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Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast.

Li P, Jin H, Yu HG - Mol. Biol. Cell (2014)

Bottom Line: Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset.We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation.Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370.

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Requirement of condensin for suppressing rDNA recombination during yeast meiosis. (A) Meiotic gene conversion (GC) rate at rDNA and ARG4. Yeast strains (2998C, 2998, 3003, 3021, and 3025) were induced to undergo meiosis at 30°C, a semipermissive temperature for condensin mutants ycs4-2, ycg1-2, and smc2-8. Tetrads were dissected and genotyped at 25°C, and the GC rate was determined by marker segregation. Top, reporter assay of rDNA gene conversion. Note that an increased GC rate was observed in all condensin and sir2Δ mutants at rDNA but not at ARG4. (B) Two-dimensional gel mapping of Holliday junction (H.J.) molecules at rDNA. Left, schematic diagram showing the position of H.J. molecules. Arrows on the 2D gels point to the formation of H.J. molecules. RFB, replication fork barrier at the rDNA. (C) Rad51 focus formation during meiosis I. Wild-type (HY3140) and PCLB2-BRN1 (HY2826) cells were induced to undergo synchronous meiosis. Aliquots were withdrawn at 3, 4, and 5 h after induction of meiosis. Nucleus surface spreads were performed, followed by indirect immunofluorescence. An anti-Rad51 polyclonal antibody was used to detect Rad51. Stages of meiosis were determined on the basis of chromosome morphology. Blue, 4′,6-diamidino-2-phenylindole–stained DNA; red, rDNA; green, Rad51. Bar, 2 μm. (D) Quantification of Rad51 focus formation in wild-type and PCLB2-BRN1 cells during meiosis I. (E) Quantification of Rad51 focus formation at the rDNA locus in wild-type and PCLB2-BRN1 cells. (F) The number of cells with Rad51 focus formation at the rDNA during meiosis I.
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Figure 2: Requirement of condensin for suppressing rDNA recombination during yeast meiosis. (A) Meiotic gene conversion (GC) rate at rDNA and ARG4. Yeast strains (2998C, 2998, 3003, 3021, and 3025) were induced to undergo meiosis at 30°C, a semipermissive temperature for condensin mutants ycs4-2, ycg1-2, and smc2-8. Tetrads were dissected and genotyped at 25°C, and the GC rate was determined by marker segregation. Top, reporter assay of rDNA gene conversion. Note that an increased GC rate was observed in all condensin and sir2Δ mutants at rDNA but not at ARG4. (B) Two-dimensional gel mapping of Holliday junction (H.J.) molecules at rDNA. Left, schematic diagram showing the position of H.J. molecules. Arrows on the 2D gels point to the formation of H.J. molecules. RFB, replication fork barrier at the rDNA. (C) Rad51 focus formation during meiosis I. Wild-type (HY3140) and PCLB2-BRN1 (HY2826) cells were induced to undergo synchronous meiosis. Aliquots were withdrawn at 3, 4, and 5 h after induction of meiosis. Nucleus surface spreads were performed, followed by indirect immunofluorescence. An anti-Rad51 polyclonal antibody was used to detect Rad51. Stages of meiosis were determined on the basis of chromosome morphology. Blue, 4′,6-diamidino-2-phenylindole–stained DNA; red, rDNA; green, Rad51. Bar, 2 μm. (D) Quantification of Rad51 focus formation in wild-type and PCLB2-BRN1 cells during meiosis I. (E) Quantification of Rad51 focus formation at the rDNA locus in wild-type and PCLB2-BRN1 cells. (F) The number of cells with Rad51 focus formation at the rDNA during meiosis I.

Mentions: Because meiotic recombination takes place at prophase I, the temporal enrichment of condensin at the rDNA led us to hypothesize that condensin regulates DSB formation at this locus. To test this hypothesis, we determined the gene conversion rate at the rDNA gene cluster using a URA3 marker incorporated at a single 25S rRNA gene on one of the homologues (Figure 2A). Yeast diploid cells were induced to undergo meiosis to produce haploid spores, which were enclosed in tetrads. Yeast tetrads were dissected, and spores were genotyped. In wild-type cells (n = 206), most tetrads had the URA3 marker segregated 2:2, and only 0.5% of tetrads showed rearrangement of the URA3 marker by gene conversion (Figure 2A). Rearranged URA3 markers showed essentially equal ratios of 1:3 and 3:1 (unpublished data). These data suggest that very little meiotic recombination occurred at the rDNA, consistent with previous findings (Gottlieb and Esposito, 1989; San-Segundo and Roeder, 1999). In contrast, in the condensin mutant ycs4-2 (n = 202), the gene conversion rate increased ∼28-fold at the semipermissive temperature of 30°C. Mutant spores showed similar ratios of 1:3 and 3:1 URA3 segregation, indicating gene conversion between rDNA homologues. Successful use of this assay required that temperature-sensitive condensin mutants be sporulated under semipermissive conditions, a state in which condensin function is only partially inactivated, resulting in the variation observed in condensin mutants ycs4-2, ycg1-2, and smc2-8 (Figure 2A). Similar to condensin mutants, sir2Δ cells showed a dramatic increase in rDNA gene conversion (Figure 2A). Because the gene conversion rate at the rDNA was significantly increased in all condensin mutants tested (Figure 2A), and because condensin did not significantly alter the gene conversion rates at two nonrepetitive sites (ARG4 and HIS4) of the yeast genome (Figure 2A and unpublished data), we conclude that condensin is required for suppression of homologue exchange at the repetitive rDNA array.


Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast.

Li P, Jin H, Yu HG - Mol. Biol. Cell (2014)

Requirement of condensin for suppressing rDNA recombination during yeast meiosis. (A) Meiotic gene conversion (GC) rate at rDNA and ARG4. Yeast strains (2998C, 2998, 3003, 3021, and 3025) were induced to undergo meiosis at 30°C, a semipermissive temperature for condensin mutants ycs4-2, ycg1-2, and smc2-8. Tetrads were dissected and genotyped at 25°C, and the GC rate was determined by marker segregation. Top, reporter assay of rDNA gene conversion. Note that an increased GC rate was observed in all condensin and sir2Δ mutants at rDNA but not at ARG4. (B) Two-dimensional gel mapping of Holliday junction (H.J.) molecules at rDNA. Left, schematic diagram showing the position of H.J. molecules. Arrows on the 2D gels point to the formation of H.J. molecules. RFB, replication fork barrier at the rDNA. (C) Rad51 focus formation during meiosis I. Wild-type (HY3140) and PCLB2-BRN1 (HY2826) cells were induced to undergo synchronous meiosis. Aliquots were withdrawn at 3, 4, and 5 h after induction of meiosis. Nucleus surface spreads were performed, followed by indirect immunofluorescence. An anti-Rad51 polyclonal antibody was used to detect Rad51. Stages of meiosis were determined on the basis of chromosome morphology. Blue, 4′,6-diamidino-2-phenylindole–stained DNA; red, rDNA; green, Rad51. Bar, 2 μm. (D) Quantification of Rad51 focus formation in wild-type and PCLB2-BRN1 cells during meiosis I. (E) Quantification of Rad51 focus formation at the rDNA locus in wild-type and PCLB2-BRN1 cells. (F) The number of cells with Rad51 focus formation at the rDNA during meiosis I.
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Figure 2: Requirement of condensin for suppressing rDNA recombination during yeast meiosis. (A) Meiotic gene conversion (GC) rate at rDNA and ARG4. Yeast strains (2998C, 2998, 3003, 3021, and 3025) were induced to undergo meiosis at 30°C, a semipermissive temperature for condensin mutants ycs4-2, ycg1-2, and smc2-8. Tetrads were dissected and genotyped at 25°C, and the GC rate was determined by marker segregation. Top, reporter assay of rDNA gene conversion. Note that an increased GC rate was observed in all condensin and sir2Δ mutants at rDNA but not at ARG4. (B) Two-dimensional gel mapping of Holliday junction (H.J.) molecules at rDNA. Left, schematic diagram showing the position of H.J. molecules. Arrows on the 2D gels point to the formation of H.J. molecules. RFB, replication fork barrier at the rDNA. (C) Rad51 focus formation during meiosis I. Wild-type (HY3140) and PCLB2-BRN1 (HY2826) cells were induced to undergo synchronous meiosis. Aliquots were withdrawn at 3, 4, and 5 h after induction of meiosis. Nucleus surface spreads were performed, followed by indirect immunofluorescence. An anti-Rad51 polyclonal antibody was used to detect Rad51. Stages of meiosis were determined on the basis of chromosome morphology. Blue, 4′,6-diamidino-2-phenylindole–stained DNA; red, rDNA; green, Rad51. Bar, 2 μm. (D) Quantification of Rad51 focus formation in wild-type and PCLB2-BRN1 cells during meiosis I. (E) Quantification of Rad51 focus formation at the rDNA locus in wild-type and PCLB2-BRN1 cells. (F) The number of cells with Rad51 focus formation at the rDNA during meiosis I.
Mentions: Because meiotic recombination takes place at prophase I, the temporal enrichment of condensin at the rDNA led us to hypothesize that condensin regulates DSB formation at this locus. To test this hypothesis, we determined the gene conversion rate at the rDNA gene cluster using a URA3 marker incorporated at a single 25S rRNA gene on one of the homologues (Figure 2A). Yeast diploid cells were induced to undergo meiosis to produce haploid spores, which were enclosed in tetrads. Yeast tetrads were dissected, and spores were genotyped. In wild-type cells (n = 206), most tetrads had the URA3 marker segregated 2:2, and only 0.5% of tetrads showed rearrangement of the URA3 marker by gene conversion (Figure 2A). Rearranged URA3 markers showed essentially equal ratios of 1:3 and 3:1 (unpublished data). These data suggest that very little meiotic recombination occurred at the rDNA, consistent with previous findings (Gottlieb and Esposito, 1989; San-Segundo and Roeder, 1999). In contrast, in the condensin mutant ycs4-2 (n = 202), the gene conversion rate increased ∼28-fold at the semipermissive temperature of 30°C. Mutant spores showed similar ratios of 1:3 and 3:1 URA3 segregation, indicating gene conversion between rDNA homologues. Successful use of this assay required that temperature-sensitive condensin mutants be sporulated under semipermissive conditions, a state in which condensin function is only partially inactivated, resulting in the variation observed in condensin mutants ycs4-2, ycg1-2, and smc2-8 (Figure 2A). Similar to condensin mutants, sir2Δ cells showed a dramatic increase in rDNA gene conversion (Figure 2A). Because the gene conversion rate at the rDNA was significantly increased in all condensin mutants tested (Figure 2A), and because condensin did not significantly alter the gene conversion rates at two nonrepetitive sites (ARG4 and HIS4) of the yeast genome (Figure 2A and unpublished data), we conclude that condensin is required for suppression of homologue exchange at the repetitive rDNA array.

Bottom Line: Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset.We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation.Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370.

Show MeSH
Related in: MedlinePlus