Limits...
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.

Show MeSH

Related in: MedlinePlus

The execution point of Ycg1 at rDNA during meiosis. (A) A schematic diagram showing the experimental procedure. Yeast cells from the ycg1-2 and ycg1-2 spo11Δ strains (HY3137 and HY4677) were induced to undergo synchronous meiosis and arrested at pachytene with GAL4.ER PGAL-NDT80. On the addition of estradiol and production of Ndt80, ycg1-2 cells were released from pachytene arrest. To inactivate the ycg1-2 allele, we cultured yeast cells at 34°C. (B–D) Quantification of Rad51 focus formation at pachytene. Yeast cells were staged at pachytene at either 25 or 34°C for 6 h after meiotic induction. Rad51 focus was determined by nucleus surface spreads and indirect immunofluorescence as in Figure 2C. (E–G) Quantification of Rad51 focus formation after pachytene release. We added 1 μM estradiol to release the cells from pachytene arrest. Samples were collected 1 h after addition of estradiol, followed by immunofluorescence microscopy. (H) Quantification of rDNA segregation during meiosis I. Yeast cells were staged at pachytene at either 25°C (permissive temperature) or 34°C (restrictive temperature) and then switched to 34 or 25°C upon the addition of estradiol. Segregation of GFP-marked rDNA was determined as shown in Figure 3C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230583&req=5

Figure 6: The execution point of Ycg1 at rDNA during meiosis. (A) A schematic diagram showing the experimental procedure. Yeast cells from the ycg1-2 and ycg1-2 spo11Δ strains (HY3137 and HY4677) were induced to undergo synchronous meiosis and arrested at pachytene with GAL4.ER PGAL-NDT80. On the addition of estradiol and production of Ndt80, ycg1-2 cells were released from pachytene arrest. To inactivate the ycg1-2 allele, we cultured yeast cells at 34°C. (B–D) Quantification of Rad51 focus formation at pachytene. Yeast cells were staged at pachytene at either 25 or 34°C for 6 h after meiotic induction. Rad51 focus was determined by nucleus surface spreads and indirect immunofluorescence as in Figure 2C. (E–G) Quantification of Rad51 focus formation after pachytene release. We added 1 μM estradiol to release the cells from pachytene arrest. Samples were collected 1 h after addition of estradiol, followed by immunofluorescence microscopy. (H) Quantification of rDNA segregation during meiosis I. Yeast cells were staged at pachytene at either 25°C (permissive temperature) or 34°C (restrictive temperature) and then switched to 34 or 25°C upon the addition of estradiol. Segregation of GFP-marked rDNA was determined as shown in Figure 3C.

Mentions: To address the timing of condensin function that is required for meiotic recombination and chromosome segregation, we determined the execution point of condensin using the ycg1-2 allele (Lavoie et al., 2004) because of its reversibility in inactivation and reactivation of the Ycg1 protein with temperature shift (Figure 6). We used the GAL4.ER PGAL-NDT80 allele (Carlile and Amon, 2008) to stage yeast cells at pachytene, which is permissible for DSB formation. On the addition of estradiol and activation of NDT80 gene expression, yeast cells resumed meiosis (Figure 6A). We found that the total number of Rad51 foci formed was comparable in arrested yeast cells at pachytene, but >60% of ycg1-2 cells showed Rad51 focus formation at the rDNA when meiosis was induced at the nonpermissive temperature (Figure 6, B–D). Furthermore, Rad51 foci persisted at the rDNA region at anaphase if ycg1-2 cells were induced to undergo meiosis at the nonpermissive temperature (Figure 6, E and F). Similarly, ∼50% of ycg1-2 cells retained Rad51 foci at rDNA even at anaphase if these cells were shifted from the permissive to the nonpermissive temperature before being released from pachytene (Figure 6, E–G). In contrast, there was little Rad51 focus formation on anaphase chromosomes when cells were switched from the nonpermissive to the permissive temperature (Figure 6, E–G), demonstrating that upon the reactivation of Ycg1 at the permissive temperature, DSBs can be repaired.


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)

The execution point of Ycg1 at rDNA during meiosis. (A) A schematic diagram showing the experimental procedure. Yeast cells from the ycg1-2 and ycg1-2 spo11Δ strains (HY3137 and HY4677) were induced to undergo synchronous meiosis and arrested at pachytene with GAL4.ER PGAL-NDT80. On the addition of estradiol and production of Ndt80, ycg1-2 cells were released from pachytene arrest. To inactivate the ycg1-2 allele, we cultured yeast cells at 34°C. (B–D) Quantification of Rad51 focus formation at pachytene. Yeast cells were staged at pachytene at either 25 or 34°C for 6 h after meiotic induction. Rad51 focus was determined by nucleus surface spreads and indirect immunofluorescence as in Figure 2C. (E–G) Quantification of Rad51 focus formation after pachytene release. We added 1 μM estradiol to release the cells from pachytene arrest. Samples were collected 1 h after addition of estradiol, followed by immunofluorescence microscopy. (H) Quantification of rDNA segregation during meiosis I. Yeast cells were staged at pachytene at either 25°C (permissive temperature) or 34°C (restrictive temperature) and then switched to 34 or 25°C upon the addition of estradiol. Segregation of GFP-marked rDNA was determined as shown in Figure 3C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230583&req=5

Figure 6: The execution point of Ycg1 at rDNA during meiosis. (A) A schematic diagram showing the experimental procedure. Yeast cells from the ycg1-2 and ycg1-2 spo11Δ strains (HY3137 and HY4677) were induced to undergo synchronous meiosis and arrested at pachytene with GAL4.ER PGAL-NDT80. On the addition of estradiol and production of Ndt80, ycg1-2 cells were released from pachytene arrest. To inactivate the ycg1-2 allele, we cultured yeast cells at 34°C. (B–D) Quantification of Rad51 focus formation at pachytene. Yeast cells were staged at pachytene at either 25 or 34°C for 6 h after meiotic induction. Rad51 focus was determined by nucleus surface spreads and indirect immunofluorescence as in Figure 2C. (E–G) Quantification of Rad51 focus formation after pachytene release. We added 1 μM estradiol to release the cells from pachytene arrest. Samples were collected 1 h after addition of estradiol, followed by immunofluorescence microscopy. (H) Quantification of rDNA segregation during meiosis I. Yeast cells were staged at pachytene at either 25°C (permissive temperature) or 34°C (restrictive temperature) and then switched to 34 or 25°C upon the addition of estradiol. Segregation of GFP-marked rDNA was determined as shown in Figure 3C.
Mentions: To address the timing of condensin function that is required for meiotic recombination and chromosome segregation, we determined the execution point of condensin using the ycg1-2 allele (Lavoie et al., 2004) because of its reversibility in inactivation and reactivation of the Ycg1 protein with temperature shift (Figure 6). We used the GAL4.ER PGAL-NDT80 allele (Carlile and Amon, 2008) to stage yeast cells at pachytene, which is permissible for DSB formation. On the addition of estradiol and activation of NDT80 gene expression, yeast cells resumed meiosis (Figure 6A). We found that the total number of Rad51 foci formed was comparable in arrested yeast cells at pachytene, but >60% of ycg1-2 cells showed Rad51 focus formation at the rDNA when meiosis was induced at the nonpermissive temperature (Figure 6, B–D). Furthermore, Rad51 foci persisted at the rDNA region at anaphase if ycg1-2 cells were induced to undergo meiosis at the nonpermissive temperature (Figure 6, E and F). Similarly, ∼50% of ycg1-2 cells retained Rad51 foci at rDNA even at anaphase if these cells were shifted from the permissive to the nonpermissive temperature before being released from pachytene (Figure 6, E–G). In contrast, there was little Rad51 focus formation on anaphase chromosomes when cells were switched from the nonpermissive to the permissive temperature (Figure 6, E–G), demonstrating that upon the reactivation of Ycg1 at the permissive temperature, DSBs can be repaired.

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