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Mus81 nuclease and Sgs1 helicase are essential for meiotic recombination in a protist lacking a synaptonemal complex.

Lukaszewicz A, Howard-Till RA, Loidl J - Nucleic Acids Res. (2013)

Bottom Line: We show in Tetrahymena, that homologous chromosomes fail to separate and JMs accumulate in the absence of Mus81 or Sgs1, whereas deletion of the MutLγ-component Mlh1 does not affect meiotic divisions.Thus, our results are consistent with Mus81 being part of an essential, if not the predominant, CO pathway in Tetrahymena.Tetrahymena shares the predominance of the Mus81 CO pathway with the fission yeast.

View Article: PubMed Central - PubMed

Affiliation: Department of Chromosome Biology, Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, A-1030 Vienna, Austria.

ABSTRACT
Mus81 resolvase and Sgs1 helicase have well-established roles in mitotic DNA repair. Moreover, Mus81 is part of a minor crossover (CO) pathway in the meiosis of budding yeast, plants and vertebrates. The major pathway depends on meiosis-specific synaptonemal complex (SC) formation, ZMM proteins and the MutLγ complex for CO-directed resolution of joint molecule (JM)-recombination intermediates. Sgs1 has also been implicated in this pathway, although it may mainly promote the non-CO outcome of meiotic repair. We show in Tetrahymena, that homologous chromosomes fail to separate and JMs accumulate in the absence of Mus81 or Sgs1, whereas deletion of the MutLγ-component Mlh1 does not affect meiotic divisions. Thus, our results are consistent with Mus81 being part of an essential, if not the predominant, CO pathway in Tetrahymena. Sgs1 may exert functions similar to those in other eukaryotes. However, we propose an additional role in supporting homologous CO formation by promoting homologous over intersister interactions. Tetrahymena shares the predominance of the Mus81 CO pathway with the fission yeast. We propose that in these two organisms, which independently lost the SC during evolution, the basal set of mitotic repair proteins is sufficient for executing meiotic recombination.

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Related in: MedlinePlus

Depletion of Mus81 and Sgs1 causes DSB-dependent meiotic segregation defects. (A) In the WT, homologous chromosomes separate in anaphase I and form two distinct chromatin masses in telophase I–interkinesis. In mus81i and sgs1i meiosis, chromosomes collapse back into a single diploid nucleus after an attempted anaphase I. Staining of the centromere marker Cna1 (orange) shows the orientation of five centromeres to each of the opposite poles and five centromeres in each daughter nucleus after complete separation of the homologs in the WT. In the mutants, collapsed nuclei contain 10 centromeres. (B) Quantification of the segregation defect. WT meiotic nuclei pass through anaphase I, whereas mutants arrest with collapsed anaphase I nuclei. Hundred cells were evaluated for each genotype and timepoint. (C) Spo11 depletion rescues the mus81i segregation defect. mus81i strains were depleted of Spo11 by SPO11 knockout. All mus81i spo11Δ cells completed anaphase I 5.5 h after induction of meiosis (n = 50 cells for each phenotype). (D) Spo11 depletion rescues the sgs1i segregation defect. sgs1i cells were depleted of Spo11 by spo11 RNAi of their mating partner. spo11 RNAi is transferable, i.e. if one of the mating partners expresses interfering RNA, both cells display the depletion phenotype. spo11 RNAi efficiency was monitored by the failure of micronuclei to elongate during meiotic prophase (see main text). The sgs1i phenotype is limited to the cell expressing siRNA and is attenuated by mating to a non-sgs1i partner. Therefore, the frequency of successful anaphases was scored in the partner affected by spo11 sgs1 double RNAi and compared with sgs1i × WT pairs. n = 100 cells for each genotype. (E) dmc1Δ mus81i and dmc1Δ sgs1i double mutants are partially rescued from the anaphase I defect and undergo defective anaphase II. Shown for each genotype are the mean values ± S.D. from three experiments with 100 nuclei counted in each. An unaccounted-for percentage of cells contained three or four nuclei, where one or both products of a first division may have undergone a second division or where nonseparating bivalents or chromosomes had formed extra nuclei. (F) Pairing in the elongated prophase nucleus. Examples of paired (FISH signals fused) and unpaired (FISH signals separate) homologous loci (red arrows) are shown. SGS1 and MUS81 genotypes carry the respective RNAi hp constructs, but RNAi was not induced; hence, they resemble the WT control. In the absence of Sgs1 but not of Mus81, homologous pairing is reduced both in the WT and in the dmc1Δ background, in which recombination takes place primarily between sisters (30). Values are means of three repeats with 100 nuclei evaluated, each. Error bars indicate standard deviation. Bars in A and F: 10 µm.
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gkt703-F3: Depletion of Mus81 and Sgs1 causes DSB-dependent meiotic segregation defects. (A) In the WT, homologous chromosomes separate in anaphase I and form two distinct chromatin masses in telophase I–interkinesis. In mus81i and sgs1i meiosis, chromosomes collapse back into a single diploid nucleus after an attempted anaphase I. Staining of the centromere marker Cna1 (orange) shows the orientation of five centromeres to each of the opposite poles and five centromeres in each daughter nucleus after complete separation of the homologs in the WT. In the mutants, collapsed nuclei contain 10 centromeres. (B) Quantification of the segregation defect. WT meiotic nuclei pass through anaphase I, whereas mutants arrest with collapsed anaphase I nuclei. Hundred cells were evaluated for each genotype and timepoint. (C) Spo11 depletion rescues the mus81i segregation defect. mus81i strains were depleted of Spo11 by SPO11 knockout. All mus81i spo11Δ cells completed anaphase I 5.5 h after induction of meiosis (n = 50 cells for each phenotype). (D) Spo11 depletion rescues the sgs1i segregation defect. sgs1i cells were depleted of Spo11 by spo11 RNAi of their mating partner. spo11 RNAi is transferable, i.e. if one of the mating partners expresses interfering RNA, both cells display the depletion phenotype. spo11 RNAi efficiency was monitored by the failure of micronuclei to elongate during meiotic prophase (see main text). The sgs1i phenotype is limited to the cell expressing siRNA and is attenuated by mating to a non-sgs1i partner. Therefore, the frequency of successful anaphases was scored in the partner affected by spo11 sgs1 double RNAi and compared with sgs1i × WT pairs. n = 100 cells for each genotype. (E) dmc1Δ mus81i and dmc1Δ sgs1i double mutants are partially rescued from the anaphase I defect and undergo defective anaphase II. Shown for each genotype are the mean values ± S.D. from three experiments with 100 nuclei counted in each. An unaccounted-for percentage of cells contained three or four nuclei, where one or both products of a first division may have undergone a second division or where nonseparating bivalents or chromosomes had formed extra nuclei. (F) Pairing in the elongated prophase nucleus. Examples of paired (FISH signals fused) and unpaired (FISH signals separate) homologous loci (red arrows) are shown. SGS1 and MUS81 genotypes carry the respective RNAi hp constructs, but RNAi was not induced; hence, they resemble the WT control. In the absence of Sgs1 but not of Mus81, homologous pairing is reduced both in the WT and in the dmc1Δ background, in which recombination takes place primarily between sisters (30). Values are means of three repeats with 100 nuclei evaluated, each. Error bars indicate standard deviation. Bars in A and F: 10 µm.

Mentions: We next studied the segregation behaviour of the abnormal mus81i and sgs1i bivalents and found that they completely failed to undergo anaphase separation. Nuclei were seen to elongate as if they were trying to separate chromosomes, but ultimately they collapsed back into a single nucleus and remained arrested in that stage (Figure 3A and B). Immunostaining with an antibody against the centromeric histone Cna1 (48) revealed that the centromeres were correctly bioriented during attempted anaphase (Figure 3A). However, while the WT formed two haploid late anaphase/telophase ensembles with 5 centromeres, mutants retained nuclei with a diploid set of 10 centromeres. This indicates that the spindle was pulling, but was unable to separate homologous chromosomes.Figure 3.


Mus81 nuclease and Sgs1 helicase are essential for meiotic recombination in a protist lacking a synaptonemal complex.

Lukaszewicz A, Howard-Till RA, Loidl J - Nucleic Acids Res. (2013)

Depletion of Mus81 and Sgs1 causes DSB-dependent meiotic segregation defects. (A) In the WT, homologous chromosomes separate in anaphase I and form two distinct chromatin masses in telophase I–interkinesis. In mus81i and sgs1i meiosis, chromosomes collapse back into a single diploid nucleus after an attempted anaphase I. Staining of the centromere marker Cna1 (orange) shows the orientation of five centromeres to each of the opposite poles and five centromeres in each daughter nucleus after complete separation of the homologs in the WT. In the mutants, collapsed nuclei contain 10 centromeres. (B) Quantification of the segregation defect. WT meiotic nuclei pass through anaphase I, whereas mutants arrest with collapsed anaphase I nuclei. Hundred cells were evaluated for each genotype and timepoint. (C) Spo11 depletion rescues the mus81i segregation defect. mus81i strains were depleted of Spo11 by SPO11 knockout. All mus81i spo11Δ cells completed anaphase I 5.5 h after induction of meiosis (n = 50 cells for each phenotype). (D) Spo11 depletion rescues the sgs1i segregation defect. sgs1i cells were depleted of Spo11 by spo11 RNAi of their mating partner. spo11 RNAi is transferable, i.e. if one of the mating partners expresses interfering RNA, both cells display the depletion phenotype. spo11 RNAi efficiency was monitored by the failure of micronuclei to elongate during meiotic prophase (see main text). The sgs1i phenotype is limited to the cell expressing siRNA and is attenuated by mating to a non-sgs1i partner. Therefore, the frequency of successful anaphases was scored in the partner affected by spo11 sgs1 double RNAi and compared with sgs1i × WT pairs. n = 100 cells for each genotype. (E) dmc1Δ mus81i and dmc1Δ sgs1i double mutants are partially rescued from the anaphase I defect and undergo defective anaphase II. Shown for each genotype are the mean values ± S.D. from three experiments with 100 nuclei counted in each. An unaccounted-for percentage of cells contained three or four nuclei, where one or both products of a first division may have undergone a second division or where nonseparating bivalents or chromosomes had formed extra nuclei. (F) Pairing in the elongated prophase nucleus. Examples of paired (FISH signals fused) and unpaired (FISH signals separate) homologous loci (red arrows) are shown. SGS1 and MUS81 genotypes carry the respective RNAi hp constructs, but RNAi was not induced; hence, they resemble the WT control. In the absence of Sgs1 but not of Mus81, homologous pairing is reduced both in the WT and in the dmc1Δ background, in which recombination takes place primarily between sisters (30). Values are means of three repeats with 100 nuclei evaluated, each. Error bars indicate standard deviation. Bars in A and F: 10 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814389&req=5

gkt703-F3: Depletion of Mus81 and Sgs1 causes DSB-dependent meiotic segregation defects. (A) In the WT, homologous chromosomes separate in anaphase I and form two distinct chromatin masses in telophase I–interkinesis. In mus81i and sgs1i meiosis, chromosomes collapse back into a single diploid nucleus after an attempted anaphase I. Staining of the centromere marker Cna1 (orange) shows the orientation of five centromeres to each of the opposite poles and five centromeres in each daughter nucleus after complete separation of the homologs in the WT. In the mutants, collapsed nuclei contain 10 centromeres. (B) Quantification of the segregation defect. WT meiotic nuclei pass through anaphase I, whereas mutants arrest with collapsed anaphase I nuclei. Hundred cells were evaluated for each genotype and timepoint. (C) Spo11 depletion rescues the mus81i segregation defect. mus81i strains were depleted of Spo11 by SPO11 knockout. All mus81i spo11Δ cells completed anaphase I 5.5 h after induction of meiosis (n = 50 cells for each phenotype). (D) Spo11 depletion rescues the sgs1i segregation defect. sgs1i cells were depleted of Spo11 by spo11 RNAi of their mating partner. spo11 RNAi is transferable, i.e. if one of the mating partners expresses interfering RNA, both cells display the depletion phenotype. spo11 RNAi efficiency was monitored by the failure of micronuclei to elongate during meiotic prophase (see main text). The sgs1i phenotype is limited to the cell expressing siRNA and is attenuated by mating to a non-sgs1i partner. Therefore, the frequency of successful anaphases was scored in the partner affected by spo11 sgs1 double RNAi and compared with sgs1i × WT pairs. n = 100 cells for each genotype. (E) dmc1Δ mus81i and dmc1Δ sgs1i double mutants are partially rescued from the anaphase I defect and undergo defective anaphase II. Shown for each genotype are the mean values ± S.D. from three experiments with 100 nuclei counted in each. An unaccounted-for percentage of cells contained three or four nuclei, where one or both products of a first division may have undergone a second division or where nonseparating bivalents or chromosomes had formed extra nuclei. (F) Pairing in the elongated prophase nucleus. Examples of paired (FISH signals fused) and unpaired (FISH signals separate) homologous loci (red arrows) are shown. SGS1 and MUS81 genotypes carry the respective RNAi hp constructs, but RNAi was not induced; hence, they resemble the WT control. In the absence of Sgs1 but not of Mus81, homologous pairing is reduced both in the WT and in the dmc1Δ background, in which recombination takes place primarily between sisters (30). Values are means of three repeats with 100 nuclei evaluated, each. Error bars indicate standard deviation. Bars in A and F: 10 µm.
Mentions: We next studied the segregation behaviour of the abnormal mus81i and sgs1i bivalents and found that they completely failed to undergo anaphase separation. Nuclei were seen to elongate as if they were trying to separate chromosomes, but ultimately they collapsed back into a single nucleus and remained arrested in that stage (Figure 3A and B). Immunostaining with an antibody against the centromeric histone Cna1 (48) revealed that the centromeres were correctly bioriented during attempted anaphase (Figure 3A). However, while the WT formed two haploid late anaphase/telophase ensembles with 5 centromeres, mutants retained nuclei with a diploid set of 10 centromeres. This indicates that the spindle was pulling, but was unable to separate homologous chromosomes.Figure 3.

Bottom Line: We show in Tetrahymena, that homologous chromosomes fail to separate and JMs accumulate in the absence of Mus81 or Sgs1, whereas deletion of the MutLγ-component Mlh1 does not affect meiotic divisions.Thus, our results are consistent with Mus81 being part of an essential, if not the predominant, CO pathway in Tetrahymena.Tetrahymena shares the predominance of the Mus81 CO pathway with the fission yeast.

View Article: PubMed Central - PubMed

Affiliation: Department of Chromosome Biology, Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, A-1030 Vienna, Austria.

ABSTRACT
Mus81 resolvase and Sgs1 helicase have well-established roles in mitotic DNA repair. Moreover, Mus81 is part of a minor crossover (CO) pathway in the meiosis of budding yeast, plants and vertebrates. The major pathway depends on meiosis-specific synaptonemal complex (SC) formation, ZMM proteins and the MutLγ complex for CO-directed resolution of joint molecule (JM)-recombination intermediates. Sgs1 has also been implicated in this pathway, although it may mainly promote the non-CO outcome of meiotic repair. We show in Tetrahymena, that homologous chromosomes fail to separate and JMs accumulate in the absence of Mus81 or Sgs1, whereas deletion of the MutLγ-component Mlh1 does not affect meiotic divisions. Thus, our results are consistent with Mus81 being part of an essential, if not the predominant, CO pathway in Tetrahymena. Sgs1 may exert functions similar to those in other eukaryotes. However, we propose an additional role in supporting homologous CO formation by promoting homologous over intersister interactions. Tetrahymena shares the predominance of the Mus81 CO pathway with the fission yeast. We propose that in these two organisms, which independently lost the SC during evolution, the basal set of mitotic repair proteins is sufficient for executing meiotic recombination.

Show MeSH
Related in: MedlinePlus