The band marked with a caret (⬎) is inferred to result from two meiotic breakages (see Results). The band in the rightmost lane reflects cross-hybridization of the ura1 probe to the S. cerevisiae URA2 gene on chromosome X (0.75 Mb). (C) Southern blot hybridization with a radioactive probe containing the rae1 gene located about 0.2 Mb from the right end of chromosome II.

reappeared. Concurrently with the disappearance of intact chromosomal DNA, broken DNA molecules migrating more rapidly appeared and were most abundant at 3–3.5 hr. Haploid pat1–114 cells also undergo meiosis, although their incomplete chromosome complement results in improper nuclear divisions and spore formation (Iino and Yamamoto, 1985; Nurse, 1985). Nevertheless, the DNA of pat1–114 haploids behaved similarly to that of diploids: at about 3–4 hr, intact DNA was less abundant and broken DNA appeared, and at about 4.5 hr intact DNA reappeared (Figure 2B). In a similarly treated pat1⫹ culture, in which meiosis is not induced, intact DNA remained, and no broken DNA appeared throughout this time course (data not shown). We conclude that S. pombe DNA is broken between premeiotic replication and the first meiotic division but then returns to the intact state. Both Meiotic Recombination and DNA Breaks Require Multiple rec Gene Products To determine whether the meiotic breakage of DNA is related to recombination, we examined several rec mutants, which are deficient in meiotic recombination but normal in other examined aspects of DNA metabolism (Ponticelli and Smith, 1989; DeVeaux et al., 1992; Li and Smith, 1997). In a rec12 mutant diploid, intact DNA molecules were observed throughout the time course, and little or no broken DNA was observed (Figure 2C). Similarly, in a rec6 mutant haploid, intact DNA and little broken DNA were observed (Figure 2D). Similar results were obtained with rec7, rec8, rec10, and rec14 mutants (data not shown). These results show that the meiotic DNA breakage is dependent upon the products of six rec genes, which are also required for meiotic recombination. Meiotic DNA breakage is thus intimately associated with meiotic recombination. Prominent Meiotic DNA Breaks Occur at a Limited Number of Sites To determine the nature of the broken DNA in rec⫹ meiotic cells, the DNA was analyzed by electrophoresis

under conditions that separate DNA molecules smaller than intact molecules; total DNA was observed by staining the gel with ethidium bromide. With this procedure, the broken DNA, most abundant at 3–4 hr after meiotic induction, appeared to be primarily in the size range of 0.1–1.5 Mb, although some of the DNA was larger than this range (Figure 3A). Remarkably, the sizes of the broken DNA did not appear to be a continuum. Rather, some discernible bands were evident, suggesting that the DNA was broken at a limited number of sites. Further evidence that the meiotic DNA breaks occur at a limited number of sites, or clusters of sites, was obtained by Southern blot hybridization of the fractionated DNA with radioactive probes from various loci in the genome. Using a probe from the ura1 gene, located about 0.75 Mb from the left end of chromosome I (5.7 Mb long; Fan et al., 1989; www.sanger.ac.uk/Projects/ S_pombe), we observed a set of discrete DNA fragments ranging from about 0.8 Mb to 1.5 Mb long (Figure 3B); this pattern of fragments was observed in four additional meiotic inductions of diploid and haploid rec⫹ strains (data not shown). These fragments were most abundant at 3–4 hr after meiotic induction, the time at which DNA fragmentation was observed above (Figures 2A and 3A). We interpret the most prominent bands as DNA molecules extending from the left telomere, through the ura1 probe, to sites of prominent breakage. For example, the band marked with an asterisk in Figure 3B had an apparent electrophoretic mobility of about 0.8 Mb, consistent with a prominent meiotic DNA break site being located just to the right (the telomere-distal side) of the ura1 gene. Other prominent bands in Figure 3B, with apparent mobilities of about 1.0, 1.3, and 1.5 Mb, indicate additional break sites, or clusters of sites, located about 200 kb apart. A less prominent band, marked with a caret, had an apparent mobility of about 0.55 Mb, too short to extend from the telomere to the ura1 probe. We infer that this fragment results from two meiotic breaks, one to the left and one to the right of the ura1 probe, consistent with this fragment being less abundant than the more prominent, singly broken fragment

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of 0.8 Mb. The other fainter bands at about 0.9, 1.2, and 1.4 Mb may also represent doubly broken fragments. If so, the prominent break sites are located about 200 kb apart, as noted above. Meiotic DNA breakage occurs at a limited number of sites in other regions of the genome as well. For example, probing with a fragment from the rae1 gene, near the right end of chromosome II, revealed prominent meiotic fragments of about 0.4, 0.6, 0.7, and 1.1 Mb (Figure 3C). Probes from loci near other chromosomal ends gave similar, but different, patterns of prominent bands with mobilities reflecting break sites, or clusters of sites, about 100–300 kb apart (data not shown). These bands were most abundant at 3–4 hr after meiotic induction but were not detectable at 0 or 6 hr. Those tested (near ura1) were also not detectable in rec6, rec7, rec12, or rec14 mutants and were diminished in intensity in rec8 and rec10 mutants (data not shown). These results imply meiosis-specific, rec gene–dependent DNA breakage at multiple but discrete sites at many places in the genome. Discussion We report here the occurrence of meiosis-induced, rec gene–dependent DNA breaks in S. pombe, only the second organism (after S. cerevisiae) in which such breaks have been reported. Zenvirth and Simchen (2000) have also reported DNA breaks during S. pombe meiosis that persist in an rhp51 mutant, lacking a homolog of the E. coli RecA and the S. cerevisiae Rad51 DNA strand exchange proteins. Similarities of Meiotic DNA Breaks in S. pombe and in S. cerevisiae In some respects, the DNA breaks in S. pombe reported here resemble those in S. cerevisiae (Roeder, 1997). In both of these distantly related yeasts, the breaks occur at or just after the time of premeiotic DNA replication and are repaired at about the time of (presumably before) the first meiotic division (Figures 1–3). In both organisms, breaks occur in haploids, indicating that homolog interactions are not necessary for their occurrence (De Massy et al., 1994; Gilbertson and Stahl, 1994; Figure 2B). At least in S. pombe, the broken DNA in haploids is returned to the intact state (Figure 2B), either by rejoining or by repair through interaction with the sister chromatid. In S. cerevisiae, the number of breaks per chromosome or genome is approximately the same as the number of recombination events, consistent with one break giving rise to one event (Baudat and Nicolas, 1997). In S. pombe, there are about 50 crossovers per meiosis (Munz et al., 1989). We estimate, from one break site every 100–300 kb (Figure 3; data not shown), about 50–150 break sites in the 14 Mb genome. At the break site near ura1, about 5%–10% of the DNA was broken at the times assayed by phosphorimager analysis of a meiosis-specific subfragment in restriction enzyme– digested DNA (Figure 3B; data not shown). The cumulative amount broken throughout meiosis may, however, be more than this. In the double-strand break repair model of Szostak et al. (1983), only one of the four chromatids is broken at one site. Thus, in S. pombe, the

site near ura1, and perhaps others, may be used for recombination in more than 20%–40% of the meioses. In both organisms, meiotic DNA breakage requires the function of multiple gene products that are also required for meiotic recombination (Roeder, 1997; Fox and Smith, 1998; Figure 2; data not shown). This relation indicates that the breaks are intimately associated with meiotic recombination, presumably in a precursor– product relation. In S. pombe, the meiosis specificity of the breaks is consistent with the meiosis specificity of the expression of the rec genes and of the phenotypes of the rec mutants (Fox and Smith, 1998). A critical component for breakage in S. cerevisiae is Spo11, which is covalently attached to the 5⬘ end of the broken DNA presumably via Tyr-135 (Bergerat et al., 1997; Keeney et al., 1997). S. pombe Rec12, a homolog of Spo11, is also essential for break formation (Figure 2C; additional data not shown), and Tyr-98, at the position homologous to Tyr-135 in Spo11, is essential for meiotic recombination (Table 1). Since in both cases a Tyr→Phe alteration was tested, a single oxygen atom in Spo11 or Rec12 is indispensable for breakage or recombination or both. These results imply that Spo11 and Rec12 act in a similar manner, though we have not determined whether a protein is bound to the meiotic broken DNA in S. pombe. Other homologous proteins are also needed for breakage and recombination in the two organisms. These include S. pombe Rec7 and Rec14, which share limited homology with S. cerevisiae Rec114 and Rec103 (⬅Ski8), respectively (Fox and Smith, 1998). Apparent Differences between Meiotic DNA Breaks in S. pombe and S. cerevisiae Although these two yeasts share some homologous proteins needed for meiotic recombination and DNA breakage, there are differences, too. S. pombe Rec6 and Rec10 are essential for wild-type levels of broken DNA (Figure 2D; data not shown), but there are no obvious homologs of these proteins in S. cerevisiae. S. pombe rec8 mutants have reduced levels of broken DNA (data not shown), but S. cerevisiae rec8 mutants, lacking a homologous protein, have wild-type levels at the sites tested (Klein et al., 1999). In S. cerevisiae, Rec102, Rec104, Mer2, and Mei4 are essential for DNA breakage (Roeder, 1997), but S. pombe has no obvious homologs of these proteins, although they may be encoded in the ⵑ10% of unreported S. pombe DNA sequences. These observations imply that, although the basic mechanism of DNA breakage by Spo11 or Rec12 may be the same, some aspect such as its regulation is different in the two organisms. This difference may be related to the presence of a full-fledged synaptonemal complex and crossover interference in S. cerevisiae but their absence in S. pombe (Roeder, 1997; Fox and Smith, 1998). A major difference in the two yeasts is the spacing between prominent meiotic DNA break sites. In two thoroughly analyzed regions on chromosome III of S. cerevisiae, breaks occur in most promoters and with a mean spacing of 2–3 kb (Baudat and Nicolas, 1997). In contrast, the prominent breaks, or clusters of breaks, that we have detected in S. pombe are spaced about 100– 300 kb apart (Figure 3; data not shown). In addition,

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where tested, DNA break sites and recombinational hotspots are coincident in S. cerevisiae (Lichten and Goldman, 1995). Although DNA breaks were readily detected elsewhere in the S. pombe genome, they were not present at detectable levels at or near the M26 hotspot (Ponticelli, 1988; Ba¨hler et al., 1991; data not shown). Prominent breaks were not detected in the 100 kb regions to the left and right of the prominent break site near ura1 (Figure 3B; data not shown). If recombination were limited to 1–2 kb around the prominent break sites, as it appears frequently to be in S. cerevisiae (Lichten and Goldman, 1995), the genetic map of S. pombe would have clusters of genes (those between break sites) separated by spaces of recombination (those containing break sites). Genes are, however, widely distributed on the S. pombe genetic map (Munz et al., 1989). These results imply that recombination occurs at a substantial distance (up to ⵑ100 kb) from the observed prominent DNA break sites. Recombination remote from these sites may stem from breaks distributed between these sites and occurring at undetectable levels. Alternatively, recombination may stem from the observed DNA breaks and occur at substantial distances from them, as is the case for the RecBCD pathway of recombination in Escherichia coli (Myers and Stahl, 1994; Smith et al., 1995). In this case, RecBCD enzyme moves from the break site and promotes recombination at a Chi hotspot at least as far as 30 kb from the break site. Similarly, a moving entity may promote meiotic recombination in S. pombe substantial distances from the break sites, perhaps at hotspots such as M26. Experimental Procedures Strains The S. pombe diploid strains used for meiotic inductions were GP338 (h⫺/h⫺ pat1-114/pat1-114 ade6-M26/ade6-M210 end1-458/ end1-458 arg1-2/⫹ ura4-294/⫹ leu1-32/⫹) and GP2740 (h⫺/h⫺ pat1114/pat1-114 rec12-117/rec12-117 ade6-M26/ade6-52 end1? arg3124/⫹ ura4-595/⫹ pro2-1/⫹). The haploid strains (all h⫺ pat1-114 ade6-M26 end1-458) were GP535 (rec⫹), GP701 (rec6-103), GP703 (rec7-102), GP704 (rec8-110), GP709 (rec10-109), and GP952 (rec14120). The rec alleles are described by Ponticelli and Smith (1989) and DeVeaux et al. (1992), and pat1-114 by Iino and Yamamoto (1985). Sources and genealogies are available upon request. To construct rec12 mutant alleles, plasmid pYL78, containing the S. cerevisiae URA3 gene and a 2.8 kb S. pombe DNA fragment with the rec12⫹ gene (Lin and Smith, 1994), was mutagenized using the Morph DNA mutagenesis kit (5 Prime→3 Prime, Boulder, CO) and the following related oligonucleotides: oJF05 (5⬘-CATTTGCAGAGATATCTATTACAGAGATGTAG), oJF03 (TATTTC; rec12-164 [Y98F]), and oJF04 (TTTTAC; rec12-165 [Y97F]). The underlined nucleotides indicate the positions of the mutated codons; the EcoRV site, indicated in bold, does not change coding but allowed identification of mutant plasmids, which were confirmed by sequencing and introduced into strain GP1459 (h⫹ rec12-152::LEU2 ade6-M26 leu1-32 ura4-294; Lin and Smith, 1994) by transformation to Ura⫹. These strains were crossed with GP1456 (h⫺ rec12-152::LEU2 ade6-52 leu1-32 ura4-294; Lin and Smith, 1994). Induction of Meiosis and Analysis of DNA Strains were grown to saturation at 25⬚C in YEL-rich medium (see Lin and Smith, 1994) supplemented with adenine (100 ␮g/ml), diluted 1:100 into EMM2* minimal medium (see Lin and Smith, 1994) with adenine (75 ␮g/ml), and grown to saturation. This culture was diluted to OD600 ⫽ 0.1 in 800 ml of EMM2* with adenine (75 ␮g/ml) and grown to OD600 ⫽ 0.3 (ⵑ1 ⫻ 107 cells/ml). The cells were harvested by centrifugation at 5000 rpm for 5 min, washed once with water,

suspended in EMM2* with adenine (10 ␮g/ml) but lacking NH4Cl, and shaken at 25⬚C for 16–20 hr to arrest the cells in G1. NH4Cl (to 5 mg/ml) and adenine (to 75 ␮g/ml) were added, and the temperature was raised to 34⬚C to induce meiosis. Samples (1 ml) were processed and analyzed for DNA content by flow cytometry as described (Li and Smith, 1997). Cells from 30 ml samples were converted to spheroplasts and embedded in agarose plugs as described (Maule, 1997) except that lyticase (Sigma) was substituted for Zymolyase. Because cells before, during, and after meiosis differ in their sensitivity to lysing enzymes, the treatment dose was adjusted to achieve maximal spheroplasting (monitored microscopically as lysis upon addition of 0.1% SDS) with minimal premature lysis (which results in mechanical breakage of the DNA). Typically, 0 hr samples were treated with lyticase (0.25 mg/ml) and Novozyme (1.25 mg/ml; Calbiochem) at 37⬚ for 1 hr; 1–2 hr samples used one-half the enzyme concentrations for 30 min; 2.5–4 hr samples used one-quarter the enzyme concentrations for 30 min; and 4.5–6 hr samples used one-half the enzyme concentrations for 45 min. Agarose plugs, ⵑ0.1 ml and containing the equivalent of ⵑ3 ⫻ 107 cells, were treated with proteinase K (1 mg/ml in 0.45 M ETDA, 10 mM Tris–HCl [pH 9.0], 1% sarkosyl) at 37⬚C for 2 days and washed twice and stored in this buffer at 4⬚C. Before electrophoresis, plugs were soaked ⬎1 hr in the running buffer (1 ⫻ TAE for Figure 2 and 0.5 ⫻ TBE for Figure 3) and molded into 0.8% “chromosomal grade” agarose (Bio-Rad) gels. Electrophoresis was at 14⬚C in a pulsed-field gel apparatus (CHEF-DR III or Mapper; Bio-Rad) using 2 V/cm, 30 min switch time, 100⬚ included angle, 48 hr for Figure 2; and 6 V/cm, linearly ramped 60–120 s switch time, 120⬚ included angle, 24 hr for Figure 3. After staining for 30 min with ethidium bromide (1 ␮g/ml), the gel was UV irradiated (0.8 J/m2), soaked for 15 min in 0.5 N NaOH, 1.5 M NaCl, and transferred to a Hybond N⫹ nylon membrane (Amersham Pharmacia). The membrane was soaked for 5 min in 0.5 M Tris–HCl (pH 7.0), rinsed in 2 ⫻ SSC, and hybridized with randomly 32P-labeled DNA. The ura1 probe was the 1.8 kb KpnI-SacI fragment of plasmid pMF48; ura1 is on cosmid c22G7 (GenBank accession number Z54328). The rae1 probe was a 1.0 kb PCR product using 25 nucleotide primers with 5⬘ ends at positions 31215 and 32229 of cosmid c16A3 (GenBank accession number AL021748). Acknowledgments We are grateful to Fred Ponticelli and Jeff Virgin for strains; Drora Zenvirth and Giora Simchen for sharing unpublished observations; Randy Schreckhise for technical assistance; Steve Henikoff for pulsed-field gel electrophoresis equipment; Paul Nurse and members of his lab at the ICRF in London for their generous hospitality to G. R. S. during a sabbatical visit; Mike Lyne and Val Woods at the Sanger Center in Cambridge, UK, for S. pombe nucleotide sequences and John Sgouros at the ICRF in London for help in analyzing them; Sue Amundsen, Luther Davis, Dan Gottschling, Jim Roberts, Mark Roth, Walt Steiner, Andrew Taylor, and Meng-Chao Yao for helpful comments on the manuscript; and Karen Brighton for preparing it. This research was supported by grants GM31693 and GM32194 and a Fogarty Center Senior International Fellowship TW02321 from the National Institutes of Health and a Research Travel Fellowship from the Burroughs Wellcome Fund to G. R. S. Received January 25, 2000; revised March 6, 2000. References Ba¨hler, J., Schuchert, P., Grimm, C., and Kohli, J. (1991). Synchronized meiosis and recombination in fission yeast: observations with pat1-114 diploid cells. Curr. Genet. 19, 445–451. Baudat, F., and Nicolas, A. (1997). Clustering of meiotic doublestrand breaks on yeast chromosome III. Proc. Natl. Acad. Sci. USA 94, 5213–5218. Bergerat, A., de Massy, B., Gadelle, D., Varoutas, P.-C., Nicolas, A., and Forterre, P. (1997). An atypical topoisomerase II from archaea with implications for meiotic recombination. Nature 386, 414–417. Cao, L., Alani, E., and Kleckner, N. (1990). A pathway for generation

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