The essential role of yeast topoisomerase III in meiosis depends on recombination
1999; Springer Nature; Volume: 18; Issue: 6 Linguagem: Inglês
10.1093/emboj/18.6.1701
ISSN1460-2075
AutoresSerge Gangloff, Bernard de Massy, L. Beaudet Arthur, Rodney Rothstein, Francis Fabre,
Tópico(s)Plant nutrient uptake and metabolism
ResumoArticle15 March 1999free access The essential role of yeast topoisomerase III in meiosis depends on recombination Serge Gangloff Corresponding Author Serge Gangloff CEA de Fontenay-aux-roses, UMR 217 CNRS-CEA, BP 6, 92265 Fontenay-aux-roses, France Search for more papers by this author Bernard de Massy Bernard de Massy Institut de Génétique Humaine, UPR 1142/CNRS, 141, rue de la Cardonille, 34396 Montpellier, France Search for more papers by this author Lane Arthur Lane Arthur Columbia University, Department of Genetics and Development, 701 West 168th Street, HHSC 1606, New York, NY, 10032 USA Search for more papers by this author Rodney Rothstein Rodney Rothstein Columbia University, Department of Genetics and Development, 701 West 168th Street, HHSC 1606, New York, NY, 10032 USA Search for more papers by this author Francis Fabre Francis Fabre CEA de Fontenay-aux-roses, UMR 217 CNRS-CEA, BP 6, 92265 Fontenay-aux-roses, France Search for more papers by this author Serge Gangloff Corresponding Author Serge Gangloff CEA de Fontenay-aux-roses, UMR 217 CNRS-CEA, BP 6, 92265 Fontenay-aux-roses, France Search for more papers by this author Bernard de Massy Bernard de Massy Institut de Génétique Humaine, UPR 1142/CNRS, 141, rue de la Cardonille, 34396 Montpellier, France Search for more papers by this author Lane Arthur Lane Arthur Columbia University, Department of Genetics and Development, 701 West 168th Street, HHSC 1606, New York, NY, 10032 USA Search for more papers by this author Rodney Rothstein Rodney Rothstein Columbia University, Department of Genetics and Development, 701 West 168th Street, HHSC 1606, New York, NY, 10032 USA Search for more papers by this author Francis Fabre Francis Fabre CEA de Fontenay-aux-roses, UMR 217 CNRS-CEA, BP 6, 92265 Fontenay-aux-roses, France Search for more papers by this author Author Information Serge Gangloff 1, Bernard de Massy2, Lane Arthur3,4, Rodney Rothstein3 and Francis Fabre1 1CEA de Fontenay-aux-roses, UMR 217 CNRS-CEA, BP 6, 92265 Fontenay-aux-roses, France 2Institut de Génétique Humaine, UPR 1142/CNRS, 141, rue de la Cardonille, 34396 Montpellier, France 3Columbia University, Department of Genetics and Development, 701 West 168th Street, HHSC 1606, New York, NY, 10032 USA 4Hi-Bred Seed, 7300 NW, 62nd Avenue, PO Box 1004, Johnston, IA, 50131-1004 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:1701-1711https://doi.org/10.1093/emboj/18.6.1701 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Yeast cells mutant for TOP3, the gene encoding the evolutionary conserved type I-5′ topoisomerase, display a wide range of phenotypes including altered cell cycle, hyper-recombination, abnormal gene expression, poor mating, chromosome instability and absence of sporulation. In this report, an analysis of the role of TOP3 in the meiotic process indicates that top3Δ mutants enter meiosis and complete the initial steps of recombination. However, reductional division does not occur. Deletion of the SPO11 gene, which prevents recombination between homologous chromosomes in meiosis I division, allows top3Δ mutants to form viable spores, indicating that Top3 is required to complete recombination successfully. A topoisomerase activity is involved in this process, since expression of bacterial TopA in yeast top3Δ mutants permits sporulation. The meiotic block is also partially suppressed by a deletion of SGS1, a gene encoding a helicase that interacts with Top3. We propose an essential role for Top3 in the processing of molecules generated during meiotic recombination. Introduction Topoisomerases play a crucial role in cellular metabolism (for a review see Duguet, 1997), and variations in topoisomerase expression levels result in pleiotropic phenotypes in bacteria and yeast (Kim and Wang, 1989; Wallis et al., 1989; Drlica, 1990). DNA topoisomerases are essential for the replication of DNA molecules (reviewed in Wang, 1996), for chromosome condensation (Adachi et al., 1991; Downes et al., 1994; Castano et al., 1996) and for the proper segregation of chromosomes at mitosis and meiosis (DiNardo et al., 1984; Holm et al., 1985, 1989; Rose et al., 1990). Another fundamental role for topoisomerases involves the relaxation of positive and negative superhelical domains of DNA that are generated by transcription (Liu and Wang, 1987; Giaever and Wang, 1988; Wu et al., 1988; Tsao et al., 1989). Additionally, the regulation of gene expression for many functions is dependent on the degree of supercoiling found in intracellular DNA (Gangloff et al., 1994a; Wang and Droge, 1996). Recently, a role for topoisomerases I and II in checkpoint control was proposed (Downes et al., 1994; Castano et al., 1996). In Saccharomyces cerevisiae, one mitochondrial and three nuclear topoisomerase activities have been described. Among the nuclear activities, DNA topoisomerases I and II (encoded by the TOP1 and TOP2 genes, respectively) are capable of relaxing both negatively and positively supercoiled DNA molecules (Goto and Wang, 1984; Thrash et al., 1984), while DNA topoisomerase III (encoded by the TOP3 gene) weakly relaxes only negatively supercoiled DNA (Kim and Wang, 1992). Little is known about the recently identified mitochondrial activity (Ezekiel et al., 1994). The genes encoding the nuclear activities (TOP1, TOP2 and TOP3) have been cloned (Goto and Wang, 1984, 1985; Thrash et al., 1985; Wallis et al., 1989) and studied extensively (for reviews see Champoux, 1994; Gangloff et al., 1994a; Hsieh et al., 1994; Lima and Mondragon, 1994; Watt and Hickson, 1994; Roca, 1995; Wang, 1996). The TOP3 gene is unique in that it encodes a type I-5′ topoisomerase in yeast. It is homologous to the Escherichia coli topA and topB genes but not to the S.cerevisiae TOP1 (I-3′) or TOP2 (II) genes. The relationships originally proposed on the basis of sequence comparison have been confirmed biochemically (Kim and Wang, 1992). Purified Top3 protein exhibits many of the properties of TopA and TopB, including relaxation of only negatively supercoiled DNA and a 5′-covalent phosphotyrosine ester linkage between the conserved tyrosine in the active site and the DNA. The enzyme is also proficient in preferentially binding single-stranded DNA and in decatenating single strands (Kim and Wang, 1992). Recently, homologs in man and mouse have been identified, and the gene has been shown to be essential during early mouse embryogenesis (Hanai et al., 1996; Fritz et al., 1997; Li and Wang, 1998; Seki et al., 1998). The top3 mutants originally were isolated because they stimulate recombination between repeated sequences (Wallis et al., 1989; Arthur, 1991; Gangloff et al., 1996). This hyper-recombinogenic phenotype is not restricted to direct repeats, since the absence of Top3 also elevates recombination between homologous ectopic genes (Bailis et al., 1992). In addition to the recombination phenotype, top3Δ mutants exhibit a cell cycle aberration characterized by an accumulation of cells containing an undivided nucleus in the neck of the bud. This cell cycle delay translates into slow growth that is believed to result from a defect in single-stranded DNA decatenation in an alternative pathway for DNA replication termination (Gangloff et al., 1994a). Among other mitotic phenotypes, top3Δ mutants are affected in the transcriptional control of several genes (Arthur, 1991). A search for suppressors of the top3Δ slow growth phenotype led to the isolation of mutations in the SGS1 gene (Gangloff et al., 1994b). The Sgs1 protein is a member of the helicase family characterized by the E.coli RecQ protein (Nakayama et al., 1985; Umezu et al., 1990). Over the past few years, Sgs1 homologs have been identified in many organisms, including yeast and man (Puranam and Blackshear, 1994; Ellis et al., 1995; Puranam et al., 1995; Yu et al., 1996; Stewart et al., 1997; Yan et al., 1998). The absence of Sgs1 suppresses all the mitotic phenotypes caused by the lack of Top3 (Gangloff et al., 1994b), which led to the hypothesis that Top3 is required to act on substrates created by Sgs1. This model is supported by the finding that Sgs1 interacts physically with Top3 in a two-hybrid system, suggesting that Top3 and Sgs1 may work together as part of a complex (Gangloff et al., 1994b). Although Top3 is involved in every aspect of DNA metabolism (Wallis et al., 1989; Arthur, 1991; Bailis et al., 1992; Gangloff et al., 1994a, 1996; Kim et al., 1995), its mitotic function is dispensable. In contrast, Top3 function is essential during meiosis, and homozygous mutant diploids are unable to form asci. In this report, we investigate the metabolic alteration responsible for the inability of top3Δ strains to sporulate. We show that the top3Δ mutants fail to complete reductional division at a step following initiation of recombination and formation of recombinant molecules. Additionally, overexpression of the E.coli topA gene restores sporulation, which suggests that a type I-5′ topoisomerase activity is essential for processing recombination intermediates generated during meiosis. Results The absence of sporulation in top3Δ mutants is not due to loss of chromosome III Diploid cells homozygous for the top3Δ deletion fail to sporulate (Wallis et al., 1989; Arthur, 1991; Gangloff et al., 1994a). Light microscopy analysis of 2000 diploid cells homozygous for top3Δ indicated that, even 7 days after transfer to sporulation medium, no visible asci could be detected. We previously have observed that diploid cells homozygous for top3Δ, at a much higher frequency than wild-type cells, become capable of mating with haploid cells and form colonies on selective medium, an event due to chromosome III loss (Arthur, 1991). In yeast, each chromosome III carries a different MAT allele at the mating type locus, and the products of both are required for entry into meiosis. A high frequency of chromosome loss may therefore lead to top3Δ's inability to sporulate. To test this hypothesis, we measured the frequency at which chromosome III is lost in a population of cells ready to enter meiosis. We used a strain (D5-4D×W1193-2C; Table I) heterozygous for LEU2, a proximal gene on chromosome III. At the t = 0 of meiosis induction, diploid cells were plated out onto rich medium. After 3 days of vegetative growth, the colonies were replicated onto synthetic medium lacking leucine. In those conditions, cells that have lost the LEU2-bearing chromosome III prior to the first cell division on the plate become unable to form colonies. This analysis was performed on several thousand colonies and revealed that <1/3000 diploid cells had lost the LEU2 marked chromosome III. This indicates that the total frequency of chromosome III loss in top3Δ mutants is <1/1500 cells, and thus cannot account for the inability of top3Δ mutants to sporulate. Table 1. Strain list All strains are isogenic to W303 (Thomas et al., 1989), except ORD2130, 2184 and 2186. The absence of TOP3 prevents the cells from undergoing reductional division To determine at which stage in meiosis top3Δ cells are blocked, we next examined the fate of the nucleus of top3Δ cells during meiosis. Meiosis was induced, and samples of wild-type and mutant cells were taken at different times, washed, fixed and incubated with 4′,6-diamidino-2-phenylindole (DAPI). Analysis by fluorescence microscopy revealed that after 12 h, 5–10% of wild-type cells have already completed both reductional and equational division, and formed four haploid staining bodies. However, in a sample of 2000 top3Δ cells, reductional division was never observed. Instead, after 2 days of incubation in sporulation medium, nuclear DAPI-stained material fragmentation is observed with subsequent loss of the intense fluorescent staining of the DNA (Figure 1). Figure 1.DAPI staining during meiosis. Nuclear division and spore formation were examined by fluorescence microscopy. Wild-type (W303) and top3Δ (U739-1A×W1193-2C) samples were taken at 0, 48 and 96 h following meiotic induction, and the DNA of ethanol-fixed cells was stained with DAPI. Binucleate (MI) and tetranucleate (MII) cells have completed first and second meiotic divisions, respectively. Download figure Download PowerPoint Double-strand breaks (DSBs) are present in top3Δ mutants In S.cerevisiae, double-strand breaks (DSBs) initiate meiotic recombination, a process necessary to achieve proper chromosome segregation (for reviews see Kleckner, 1996; Roeder, 1997). To investigate whether top3Δ homozygous diploids enter meiosis, the occurrence of DSBs at time t = 0, t = 8 and t = 24 h in sporulation medium was analyzed (de Massy and Nicolas, 1993; de Massy et al., 1994). Interestingly, DSBs at both the CYS3 and the ARG4 loci were detected. The levels of breaks as well as their kinetics of appearance and processing are comparable with those observed in wild-type controls (Figure 2). Global analysis of DSB formation along the 340 kb of chromosome III indicated that the pattern of DSBs formed on this chromosome is similar in the top3Δ mutants and the wild-type controls, suggesting that the results observed at the ARG4 and CYS3 loci reflect a general trend in the genome (data not shown). Figure 2.Detection of meiotic DSBs at the ARG4 and CYS3 loci. Cells were taken at 0, 8 and 24 h after transfer to sporulation medium. Genomic DNA extracted from wild-type (ORD2130), top3Δ/TOP3 (ORD2186) and top3Δ/top3Δ (ORD2184) meiotic cells was digested with HindIII and probed sequentially with DNA corresponding to known meiotic recombination hotspots. Left panel: hybridization with the radiolabeled EcoRI fragment encompassing the 3′ region of the CYS3 gene detects the CYS3 DSBs on chromosome I (see Figure 1 in de Massy et al., 1994). Right panel: in addition to the CYS3 signal, the radiolabeled EcoRV–BglII (1016 bp) ARG4 internal fragment (see Figure 3) reveals the ARG4 DSBs on chromosome VIII. The vertical bars on the sides indicate the positions of the CYS3 and the ARG4 meiotic DSBs sites. Download figure Download PowerPoint Recombinant molecules are formed, but generate a lethal substrate To analyze the progression of recombination after the introduction of a DSB, we examined the formation of recombinant molecules by using arg4 heteroalleles that differ from each other by their restriction pattern (Figure 3), The presence of recombinant molecules does not, however, imply that Holliday junctions are resolved actively. In the arg4-ΔEcoRV/arg4-ΔBglII diploid cells, the great majority of the convertants at the ARG4 locus involve only the EcoRV restriction site which lies close to the upstream region of the gene where the DSBs occur. After a double digestion with EcoRV and BglII, and in the absence of a pre-treatment with DNA cross-linking agents, passive migration of the junction releases linear DNA fragments. Using this experimental design, the two parental fragments were detected. However, two additional signals corresponding to arg4 recombinant molecules were also detected by Southern analysis in both wild-type and top3Δ mutants (see 1 and 7 kb fragments, Figure 3). Together, these results and those presented in the previous section indicate that top3Δ cells are progressing through meiosis and that the Top3 function is not required for the formation of either DSBs or recombinant molecules. Figure 3.Physical map of the ARG4 region and detection of recombinant molecules. (A) Physical map of the alleles present at the ARG4 locus. The EcoRV and BglII heteroallelic mutations in the ARG4 genes are indicated. The position of the ARG4 EcoRV–BglII probe is indicated by the black box, and the ARG4 open reading frame and direction of transcription are indicated by the horizontal arrow. The sizes of the parental (P1 and P2) and expected recombinant fragments (Rec1 and Rec2) resulting from the EcoRV + BglII digest are shown underneath. (B) At times 0 and 24 h after meiotic induction, genomic DNA extracted from wild-type (ORD2130), top3Δ/TOP3 (ORD2186) and top3Δ/top3Δ (ORD2184) cells was digested with EcoRV and BglII and probed with the EcoRV–BglII (1016 bp) ARG4 internal fragment. As in Figure 2 (right), the intense unlabeled signal corresponds to the CYS3 parental band. Download figure Download PowerPoint In yeast cells, meiotic levels of recombination can be induced before commitment to reductional division in a return to growth (RTG) experiment (Sherman and Roman, 1963). In top3Δ mutants, although DSBs and recombinant molecules are observed, no stimulation of recombination could be measured through the formation of Arg+ prototrophs. At t = 0, we recovered four and 20 Arg+ clones for 106 cells in wild-type and top3Δ mutants, respectively. After 24 h in the sporulation medium, 17 000 Arg+ recombinants were recovered for 106 wild-type cells, compared with only 25 for top3Δ mutants. We interpret this result as a failure of top3Δ cells to resume vegetative growth following meiotic recombination initiation. This is not due to a general deficiency in DSB repair since mitotic top3Δ mutants switch mating type in the presence of the HO endonuclease (S.Gangloff, unpublished result). Bypassing recombination between homologs restores sporulation To determine whether the top3Δ arrest in meiosis I is related to recombination, we examined whether cells that do not undergo meiotic recombination are capable of sporulating in the absence of Top3. The spo11Δ mutation abolishes meiotic DSB formation (Klapholz et al., 1985; Bergerat et al., 1997; Keeney et al., 1997) and, thus, recombination. Since spo11Δ mutants do not sporulate efficiently and produce mostly inviable spores due to non-disjunction, we tested our hypothesis in strains homozygous for both the spo11Δ and the spo13Δ mutations. spo13Δ mutants bypass meiosis I and produce asci containing two diploid spores (dyads) that are viable even in the absence of recombination (Malone and Esposito, 1981; Klapholz et al., 1985). Isogenic diploid strains were constructed in which the spo11Δ, spo13Δ and top3Δ mutations are homozygous (Table I). These diploid cells were analyzed for their ability to undergo meiosis and form spores. The results summarized in Table II reveal that TOP3 is not required to form viable spores in the absence of recombination: while the top3Δ spo13Δ cells form no asci, the triple mutant top3Δ spo11Δ spo13Δ sporulates and forms dyads with a viability similar to that of spo11Δ spo13Δ controls. We conclude that the meiotic arrest of top3Δ spo13Δ cells depends on the activity of Spo11 and, therefore, on meiotic recombination. This result suggests that resolution of entangled homologs after recombination is probably responsible for the sporulation defect observed in top3Δ mutants. This hypothesis is also in agreement with the observation that nuclear division occurs within the same time frame in the presence or absence of TOP3 when recombination is abolished. As expected, two DAPI-staining bodies could be observed in 2000 cells after 2 days (Figure 4) or even after 6 days of incubation (data not shown) when the SPO11 controlled initiation of recombination is functional. Furthermore, the timing of dyad formation is independent of TOP3, which also suggests that the absence of this topoisomerase does not greatly affect pre-meiotic DNA synthesis. Figure 4.Effect of recombination initiation on top3Δ meiotic product formation. The same type of experiment as that described in Figure 1 was performed on spo11Δ spo13Δ double mutants (D50-1D×D50-3D) and on spo11Δ spo13Δ top3Δ (D52 = D50-3C×D51-3D) triple mutants. Here, binucleate cells correspond to cells that have undergone a single equational division. Download figure Download PowerPoint Table 2. Sporulation and spore viability Strain Strain name or cross Percentage of asci formed Spore viability 2 days 4 days 6 days Wild-type (W303) 45 80 85 95% top3Δ (U739-1A×W1193-2C) 0 0 0 0 spo11Δ top3Δ (D65-9A×D51-1A) 0 0 0 0 spo13Δ top3Δ (D50bis-9D×D65-2B) 0 0 0 0 spo11Δ spo13Δ* (D50-1D×D50-3D) 8 15 20 79% top3Δ spo11Δ spo13Δ* (D52) 8 15 20 78% sgs1Δ (D17) 1 5 30 74% top3Δ sgs1Δ (D18) 0 0.1 2 67% sgs1Δ spo13Δ* (D66-6C×D67-6B) 0 2 15 ND sgs1Δ spo11Δ spo13Δ* (D66-6C×D67-9C) 10 15 20 86% Diploid cells, homozygous for the mutations listed, were pre-grown on YPD at 30°C (Sherman et al., 1986) and inoculated into 10 ml of SPS medium. When the cells reach stationary phase, they are washed with water and resuspended in 10 ml of 1% potassium acetate medium. Aliquots of 1 ml are taken at various time points, and the proportion of tetrads [or dyads (*) for spo11Δ spo13Δ mutants] is determined by dividing the number of four (two)-spored asci by the total number of cells counted. Cell viability was determined after dissection, by dividing the number of spores forming colonies by the total number of spores dissected. For each time point, a minimum of 200 full tetrads (or dyads) were scored. ND, not determined. Overexpression of TopA suppresses the sporulation deficiency of top3Δ mutants Wallis et al. (1989) have shown that expression in yeast of the E.coli topA gene, encoding a functionally related type I-5′ topoisomerase, is capable of complementing the slow growth phenotype of top3Δ null mutants. Thus, we tested whether expression of topA (YEptopA-PGAL1) would also complement the sporulation defect of top3Δ mutant strains. Diploid cells homozygous for the top3Δ deletion and containing the topA plasmid were tested for spore formation. The diploid strains were pre-grown on galactose medium to induce the expression of topA and then replica-plated to sporulation medium containing 0.1% galactose. Under these conditions, three- and four-spored asci accounted for 20% of the cells present after 3 days of induction at 30°C compared with 50% for wild-type diploid controls transformed by YEptopA-PGAL1 plasmid (Table III). When four-spored asci from the top3Δ/top3Δ YEptopA-PGAL1 diploids were dissected on either YPD or YPGal, the spore viability was poor. From a total of 71 tetrads dissected, only 110 viable colonies (39%) were recovered, while 89% viability was observed for isogenic wild-type strains containing the topA plasmid. Of the 71 top3Δ/top3Δ YEptopA-PGAL1 tetrads, only five yielded four viable spores. Among tetrads where only three spores form colonies, some spores were found to grow on medium lacking the two amino acids corresponding to the auxotrophic markers used to disrupt the two copies of the TOP3 gene. This implies that the two chromosomes bearing the top3 deletion did not disjoin and segregated to the same cell. These observations indicate that overexpression of a type I-5′ topoisomerase activity can restore sporulation in cells lacking Top3. However, the efficiency of the process is poor, and chromosome non-disjunction may be important in spore inviability. This result suggests that the essential enzymatic activity necessary for performing meiosis is shared with type I-5′ enzymes, but absent from the type I-3′ or II counterparts. Table 3. Effects of TopA overexpression on sporulation Genotype Sporulation efficiency Tetrads analyzed Four viable spores Three viable spores Two and one viable spores Spore viability top3Δ + topA 20% 71 5 11 57 39% wt + topA 50% 46 38 2 6 89% NB: top3Δ strains do not form spores on galactose. The absence of SGS1 allows some sporulation in top3Δ mutants The SGS1 gene encodes a protein containing a DNA helicase domain conserved from bacteria to man (Rothstein and Gangloff, 1995). Mutations in either BLM or WRN, two human genes encoding proteins structurally and functionally homologous to Sgs1, lead to genome instability and a high incidence of cancer (Ellis et al., 1995; Yu et al., 1996). We have shown previously that Sgs1 interacts with Top3 in a two-hybrid system (Gangloff et al., 1994b). Interestingly, TOP3 and SGS1 also interact genetically, since the absence of SGS1 suppresses the slow growth, the altered cell cycle distribution and the hyper-recombination phenotype of top3Δ mutants (Gangloff et al., 1994b). Because of the potential for Sgs1 and Top3 to act together in a complex (Gangloff et al., 1994b), we analyzed the possibility that unprocessed structures introduced by Sgs1 in the absence of Top3 are responsible for the sporulation defect. As shown in Table II, top3Δ sgs1Δ double mutants sporulate very poorly, but produce viable spores. Out of 18 top3Δ sgs1Δ tetrads dissected, the overall spore viability was 67%, with five, six, three and four tetrads giving rise to four, three, two and one viable spore, respectively. We found that, unlike top3Δ strains, sgs1Δ mutant strains complete the meiotic process, but with a long delay in the appearance of four-spored asci and a very low yield (Figure 5). Out of 33 sgs1Δ tetrads dissected, the overall spore viability was 74%, with 14, eight, seven and four tetrads giving rise to four, three, two and one viable spore, respectively, indicating that sgs1Δ is epistatic to top3Δ with respect to spore viability. Figure 5.Sporulation kinetics of wild-type and sgs1Δ cells. The percentage of four-spored asci was determined for wild-type and sgs1Δ mutants as a function of time following induction of meiosis. Download figure Download PowerPoint We next examined whether the 48 h delay in the meiotic process is related, similarly to the arrest of top3Δ mutants, to the recombination process. We therefore constructed sgs1Δ spo11Δ spo13Δ triple mutant diploid cells and monitored both nuclear division and spore (dyad) formation. In the spo11Δ spo13Δ background, the kinetics of spore formation and the timing of equational division are independent of the presence of SGS1, which indicates that Sgs1 is also involved in the meiotic recombination process (Figure 6). Figure 6.Effect of recombination initiation on sgs1Δ meiotic product formation. Nuclear division and spore formation were examined in the sgs1Δ (D47), spo11Δ spo13Δ sgs1Δ (D66-6C×D67-9C) and spo13Δ sgs1Δ (D66-6C×D67-6B) strains throughout meiosis by staining DNA with DAPI. In the absence of the SPO13 function, only dyads are obtained. In the sgs1Δ mutants, tetrads containing four haploid spores start to appear after 5 days of incubation. Download figure Download PowerPoint Discussion The absence of the type I-5′ topoisomerase Top3 results in a pleiotropic mitotic phenotype (Wallis et al., 1989; Gangloff et al., 1994a,b, 1996). Vegetative cells are viable but meiosis does not proceed. This indicates that Top3 plays either a specific and essential role in meiosis or that another protein can partially substitute for Top3 during the mitotic but not the meiotic cycle. The experiments described in this study were designed to address the meiotic role of Top3. Since the Sgs1 helicase interacts with Top3, and since sgs1Δ is epistatic to top3Δ with respect to mitotic top3Δ phenotypes, we have extended this study to the sgs1Δ and sgs1Δ top3Δ mutants. Aberrant gene expression is not a likely cause for the sporulation defect TOP3 is known to control the expression of several genes (Arthur, 1991). IME1 and/or IME2, which belong to the cascade of regulatory genes involved in the control of entry into meiosis (Sia and Mitchell, 1995), could depend on TOP3 for their expression. This is not the case since overexpression of these genes was not found to alleviate the sporulation defect of top3Δ cells (unpublished results). Although it cannot be formally excluded that TOP3 controls the expression of other meiosis-specific genes, altered gene expression is not a probable candidate for the sporulation defect since spo11Δ spo13Δ top3Δ triple mutants do sporulate similarly to spo11Δ spo13Δ strains. Recombination is responsible for the top3Δ meiotic defect Analysis of top3Δ cells at the cellular level indicates that very few, if any, cells are capable of completing the first meiotic division (MI). Since this is the part of meiosis where recombination takes place, several aspects of this process were analyzed. Initiation, as evidenced by formation of DSBs, occurs at a similar frequency in both normal and top3Δ cells. Additionally, comparable amounts of recombinant molecules could be detected in wild-type and mutants, suggesting that TOP3 is not involved in the formation of these recombinant molecules (Figure 3). Although detection of intragenic recombinant molecules does not require that the Holliday junctions are resolved, it is still possible that Top3 may affect the recombination process itself. It has been shown previously that meiotic recombinant molecules can be detected by genomic blot analysis in mutants such as rad51, rad52, rad55 and rad57 that are deficient in homologous recombination (Borts et al., 1986; Shinohara et al., 1992; Schwacha and Kleckner, 1997). However, in these studies, the level of recombinant molecules was much lower than in control cells, which is clearly not the case for top3Δ. Cells mutant for TOP3 appear to proceed into an irreversible phase leading to cell death (Figure 1). In addition, RTG analysis did not indicate any increase in the recovery of Arg+ recombinants with top3Δ, suggesting a viability loss of the meiotic cells following DSBs. Finally, we used spo11Δ, an early meiotic mutation that prevents DSB formation (
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