During meiosis DNA double-strand breaks initiate recombination in the distantly related budding and fission yeasts and perhaps in most eukaryotes. Repair of broken meiotic DNA is essential for formation of viable gametes. We report here distinct but overlapping sets of proteins in these yeasts required for formation and repair of double-strand breaks. Meiotic DNA breakage in Schizosaccharomyces pombe did not require Rad50 or Rad32, although the homologs Rad50 and Mre11 are required in Saccharomyces cerevisiae; these proteins are required for meiotic DNA break repair in both yeasts. DNA breakage required the S. pombe midmeiosis transcription factor Mei4, but the structurally unrelated midmeiosis transcription factor Ndt80 is not required for breakage in S. cerevisiae. Rhp51, Swi5, and Rad22 + Rti1 were required for full levels of DNA repair in S. pombe, as are the related S. cerevisiae proteins Rad51, Sae3, and Rad52. Dmc1 was not required for repair in S. pombe, but its homolog Dmc1 is required in the well-studied strain SK1 of S. cerevisiae. Additional proteins required in one yeast have no obvious homologs in the other yeast. The occurrence of conserved and nonconserved proteins indicates potential diversity in the mechanism of meiotic recombination and divergence of the machinery during the evolution of eukaryotes.

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IN most eukaryotes homologous recombination occurs at high levels during meiosis to aid the proper segregation of homologs at the first meiotic division and to increase genetic diversity among gametes, the products of meiosis. The two meiotic cell divisions reduce the diploid number of chromosomes in the precursor cells to the haploid number in the gametes. The general mechanism of reductional segregation of homologs is highly conserved: in most eukaryotes recombination between homologs provides a physical connection between them that imparts tension when the homologs are properly arranged to segregate to opposite poles of the cell (NICKLAS 1997). In the absence of recombination, homologs frequently missegregate, resulting in aneuploid gametes; the subsequent zygotic progeny are frequently sick or dead, underscoring the importance of understanding meiotic recombination.

The general mechanism of meiotic recombination may also be highly conserved: double-strand (ds) breaks in DNA may initiate meiotic recombination in most or all eukaryotes, and repair of these breaks by interaction with a homolog can produce recombinants. Meiotic ds breaks associated with recombination were first observed in the budding yeast Saccharomyces cerevisiae (SUN et al. 1989; CAO et al. 1990). Such breaks have been observed more recently in a second organism, the distantly related fission yeast Schizosaccharomyces pombe (CERVANTES et al. 2000). There are two lines of indirect evidence for meiotic ds breaks in other organisms. First, various eukaryotes encode homologs of the Spo11 protein, which is essential for meiotic DNA ds break formation in S. cerevisiae (KEENEY et al. 1997). Where tested in other organisms, these proteins are essential for meiotic recombination or viable gamete formation (KEENEY 2001), and in S. pombe the homolog, called Rec12, is also essential for meiotic DNA ds break formation (CERVANTES et al. 2000). Second, in mice a modified histone, -H2AX, thought to associate with chromatin specifically near ds breaks, appears as foci on meiotic chromosomes at the time expected for recombination; these foci are Spo11 dependent (ROGAKOU et al. 1998; MAHADEVAIAH et al. 2001).

The Spo11 protein of S. cerevisiae makes meiotic DNA ds breaks by cleaving phosphodiester bonds at nearby positions on complementary strands and becomes covalently linked to the 5' DNA ends via phosphotyrosine bonds (KEENEY et al. 1997; KEENEY 2001). The tyrosine thought to make this bond, Tyr-135, is within an amino acid sequence that is similar in Spo11 proteins from diverse organisms. The corresponding tyrosine of S. pombe Rec12, Tyr-98, is essential for meiotic recombination (CERVANTES et al. 2000). These observations suggest that the chemistry of meiotic DNA ds break formation is highly conserved.

Other aspects of the mechanism of DNA ds break formation in meiosis appear to be less conserved. S. cerevisiae Spo11 does not make meiotic DNA breaks on its own: nearly a dozen other proteins are required for ds break formation (KEENEY 2001). Some, but not all, of these other proteins have homologs in S. pombe and other organisms. Similarly, some proteins in addition to Rec12 that are required for meiotic ds break formation in S. pombe do not have obvious homologs in S. cerevisiae (DAVIS and SMITH 2001; see DISCUSSION). These nonconserved, additional proteins may regulate the timing and positioning of ds break formation by the conserved Spo11-related proteins.

The repair of ds breaks is complex and not yet as well understood as their formation. In S. cerevisiae the 5' ends at ds breaks are resected a few hundred nucleotides; the resultant 3' single-strand (ss) tails invade a homolog, producing a joint molecule that contains hybrid DNA (SCHWACHA and KLECKNER 1994, 1995; HUNTER and KLECKNER 2001). Resection requires the Rad50·Mre11· Xrs2 complex, which has exonuclease activity, but this activity digests in the 3' 5' direction, as does the homologous protein Rad50·Mre11·Nbs1 from humans (HABER 1998). Presumably, a different nuclease, or a controlling factor, complexed with Rad50·Mre11·Xrs2 is responsible for meiotic resection in S. cerevisiae. Joint molecule formation requires the S. cerevisiae Rad51 and Dmc1 proteins, homologs of the Escherichia coli RecA strand-transfer protein (SCHWACHA and KLECKNER 1994, 1995; SUNG et al. 2000; SUGAWARA et al. 2003; WOLNER et al. 2003). This step is aided by several additional proteins, including Rad52, in S. cerevisiae. Proteins required for the resolution of joint molecules into recombinant molecules have not been firmly established, but recent evidence implicates the Mus81·Eme1 complex in resolution of joint molecules containing Holliday junctions into crossover recombinants in S. pombe (BODDY et al. 2001; GAILLARD et al. 2003; OSMAN et al. 2003; SMITH et al. 2003).

To assess the generality of the mechanism of meiotic recombination, we investigated the protein requirements for formation and repair of DNA ds breaks during meiosis in S. pombe and compared the results with published data from S. cerevisiae. We find here that certain homologous proteins are required for breakage or repair in both yeasts; other proteins, however, are required in one yeast but not in the other. These results imply that, although the basic ds break-join mechanism occurs in both yeasts, certain aspects of this mechanism, such as its regulation, differ in these distantly related organisms.

Strains and culture media:

The S. pombe strains used for meiotic DNA analysis and their genotypes are listed in Table 1 . Sources of alleles inactivating recombination genes are in Table 1, footnote a. The alleles in the strains listed or in the relevant parent were confirmed by nucleotide sequencing of appropriate DNA fragments generated by PCR. Genealogies are available upon request. Culture media are described by GUTZ et al. (1974).

View this table: In this window In a new window   TABLE 1 S. pombe strains for meiotic DNA break determination

 

Induction of meiosis and analysis of DNA:

Strains were grown, induced for meiosis, and analyzed for cellular DNA content and meiotic DNA breakage as described by YOUNG et al. (2002). In brief, cells were grown at 25° in appropriately supplemented Edinburgh minimal medium 2 (EMM2), arrested in G₁ by starvation for nitrogen, and induced for meiosis by adding NH₄Cl and raising the temperature to 34° to inactivate the pat1-114 (Ts) protein kinase. At the indicated times cells were harvested by centrifugation, suspended in agarose plugs, and lysed with enzymes. After successive treatments with proteinase K and, when indicated, NotI restriction enzyme, the DNA in the plugs was subjected to pulsed-field gel electrophoresis. NotI-digested DNA was analyzed by Southern blot hybridization using radioactive probes homologous to the ends of the NotI fragment J; results with the right end probe (data not shown) were similar to those with the left end probe shown in Figure 2. Each mutant was induced two to five times with similar results.

View larger version (238K): In this window In a new window Download PPT slide   FIGURE 2.— Meiotic DNA breakage at prominent sites on the 501-kb NotI fragment J of chromosome I. Cells with the indicated genotype were induced for meiosis and harvested at the indicated times (hours); the DNA was digested with NotI and analyzed by pulsed-field gel electrophoresis and Southern blot hybridization using a probe from the left end of the NotI fragment J as described in MATERIALS AND METHODS. The topmost band is the unbroken NotI fragment J; lower bands reflect meiosis-specific breaks at the prominent meiotic break sites, such as mbs1 and mbs2 (top and bottom carets, respectively), described by YOUNG et al. (2002). The percentages of meiosis-specific DNA breakage (starred lane minus 1-hr lane) at mbs1 and mbs2 are indicated. The inductions correspond to those in Figure 1. (A) Wild-type (rec+ rad+, etc.) strain GP535. (B) rad50 strain GP3652. (C) rad32 strain GP2496. (D) rhp51 strain GP3581. (E) dmc1 strain GP3607. (F) rhp51 dmc1 strain GP4233. (G) swi5-39 strain GP813. (H) rad22 rti1 strain GP3269. (I) mus81 strain GP3574. (J) mei4 strain GP3650. Similar results were obtained in other experiments using these or other strains containing the indicated mutations (data not shown).

 

Viable spore yields and meiotic recombinant frequencies:

Haploid pat1⁺ strains were grown to saturation in yeast extract liquid medium supplemented with adenine (100 µg/ml). Cells were mated and allowed to undergo meiosis at the indicated temperature on SPA sporulation agar or EMM2 minimal agar supplemented with all required nutrients. Haploid pat1-114 strains were grown and induced for meiosis as described above. The ascus-cell suspensions were harvested after 2–3 days (pat1⁺ strains) or at 24 hr (pat1-114 strains) and treated with glusulase and EtOH to kill vegetative cells as described by GUTZ et al. (1974). Total spore yields were determined by plating on yeast extract agar (YEA) supplemented with adenine (100 µg/ml), and Ade⁺ spore yields were determined by plating on YEA-rich agar supplemented with guanine (200 µg/ml), which inhibits uptake of adenine (CUMMINS and MITCHISON 1967). Viable spore yields are expressed as the number of viable spores produced divided by the number of viable pat1⁺ cells of the less numerous parent in the mating mixture or by the number of viable pat1-114 cells induced. Ade⁺ recombinant frequencies are expressed as the Ade⁺ spore yield divided by the total viable spore yield.

We prepared DNA from synchronously induced meiotic cells and assayed it for breakage using two types of pulsed-field gel electrophoresis (CERVANTES et al. 2000; YOUNG et al. 2002; see MATERIALS AND METHODS). First, we analyzed the DNA under conditions that separate the three intact chromosomal DNAs—5.7, 4.6, and 3.5 Mb for chromosomes I, II, and III, respectively. DNA from wild-type (i.e., rec⁺ rad⁺, etc.) cells before and up to 2 hr after induction of meiosis was mostly intact (Figure 1A) . Between 3 and 4 hr after meiotic induction, i.e., after meiotic DNA replication was complete (data not shown; CERVANTES et al. 2000), intact chromosomal DNA largely disappeared, and more rapidly migrating (broken) DNA appeared. After 4 hr, intact chromosomal DNA reappeared, 1 hr before the first meiotic division (see, for example, LI and SMITH 1997). Broken DNA does not appear in rec12 or in seven other rec mutants that are deficient in meiotic recombination, indicating that this breakage is intimately associated with meiotic recombination (CERVANTES et al. 2000; YOUNG et al. 2002).

View larger version (271K): In this window In a new window Download PPT slide   FIGURE 1.— Breakage and repair of meiotic chromosomal DNA. S. pombe cells with the indicated genotype were induced for meiosis and harvested after the indicated times (hours); DNA was analyzed by pulsed-field gel electrophoresis and stained with ethidium bromide as described in MATERIALS AND METHODS. The bands in the mitotic (0 hr) lanes are, from top to bottom, the loading wells and chromosomes I, II, and III (5.7, 4.6, and 3.5 Mb, respectively). The smear (bracketed) below chromosome III is meiosis-specific broken DNA that appears transiently in wild-type cells but accumulates in some mutants, such as rad50. The broken bracket in J indicates a low level of mechanically broken DNA (not meiosis specific) that occasionally appears, such as in this induction of mei4. (A) Wild-type (rec+ rad+, etc.) strain GP535. (B) rad50 strain GP3652. (C) rad32 strain GP2496. (D) rhp51 strain GP3581. (E) dmc1 strain GP3607. (F) rhp51 dmc1 strain GP4233. (G) swi5-39 strain GP813. (H) rad22 rti1 strain GP3269. (I) mus81 strain GP3574. (J) mei4 strain GP3650. Similar results were obtained in other experiments using these or other strains containing the indicated mutations (data not shown).

 
Second, we analyzed the DNA after digestion with the rare-cutting restriction enzyme NotI, followed by electrophoresis and Southern blot hybridization with a probe homologous to the end of the 501-kb fragment J of chromosome I (FAN et al. 1989). We reported previously that this fragment contains six meiosis-specific break sites (mbs; or clusters of sites), including the prominent sites mbs1 and mbs2 (YOUNG et al. 2002). Breakage at one or another of these sites produces DNA fragments that migrate more rapidly than the intact fragment J. In accord with the analysis of DNA without restriction enzyme digestion, DNA broken at these sites appeared between 2.5 and 5 hr after meiotic induction but not before or after (Figure 2A) . DNA broken at these sites does not appear in rec mutants, showing that breakage at these specific sites, like general chromosomal DNA breakage, is recombination associated (CERVANTES et al. 2000; YOUNG et al. 2002). We used these two assays to analyze meiotic DNA breakage and repair in mutants suggested by previous studies to be altered in breakage, repair, or both.

S. pombe Rad50 and Rad32 proteins are required for meiotic DNA repair but not for meiotic DNA breakage:

The S. cerevisiae Rad50·Mre11·Xrs2 complex has a dual role in meiotic recombination: it is required for both DNA ds breakage and repair (HABER 1998). The S. pombe Rad50 and Rad32 proteins have 35 and 45% amino acid sequence identity with S. cerevisiae Rad50 and Mre11, respectively (TAVASSOLI et al. 1995; HARTSUIKER et al. 2001). S. pombe rad50 and rad32 mutants are defective in repair of mitotic DNA ds breaks and in formation of viable gametes (spores; TAVASSOLI et al. 1995; HARTSUIKER et al. 2001; see below). We therefore assayed these mutants for meiotic DNA breakage and repair.

In both rad50 and rad32 mutants broken DNA began to appear 3 hr after meiotic induction, was abundant by 4 hr, and remained detectable for up to 8 hr (Figure 1, B and C). In some inductions the intact chromosomal DNA did not disappear to the extent seen in wild-type inductions at 4 hr. Since in vegetative cultures of rad50 and rad32 mutants up to 75% of the cells do not divide (TAVASSOLI et al. 1995; HARTSUIKER et al. 2001), we presume that the residual intact chromosomal DNA reflects these cells, which may not enter meiosis. Breakage at mbs1 and mbs2 was also evident in both mutants at 3 hr and persisted for at least 8 hr after induction (Figure 2, B and C). The frequency of DNA breakage at mbs1 was 6% in wild-type cells (Figure 2A) and 11% in rad50S mutants in which broken DNA accumulates (YOUNG et al. 2002), but was only 2% in the rad50 and rad32 mutants. We presume this reduction reflects dead cells in the mutant cultures, as noted above. We cannot exclude, however, the possibility that Rad50 and Rad32 are required for the full, wild-type level of breakage. Nevertheless, these physical analyses showed that meiotic DNA breakage, including breakage at or near mbs1 and mbs2, did occur in rad50 and rad32 mutants. Little, if any, of this broken DNA was repaired in a timely manner.

These observations predict that formation of viable spores, which requires intact chromosomes, would be much reduced, as previously reported for rad32 mutants (TAVASSOLI et al. 1995), but would be rescued by a mutation blocking meiotic DNA breakage. To test this prediction, we measured the viable spore yields of rad50 and rad32 mutants with or without additional mutations in rec6 and rec12, whose products are required for meiotic DNA breakage (CERVANTES et al. 2000). The results in Table 2 support these predictions. The rad50 or rad32 mutation or their combination reduced viable spore yields by a factor of 1000, relative to wild type, and the rec6 or rec12 mutation increased the yields of the rad50 and rad32 mutants by 70-fold. In rec6 or rec12 mutants, the yields were reduced by a factor of 6–10 relative to wild type, at least in part because of faulty chromosome segregation (PONTICELLI and SMITH 1989; SHARIF et al. 2002; DAVIS and SMITH 2003). The rec6 or rec12 mutation increased the yields of the rad50 and rad32 mutants to within a factor of 2 or 3 of the yields of the rec6 and rec12 single mutants; the slightly lower yields of the double mutants may reflect reduced spore germination or reduced mitotic viability of rad50 and rad32 mutants, as noted above (TAVASSOLI et al. 1995; HARTSUIKER et al. 2001).

View this table: In this window In a new window   TABLE 2 Genetic evidence that Rad32 and Rad50 are not required for meiotic DNA breakage: rec mutations blocking DNA breakage suppress the low viable spore yield of rad32 and rad50 deletion mutants

 
In summary, the physical and genetic data reported here show that the S. pombe Rad50 and Rad32 proteins are not essential for meiotic DNA breakage but are needed for repair of these breaks.

S. pombe Rhp51 protein is required for meiotic DNA repair but Dmc1 protein is not:

Two homologs of the E. coli RecA strand-transfer protein, designated Rad51 and Dmc1, are required for repair of meiotic broken DNA in S. cerevisiae (ROEDER 1997). We tested the S. pombe mutants lacking the homologs, designated Rhp51 and Dmc1 (MURIS et al. 1993; SHINOHARA et al. 1993; FUKUSHIMA et al. 2000), for meiotic DNA breakage and repair. In both rhp51 and dmc1 mutants bulk DNA breakage occurred at 3 hr (Figure 1, D and E), as in wild-type cells (Figure 1A). In the dmc1 mutant repair occurred at 5 hr, as in wild-type cells, but in the rhp51 mutant broken DNA remained, as in the rad50 and rad32 mutants discussed above. ZENVIRTH and SIMCHEN (2000) also concluded that meiotic DNA is broken but not repaired in an rhp51 mutant, and SHIMADA et al. (2002) also showed that whole chromosomes are broken and repaired with similar kinetics in dmc1⁺ and dmc1 strains. Analysis of NotI-digested DNA revealed a pattern similar to that of bulk chromosomal DNA: in both mutants DNA was broken at mbs1 and mbs2, and it was repaired in the dmc1 mutant but not in the rhp51 mutant (Figure 2, D and E). The rhp51 dmc1 double mutant behaved much like the rhp51 single mutant (Figures 1F and 2F); breakage in the double mutant was slightly later than that in either single mutant, perhaps because DNA replication was later in the double mutant in this experiment (data not shown). These results show that Rhp51 plays a major role in the repair of meiotic broken DNA, but Dmc1 plays at most a minor role (see DISCUSSION).

The small protein Swi5 is required for meiotic DNA repair:

S. pombe swi5 mutants were isolated on the basis of their reduced frequency of mating-type switching (GUTZ and SCHMIDT 1985). The swi5 gene encodes a polypeptide of 85 amino acids with 20% amino acid sequence identity to S. cerevisiae Sae3, which is required for meiotic DNA break repair (MCKEE and KLECKNER 1997; AKAMATSU et al. 2003; see DISCUSSION). The frequencies of intragenic and intergenic meiotic recombination in swi5 mutants are also reduced by a factor of 5 for the intervals reported (SCHMIDT et al. 1987; DEVEAUX et al. 1992; SCHMIDT 1993). Consequently, we examined meiotic DNA breakage and repair in the swi5-39 mutant, which bears a nonsense mutation in codon 38 (AKAMATSU et al. 2003). Bulk chromosomal DNA breakage and breakage at mbs1 and mbs2 occurred at 3.5 hr (Figures 1G and 2G), approximately as in wild type. Much of the broken DNA persisted for at least 12 hr, and there was little, if any, recovery of intact chromosomal DNA by this time (Figures 1G and 2G; data not shown); repair in wild-type cells was essentially complete by 5 hr (Figures 1A and 2A). Thus, Swi5 is required for repair, but not for formation, of meiotic DNA breaks.

The deficiency of meiotic DNA repair in the swi5-39 mutant appeared similar to that of rad50 and rad32 mutants. Viable spore yield is strongly reduced in rad50 and rad32 mutants (TAVASSOLI et al. 1995; HARTSUIKER et al. 2001; Table 2), but, paradoxically, spore viability is as high in swi5 mutants as in wild type under the conditions reported (malt extract medium at 25°; SCHMIDT 1993). To address this paradox, we measured meiotic recombination and spore viability under the conditions used for meiotic DNA break determination (EMM2 at 34°) and, for comparison, at the standard temperature of 25°. The results in Table 3 show that, with respect to ade6 intragenic recombination and viable spore yield, the swi5-39 mutant is not strongly temperature sensitive. In standard matings of pat1⁺ haploids, the swi5-39 mutation reduced ade6 recombination by a factor of 10, and the viable spore yields by a factor of 3, relative to swi5⁺. Spore viability, measured after micromanipulation of ascal tetrads or random spores, was also reduced by a factor of 3 in swi5-39 relative to swi5⁺. In meiotic inductions of pat1-114 haploids, viable spore yields were similar in swi5⁺ and swi5-39 mutants, 1 viable spore/100 cells induced, as reported previously for swi5⁺ cells (IINO and YAMAMOTO 1985). Comparing pat1⁺ strains, these levels of viable spore yield and spore viability are much higher than those of rad50 and rad32 mutants (TAVASSOLI et al. 1995; HARTSUIKER et al. 2001; Table 2), even though the level of broken DNA accumulated in meiosis appears at least as high in the swi5-39 mutant as in rad50 or rad32 mutants (Figures 1 and 2). Perhaps in swi5 mutants, but not in rad50 or rad32 mutants, broken DNA is repaired late in meiosis or shortly after spore germination, thereby giving rise to higher levels of viable spores.

View this table: In this window In a new window   TABLE 3 swi5 mutation reduces meiotic recombination and spore viability

 

Rad22 and Rti1, two Rad52 homologs, are required for meiotic DNA repair:

The S. cerevisiae Rad52 protein is required for repair of meiotic DNA breaks; in its absence the broken DNA is hyperresected from its ends (SOUSTELLE et al. 2002). S. pombe contains two genes, designated rad22 (or rad22A) and rti1 (or rad22B), whose products over extensive portions have, respectively, 33 and 37% amino acid sequence identity to Rad52 (OSTERMANN et al. 1993; SUTO et al. 1999; VAN DEN BOSCH et al. 2001). Disruption of both S. pombe genes strongly reduces spore viability; single gene disruptions have only a mild effect (VAN DEN BOSCH et al. 2001). We therefore examined the rad22 rti1 double mutant for meiotic DNA breakage and repair.

In the rad22 rti1 double mutant, meiotic DNA breakage occurred at 3 hr, as in wild type, as assayed by analysis of either bulk chromosomal DNA (Figure 1H) or DNA broken at mbs1 or mbs2 (Figure 2H). Some broken DNA persisted beyond the time of its repair in wild type at 4.5 hr; bulk chromosomal DNA fragments were visible as late as 8 hr (Figure 1H). Intact DNA reappeared at 5 hr in the rad22 rti1 mutant at low level (Figure 1H) but in wild type at high level (Figure 1A). DNA fragments broken at mbs1 or mbs2 became less abundant by 6–10 hr (Figure 2H); this disappearance may reflect degradation or repair of the broken DNA or both. We conclude that neither Rad22 nor Rti1 is required for meiotic DNA breakage, but one or both proteins is required for high-level repair.

Mus81 is required after meiotic DNA breakage:

The S. pombe Mus81·Eme1 protein complex is required for production of viable spores and can cleave branched DNA molecules (BODDY et al. 2001; GAILLARD et al. 2003; OSMAN et al. 2003). Like rad50 and rad32 mutants (Table 2), mus81 and eme1 mutants produce few viable spores, and introduction of a rec6 or rec12 mutation increases viable spore yields to the level of the rec mutants (BODDY et al. 2001). These are the properties expected of mutants lacking an enzyme that cleaves joint molecules. As expected from this view, DNA breakage in a mus81 mutant occurred at the normal time (3 hr), and the broken DNA largely disappeared at approximately the normal time (5 hr; Figures 1I and 2I). Intact chromosomal DNA, however, did not consistently reappear in all experiments; the DNA may have been degraded, or it may have been in branched structures that failed to enter the gel. (We noted that in some inductions of the mus81 mutant, like those of the rad50 and rad32 mutants, intact chromosomal DNA did not entirely disappear, perhaps due to dead cells in some cultures, as noted previously.)

A meiosis-specific transcription factor, Mei4, is required for meiotic DNA breakage:

In S. cerevisiae, the midmeiosis transcription factor Ndt80 is required for the proper completion of repair, but not formation, of broken meiotic DNA; presumably, the product of an unknown gene activated by Ndt80 resolves joint molecules into crossover recombinant molecules (XU et al. 1995; ALLERS and LICHTEN 2001). In S. pombe the Mei4 forkhead-like transcription factor is induced early in meiosis, beginning at 2 hr but reaching its maximum level at 5 hr, and activates genes whose products function later in meiosis, which, for most genes, is >4 hr after induction (HORIE et al. 1998; ABE and SHIMODA 2000). Because of this functional similarity, we examined meiotic DNA in a mei4 mutant. Bulk chromosomal DNA remained largely intact for up to 8 hr after induction; little or no broken DNA was visible by assay of either bulk chromosomal DNA or DNA broken at mbs1 or mbs2 (Figures 1J and 2J). DNA replication, assayed by flow cytometry (LI and SMITH 1997), was complete and occurred at the normal time, 2 hr after induction (data not shown), as reported previously for wild-type cells (CERVANTES et al. 2000; YOUNG et al. 2002). H. MURAKAMI (personal communication) has also observed that bulk chromosomal DNA remains largely intact in a mei4 mutant during meiosis. We conclude that Mei4, or more likely the product(s) of one or more genes controlled by it, is required for meiotic DNA breakage but for not replication.

We have reported here both similarities and differences in the proteins required for meiotic DNA breakage and repair in S. pombe (Sp) and those required in the distantly related yeast S. cerevisiae (Sc). Some proteins with extensive amino acid sequence identity appear to have similar or identical functions in the two yeasts. For example, the SpRec12 and ScSpo11 proteins share an essential Tyr in a conserved sequence motif and are essential for meiotic DNA breakage; the essential Tyr is likely at the active site (KEENEY et al. 1997; CERVANTES et al. 2000; KEENEY 2001). Similarly, the SpRhp51 and ScRad51 proteins resemble the E. coli RecA strand transfer protein and are required for repair of meiotic broken DNA (ABOUSSEKHRA et al. 1992; SHINOHARA et al. 1992, 1993; MURIS et al. 1993; Figures 1D and 2D). Thus, the basic mechanisms of meiotic DNA breakage by topoisomerase-like cleavage and of repair by strand exchange with homologous DNA appear to be conserved between these two yeasts and possibly most or all eukaryotes (KEENEY 2001).

Other proteins, however, with similar sequences have different functions in the two yeasts (Table 4) ; examples include the SpRad50·Rad32·Nbs1 and the ScRad50·Mre11·Xrs2 complexes discussed below. In addition, some proteins required for meiotic DNA breakage or repair have been identified in one yeast but not in the other (Table 5) , in spite of their complete or nearly complete genome sequences being known (GOFFEAU et al. 1996; WOOD et al. 2002). Below, we discuss these similarities and differences and their implications for meiotic recombination.

View this table: In this window In a new window   TABLE 4 Summary of meiotic DNA breakage and repair in S. pombe mutants studied here and in related S. cerevisiae mutants

 

View this table: In this window In a new window   TABLE 5 Meiotic DNA breakage and repair proteins identified in one yeast but not obviously recognizable in the other

 

Proteins required for meiotic DNA breakage:

At least three proteins required for meiotic DNA breakage appear to be conserved between S. pombe and S. cerevisiae. These are SpRec12 and ScSpo11; SpRec7 and ScRec114; and SpRec14 and ScSki8 (also called Rec103). Mutations in these genes abolish meiotic DNA breakage and recombination (reviewed in DAVIS and SMITH 2001; KEENEY 2001). As noted above, Spo11 and its homologs likely contain the active site that cleaves the DNA; the other proteins may form with Spo11 a complex essential for its activity. Additional proteins apparently unique to each yeast are also essential for meiotic breakage and recombination (Table 5): these include Rec6, Rec10, and Rec15 in S. pombe and Rec102, Rec104, Rec107 (also called Mer2), and Mei4 in S. cerevisiae. (SpMei4 and ScMei4 are not related.) The absence of identifiable homologs for these proteins in interspecies comparisons may reflect a lack of amino acid sequence conservation for proteins with conserved function. Alternatively, some aspect of breakage and recombination, such as regulation of the distribution of breakage and recombination along chromosomes, may differ in these two yeasts. This hypothetical difference in regulation may be related to the presence of a full-fledged synaptonemal complex and crossover interference in S. cerevisiae but to their absence in S. pombe (KOHLI and BäHLER 1994). Consistent with this suggestion, ScHop1 and Red1, components of the synaptonemal complex, are required for high-level breakage and recombination in S. cerevisiae (KEENEY 2001), but S. pombe lacks obvious homologs.

A major difference in the proteins required for meiotic DNA breakage is the requirement for the SpRad50·Rad32·Nbs1 and ScRad50·Mre11·Xrs2 complexes. [We assume that the Sp polypeptides form a functional complex, as do the Sc and human proteins. After completion of this work, CHAHWAN et al. (2003) and UENO et al. (2003) reported that the SpNbs1 polypeptide appears to be functionally similar to ScXrs2, although they share only very limited amino acid sequence identity. The rad50, rad32, and nbs1 single, double, and triple mutants have nearly identical sensitivities to DNA-damaging agents, and tagged versions of Nbs1 and Rad32 co-immunoprecipitate.] The Sc complex is required for meiotic DNA breakage (ALANI et al. 1990; JOHZUKA and OGAWA 1995), but the Sp complex is not (Figure 1, B and C; Figure 2, B and C). Similarly, the Sc complex is required for nonhomologous end-joining (PAQUES and HABER 1999), but the Sp complex is not (MANOLIS et al. 2001). Both complexes are required, however, for mitotic and meiotic ds DNA break repair, as discussed below. As expected, S. pombe rad50 and rad32 mutants form very few viable spores, but this phenotype is largely suppressed by a rec6 or rec12 mutation (Table 2), each of which blocks meiotic DNA breakage (CERVANTES et al. 2000; YOUNG et al. 2002). In the basidiomycete Coprinus cinereus Rad50 also appears dispensable for meiotic DNA breakage: rad50 mutants arrest in meiosis I prophase, but this arrest is alleviated by a spo11 mutation, and gamma-irradiation of the rad50 spo11 double mutant restores the arrest (S. ACHARYA and M. ZOLAN, personal communication).

ScMre11 binds to meiotic chromosomes in a Spo11-dependent manner, and it has been speculated that ScRad50·Mre11·Xrs2 must be in place for it to modify chromatin or to effect subsequent repair before Spo11 makes DNA breaks (BORDE et al. 2004). If so, SpRec12 is apparently not regulated in the same way by Rad50 and Rad32, since Rec12-dependent DNA breakage occurs in the absence of Rad50 or Rad32. S. pombe may represent a simpler scheme in which meiotic DNA breakage is not coupled to the potential for repair.

Proteins required for meiotic DNA break repair:

Several proteins required for high levels of meiotic DNA break repair appear to be conserved: these include SpRad50·Rad32 and ScRad50·Mre11; SpRhp51 and ScRad51; and SpRad22 and Rti1 and ScRad52. In S. cerevisiae Rad50·Mre11·Xrs2 is required for resection of ds DNA ends to produce 3'-ended ss tails, to which Rad51 binds and promotes strand exchange with a homologous duplex (ROEDER 1997; HABER 1998). In assays using purified components, Rad52 aids Rad51 in formation of joint molecules (SUNG et al. 2000); in cells, Rad52 appears to promote binding of Rad51 to ss DNA adjacent to a ds break (SUGAWARA et al. 2003; WOLNER et al. 2003). In rad51 and rad52 mutant meiotic cells, ds DNA ends are hyperresected; presumably, joint molecules are not formed (SHINOHARA et al. 1992; SOUSTELLE et al. 2002). Resection of ds DNA ends and formation of joint molecules have not, to our knowledge, been assessed in S. pombe. Nevertheless, the persistence of broken DNA in the corresponding S. pombe mutant meiotic cells (Figures 1H and 2H) and the extensive amino acid sequence similarities between the corresponding proteins are consistent with the view that these proteins have similar functions in the two yeasts.

A second RecA homolog, Dmc1, may have a different role in the two yeasts. S. pombe dmc1 mutants repaired broken DNA at the normal time (Figures 1E and 2E), have high spore viability (75%), and have only modestly reduced meiotic recombinant frequencies (30% of wild type; FUKUSHIMA et al. 2000; GRISHCHUK and KOHLI 2003). In S. cerevisiae dmc1 mutants derived from strain SK1, meiotic ds DNA ends are hyperresected, joint molecules and recombinant molecules are made at only low levels, and cells arrest at the pachytene stage of meiosis (BISHOP et al. 1992; SCHWACHA and KLECKNER 1994). S. cerevisiae dmc1 mutants derived from a different strain, however, progress through meiosis and form spores in which the recombinant frequency is 40% of wild type (ROCKMILL 1995). Thus, Dmc1 appears to play a major role in meiotic recombination in some S. cerevisiae strains but only a minor role in other S. cerevisiae strains and in S. pombe. Nevertheless, in both S. cerevisiae and S. pombe transcription of the dmc1 gene is very strongly induced early in meiosis (BISHOP et al. 1992; FUKUSHIMA et al. 2000), suggesting an important role in meiosis. Since S. pombe dmc1 rhp51 double mutants have very low viable spore yields and recombinant frequencies (GRISHCHUK and KOHLI 2003) and accumulate broken meiotic DNA (Figures 1F and 2F), Dmc1 appears to provide an alternative to Rhp51 for DNA strand exchange, as in S. cerevisiae (ROEDER 1997). The role of the seemingly minor Dmc1 alternative in S. pombe remains to be elucidated.

The small protein Swi5 appears to be essential for the timely repair of meiotic broken DNA (Figures 1G and 2G). In swi5 mutants intragenic and intergenic recombinants, likely reflecting gene conversions and crossovers, respectively, arise at frequencies 5–10 times lower than those in swi5⁺ cells (SCHMIDT et al. 1987; DEVEAUX et al. 1992; SCHMIDT 1993; Table 3). Swi5 thus plays a major role in the repair of meiotic broken DNA and recombination. Mating-type switching in S. pombe is a type of homologous recombination that involves a ss DNA break, which may be converted to a ds break by replication (BEACH 1983; ARCANGIOLI 1998; ARCANGIOLI and DE LAHONDES 2000). swi5 mutants have nearly normal levels of broken DNA at the mat1 locus (EGEL et al. 1984) but switch at low frequencies (GUTZ and SCHMIDT 1985). These low-frequency recombinants appear to have the normal structure. Thus, Swi5 appears to be important but not absolutely essential for repairing broken DNA in both meiotic and mating-type-switching recombination.

The activity of the remarkably small Swi5 protein—85 amino acids (AKAMATSU et al. 2003)—remains to be elucidated, but recent evidence suggests that it may be involved in DNA strand exchange. Swi5 indirectly interacts physically with Rhp51 via Sfr1 (AKAMATSU et al. 2003). Swi5 shares 20% sequence identity with a 102-amino-acid form of S. cerevisiae Sae3, predicted from conceptual RNA splicing and translation (V. WOOD, personal communication). S. cerevisiae dmc1, sae3, and dmc1 sae3 double mutants have similar phenotypes: in the SK1 background they accumulate hyperresected meiotic broken DNA and arrest in the first meiotic prophase (MCKEE and KLECKNER 1997). The different phenotypes of S. pombe rhp51, dmc1, and swi5 mutants suggest, within this framework, complex interactions among strand exchange activities.

The Mus81·Eme1 complex of S. pombe has many of the properties expected for a Holliday junction resolvase. The protein cleaves Holliday junctions at or near their branch point with a preference for junctions already containing a nick (BODDY et al. 2001; GAILLARD et al. 2003). Mutants lacking either subunit form few viable spores; a rec6 or rec12 mutation increases spore viability to the level of the single rec mutants (BODDY et al. 2001), paralleling the phenotypes of the rad50 and rad32 mutants (Table 2). In the mus81 mutant, meiotic DNA breakage occurred at the normal time; the broken DNA disappeared, but intact chromosomal DNA did not consistently reappear (Figures 1I and 2I). This is the behavior expected in the absence of resolution of joint molecules. Crossing over, but not gene conversion, is severely reduced in mus81 mutants (OSMAN et al. 2003; SMITH et al. 2003). These data are thus consistent with Mus81·Eme1 being an essential Holliday junction resolvase in S. pombe. The corresponding protein in S. cerevisiae, Mus81·Mms4, also cleaves branched DNA molecules near their branch point, but mutants lacking the protein have nearly normal meiotic recombinant frequencies among the spores produced; few cells, however, produce spores (INTERTHAL and HEYER 2000; DE LOS SANTOS et al. 2003). Thus, SpMus81·Eme1 plays a more critical role in meiotic recombination than does ScMus81·Mms4. S. pombe may have a single pathway for resolution of joint molecules into recombinants, and S. cerevisiae may have two or more pathways (DE LOS SANTOS et al. 2003).

The S. pombe Rad22 and Rti1 proteins are required for high levels of meiotic DNA break repair (Figures 1H and 2H), as is the related S. cerevisiae Rad52 protein (SOUSTELLE et al. 2002). As expected, the S. pombe double mutant has very low spore viability: 3% compared to 92% for wild type in the same study (VAN DEN BOSCH et al. 2001). Curiously, the meiotic ade6 intragenic recombinant frequency among the few viable spores is near that of wild type (VAN DEN BOSCH et al. 2002). Perhaps a small subpopulation of cells repairs broken DNA normally, as suggested by the reappearance of a low level of intact DNA in the rad22 rti1 mutant (Figure 1H); this subpopulation may form viable spores with normal recombinant frequencies. Mitotic S. pombe cells also have a subpopulation with a higher-than-average frequency of recombination, and the relative size of this subpopulation is increased in rad2 mutants (GROSSENBACHER-GRUNDER 1985). The distinguishing features of these hypothetical subpopulations are unknown.

Transcriptional regulation of genes for meiotic DNA breakage and repair:

In both S. pombe and S. cerevisiae 10% of all genes are induced during meiosis by a complex interplay of physiological changes that signal starvation and transcriptional regulators that in part signal diploidy (CHU et al. 1998; MATA et al. 2002). Many of the genes whose products are involved in meiotic DNA breakage and repair are induced. In S. pombe all of the genes whose products are known to be required for meiotic DNA breakage (rec6, -7, -8, -10, -11, -12, -14, and -15 and mei4) are induced during meiosis (FOX and SMITH 1998; HORIE et al. 1998; MATA et al. 2002); of those tested (rec6, -7, -8, -10, -11, and -15), induction of all but rec10 depends upon the Rep1 (also called Rec16) protein (LI and SMITH 1997; DING and SMITH 1998), which likely forms a complex with the Cdc10 transcriptional activator (SUGIYAMA et al. 1994). As expected, rep1 (rec16) mutants are strongly deficient in meiotic recombination (DEVEAUX et al. 1992; DING and SMITH 1998); meiotic DNA breakage has not been tested in rep1 (rec16) mutants but is expected not to occur owing to the failure of induction of genes such as rec6.

A second S. pombe transcriptional activator, Mei4, is required for the induction of genes midway through meiosis and for completion of the first meiotic division (HORIE et al. 1998; ABE and SHIMODA 2000). Mei4 is not required for the induction of the rec genes tested (rec6, -7, -8, -10, and -12; ABE and SHIMODA 2000), however, so we expected that breaks would be made but perhaps not repaired; S. cerevisiae mutants lacking the structurally unrelated midmeiosis transcriptional activator Ndt80 make breaks but not crossover recombinant DNA (XU et al. 1995; ALLERS and LICHTEN 2001). Instead, we found that breaks were not made in S. pombe mei4 mutants (Figures 1J and 2J). This result implies that Mei4 controls the expression of one or more early meiotic genes required for DNA breakage. Like Ndt80, it may also control one or more later meiotic genes for the proper repair of broken DNA.

The results discussed here illustrate the diversity of proteins required for meiotic DNA breakage and repair and suggest divergence of the mechanism or control of meiotic recombination. Such divergence is concordant with the divergence of other aspects of meiosis among eukaryotes—for example, the presence or absence of a synaptonemal complex and the arrest of meiosis for years in some organisms (MOENS 1987). Further investigations are required to reveal the universal features of DNA metabolism during meiotic recombination.

We thank Jirair Bedoyan, Nick Boddy, Marcella Cervantes, Edgar Hartsuiker, Jürg Kohli, Hiroshi Nojima, Albert Pastink, Chikashi Shimoda, Jeff Virgin, and Felicity Watts for S. pombe strains; Jirair Bedoyan for unpublished observations on rad32 strains; Hiroshi Iwasaki, Michael Lichten, Hiroshi Murakami, Alain Nicolas, Paul Russell, Val Wood, and Mimi Zolan for valuable discussions and unpublished information; Chad Ellermeier and Claudia Rubio for technical assistance; Sue Amundsen, Gareth Cromie, Luther Davis, Joe Farah, Walt Steiner, and Andrew Taylor for comments on the manuscript; and Jeremy Mseitif for preparing it. This research was supported by research grant GM-32194 from the National Institutes of Health.

¹ Present address: Department of Environmental Health, University of Washington, Seattle, WA 98105.

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				J. A. Farah, G. A. Cromie, and G. R. Smith Ctp1 and Exonuclease 1, alternative nucleases regulated by the MRN complex, are required for efficient meiotic recombination PNAS, June 9, 2009; 106(23): 9356 - 9361. [Abstract] [Full Text] [PDF]

				S. R. Ferrari, J. Grubb, and D. K. Bishop The Mei5-Sae3 Protein Complex Mediates Dmc1 Activity in Saccharomyces cerevisiae J. Biol. Chem., May 1, 2009; 284(18): 11766 - 11770. [Abstract] [Full Text] [PDF]

				E. Hartsuiker, K. Mizuno, M. Molnar, J. Kohli, K. Ohta, and A. M. Carr Ctp1CtIP and Rad32Mre11 Nuclease Activity Are Required for Rec12Spo11 Removal, but Rec12Spo11 Removal Is Dispensable for Other MRN-Dependent Meiotic Functions Mol. Cell. Biol., April 1, 2009; 29(7): 1671 - 1681. [Abstract] [Full Text] [PDF]

				S. N. Acharya, A. M. Many, A. P. Schroeder, F. M. Kennedy, O. P. Savytskyy, J. T. Grubb, J. A. Vincent, E. A. Friedle, M. Celerin, D. S. Maillet, et al. Coprinus cinereus rad50 Mutants Reveal an Essential Structural Role for Rad50 in Axial Element and Synaptonemal Complex Formation, Homolog Pairing and Meiotic Recombination Genetics, December 1, 2008; 180(4): 1889 - 1907. [Abstract] [Full Text] [PDF]

				E. Doll, M. Molnar, G. Cuanoud, G. Octobre, V. Latypov, K. Ludin, and J. Kohli Cohesin and Recombination Proteins Influence the G1-to-S Transition in Azygotic Meiosis in Schizosaccharomyces pombe Genetics, October 1, 2008; 180(2): 727 - 740. [Abstract] [Full Text] [PDF]

				G. A. Cromie, R. W. Hyppa, and G. R. Smith The Fission Yeast BLM Homolog Rqh1 Promotes Meiotic Recombination Genetics, July 1, 2008; 179(3): 1157 - 1167. [Abstract] [Full Text] [PDF]

				Y. Akamatsu, Y. Murayama, T. Yamada, T. Nakazaki, Y. Tsutsui, K. Ohta, and H. Iwasaki Molecular Characterization of the Role of the Schizosaccharomyces pombe nip1+/ctp1+ Gene in DNA Double-Strand Break Repair in Association with the Mre11-Rad50-Nbs1 Complex Mol. Cell. Biol., June 1, 2008; 28(11): 3639 - 3651. [Abstract] [Full Text] [PDF]

				K. Hirota, W. W. Steiner, T. Shibata, and K. Ohta Multiple Modes of Chromatin Configuration at Natural Meiotic Recombination Hot Spots in Fission Yeast Eukaryot. Cell, November 1, 2007; 6(11): 2072 - 2080. [Abstract] [Full Text] [PDF]

				Y. Murakami-Tonami, C. Yamada-Namikawa, A. Tochigi, N. Hasegawa, H. Kojima, M. Kunimatsu, M. Nakanishi, and H. Murakami Mei4p coordinates the onset of meiosis I by regulating cdc25+ in fission yeast PNAS, September 11, 2007; 104(37): 14688 - 14693. [Abstract] [Full Text] [PDF]

				Z. Fu, K. A. Larson, R. K. Chitta, S. A. Parker, B. E. Turk, M. W. Lawrence, P. Kaldis, K. Galaktionov, S. M. Cohn, J. Shabanowitz, et al. Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase Mol. Cell. Biol., November 15, 2006; 26(22): 8639 - 8654. [Abstract] [Full Text] [PDF]

				K. Ogino and H. Masai Rad3-Cds1 Mediates Coupling of Initiation of Meiotic Recombination with DNA Replication: Mei4-DEPENDENT TRANSCRIPTION AS A POTENTIAL TARGET OF MEIOTIC CHECKPOINT J. Biol. Chem., January 20, 2006; 281(3): 1338 - 1344. [Abstract] [Full Text] [PDF]

				J. A. Farah, G. Cromie, L. Davis, W. W. Steiner, and G. R. Smith Activation of an Alternative, Rec12 (Spo11)-Independent Pathway of Fission Yeast Meiotic Recombination in the Absence of a DNA Flap Endonuclease Genetics, December 1, 2005; 171(4): 1499 - 1511. [Abstract] [Full Text] [PDF]

				C. Ellermeier and G. R. Smith Cohesins are required for meiotic DNA breakage and recombination in Schizosaccharomyces pombe PNAS, August 2, 2005; 102(31): 10952 - 10957. [Abstract] [Full Text] [PDF]

				J. A. Farah, G. Cromie, W. W. Steiner, and G. R. Smith A Novel Recombination Pathway Initiated by the Mre11/Rad50/Nbs1 Complex Eliminates Palindromes During Meiosis in Schizosaccharomyces pombe Genetics, March 1, 2005; 169(3): 1261 - 1274. [Abstract] [Full Text] [PDF]

				C. Ellermeier, H. Schmidt, and G. R. Smith Swi5 Acts in Meiotic DNA Joint Molecule Formation in Schizosaccharomyces pombe Genetics, December 1, 2004; 168(4): 1891 - 1898. [Abstract] [Full Text] [PDF]

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