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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.

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

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Requirement of condensin for repair of an artificial rDNA break during meiosis. (A) Schematic diagram showing the generation of an inducible DSB at the rDNA. (B) PCR-based assay of chromosome breakage at the rDNA (HY3050 and HY 4490). I-SceI fragment was amplified with one primer from the rDNA and another from the selectable marker Clonat, as indicated in A. The control fragment was from ACT1. Right, quantification of break formation at the rDNA. (C) Rad52 focus formation in yeast cells with an inducible rDNA break during meiosis. Yeast strains (HY3831, HY3887, HY3899, HY3900, and HY3901) were induced to undergo synchronous meiosis; Rad52 focus formation was determined by fluorescence microscopy. (D) Duration of Rad52 focus in meiotic yeast cells with an induced rDNA break (HY3831 and HY3887). (E) Localization of Rad52 foci in live meiotic cells. Three classes of Rad52-GFP (green) localization were found: inside the nucleolus (Nop1-RFP, red), at the periphery of the nucleolus, and outside of the nucleolus. (F) Segregation of rDNA in the presence of an inducible DSB. Yeast strains HY3050, HY3456, HY3458, HY4488, HY4489, and HY4490 all harbored the I-SceI recognition sequence, but only HY3050, HY3456, and HY3458 produced I-SceI.
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Figure 7: Requirement of condensin for repair of an artificial rDNA break during meiosis. (A) Schematic diagram showing the generation of an inducible DSB at the rDNA. (B) PCR-based assay of chromosome breakage at the rDNA (HY3050 and HY 4490). I-SceI fragment was amplified with one primer from the rDNA and another from the selectable marker Clonat, as indicated in A. The control fragment was from ACT1. Right, quantification of break formation at the rDNA. (C) Rad52 focus formation in yeast cells with an inducible rDNA break during meiosis. Yeast strains (HY3831, HY3887, HY3899, HY3900, and HY3901) were induced to undergo synchronous meiosis; Rad52 focus formation was determined by fluorescence microscopy. (D) Duration of Rad52 focus in meiotic yeast cells with an induced rDNA break (HY3831 and HY3887). (E) Localization of Rad52 foci in live meiotic cells. Three classes of Rad52-GFP (green) localization were found: inside the nucleolus (Nop1-RFP, red), at the periphery of the nucleolus, and outside of the nucleolus. (F) Segregation of rDNA in the presence of an inducible DSB. Yeast strains HY3050, HY3456, HY3458, HY4488, HY4489, and HY4490 all harbored the I-SceI recognition sequence, but only HY3050, HY3456, and HY3458 produced I-SceI.

Mentions: To investigate further how condensin regulates DSB processing, we generated an artificial DSB at the rDNA using the I-SceI system (Figure 7A). Artificial DSBs by the I-SceI site-specific endonuclease appear to be processed as endogenous Spo11-mediated DSBs (Fukuda et al., 2008) but would simplify our assay of condensin function in DSB repair. We abolished the formation of endogenous DSBs by the spo11Δ allele and then inserted one copy of the recognition sequence of I-SceI into the 25S rRNA gene where the URA3 marker was incorporated (Figure 2A). On the induction of I-SceI by the meiosis-specific REC8 promoter, I-SceI cut its recognition sequence with ∼80% efficiency, as determined by a PCR-based assay of chromosome breakage (Figure 7B). By fluorescence microscopy, we observed both Rad51 and Rad52 focus formation at the rDNA region (Figure 7C and Supplemental Figure S4, A–C), indicating the formation of rDNA breaks.


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 repair of an artificial rDNA break during meiosis. (A) Schematic diagram showing the generation of an inducible DSB at the rDNA. (B) PCR-based assay of chromosome breakage at the rDNA (HY3050 and HY 4490). I-SceI fragment was amplified with one primer from the rDNA and another from the selectable marker Clonat, as indicated in A. The control fragment was from ACT1. Right, quantification of break formation at the rDNA. (C) Rad52 focus formation in yeast cells with an inducible rDNA break during meiosis. Yeast strains (HY3831, HY3887, HY3899, HY3900, and HY3901) were induced to undergo synchronous meiosis; Rad52 focus formation was determined by fluorescence microscopy. (D) Duration of Rad52 focus in meiotic yeast cells with an induced rDNA break (HY3831 and HY3887). (E) Localization of Rad52 foci in live meiotic cells. Three classes of Rad52-GFP (green) localization were found: inside the nucleolus (Nop1-RFP, red), at the periphery of the nucleolus, and outside of the nucleolus. (F) Segregation of rDNA in the presence of an inducible DSB. Yeast strains HY3050, HY3456, HY3458, HY4488, HY4489, and HY4490 all harbored the I-SceI recognition sequence, but only HY3050, HY3456, and HY3458 produced I-SceI.
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Figure 7: Requirement of condensin for repair of an artificial rDNA break during meiosis. (A) Schematic diagram showing the generation of an inducible DSB at the rDNA. (B) PCR-based assay of chromosome breakage at the rDNA (HY3050 and HY 4490). I-SceI fragment was amplified with one primer from the rDNA and another from the selectable marker Clonat, as indicated in A. The control fragment was from ACT1. Right, quantification of break formation at the rDNA. (C) Rad52 focus formation in yeast cells with an inducible rDNA break during meiosis. Yeast strains (HY3831, HY3887, HY3899, HY3900, and HY3901) were induced to undergo synchronous meiosis; Rad52 focus formation was determined by fluorescence microscopy. (D) Duration of Rad52 focus in meiotic yeast cells with an induced rDNA break (HY3831 and HY3887). (E) Localization of Rad52 foci in live meiotic cells. Three classes of Rad52-GFP (green) localization were found: inside the nucleolus (Nop1-RFP, red), at the periphery of the nucleolus, and outside of the nucleolus. (F) Segregation of rDNA in the presence of an inducible DSB. Yeast strains HY3050, HY3456, HY3458, HY4488, HY4489, and HY4490 all harbored the I-SceI recognition sequence, but only HY3050, HY3456, and HY3458 produced I-SceI.
Mentions: To investigate further how condensin regulates DSB processing, we generated an artificial DSB at the rDNA using the I-SceI system (Figure 7A). Artificial DSBs by the I-SceI site-specific endonuclease appear to be processed as endogenous Spo11-mediated DSBs (Fukuda et al., 2008) but would simplify our assay of condensin function in DSB repair. We abolished the formation of endogenous DSBs by the spo11Δ allele and then inserted one copy of the recognition sequence of I-SceI into the 25S rRNA gene where the URA3 marker was incorporated (Figure 2A). On the induction of I-SceI by the meiosis-specific REC8 promoter, I-SceI cut its recognition sequence with ∼80% efficiency, as determined by a PCR-based assay of chromosome breakage (Figure 7B). By fluorescence microscopy, we observed both Rad51 and Rad52 focus formation at the rDNA region (Figure 7C and Supplemental Figure S4, A–C), indicating the formation of rDNA breaks.

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