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Role of SAGA in the asymmetric segregation of DNA circles during yeast ageing.

Denoth-Lippuner A, Krzyzanowski MK, Stober C, Barral Y - Elife (2014)

Bottom Line: Reciprocally, this causes retention and accumulation of NPCs, which affects the organization of ageing nuclei.Thus, SAGA prevents the spreading of DNA circles by linking them to NPCs, but unavoidably causes accumulation of circles and NPCs in the mother cell, and thereby promotes ageing.Together, our data provide a unifying model for the asymmetric segregation of DNA circles and how age affects nuclear organization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Department of Biology, ETH Zürich, Zürich, Switzerland.

ABSTRACT
In eukaryotes, intra-chromosomal recombination generates DNA circles, but little is known about how cells react to them. In yeast, partitioning of such circles to the mother cell at mitosis ensures their loss from the population but promotes replicative ageing. Nevertheless, the mechanisms of partitioning are debated. In this study, we show that the SAGA complex mediates the interaction of non-chromosomal DNA circles with nuclear pore complexes (NPCs) and thereby promotes their confinement in the mother cell. Reciprocally, this causes retention and accumulation of NPCs, which affects the organization of ageing nuclei. Thus, SAGA prevents the spreading of DNA circles by linking them to NPCs, but unavoidably causes accumulation of circles and NPCs in the mother cell, and thereby promotes ageing. Together, our data provide a unifying model for the asymmetric segregation of DNA circles and how age affects nuclear organization.

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The SAGA complex mediates retention of DNA circles including ERCs.(A) Representative images and quantifications of plasmid propagation frequencies (pf) in wt and different mutant cells (mean ± SD N ≥ 3 clones). (B) Mother-bud distribution of the rDNA containing plasmid pDS163 in wt, gcn5∆ and sgf73∆ mutant cells (mean ± SD N = 10 independent experiments). (C) Detection of ERC levels in wt, sgf73∆ and gcn5∆ mutant cells by Southern blotting (« depict ERCs) in 11 and 14 generations old cells. (D) Quantifications of half-sectors representing recombination events within the rDNA repeats in wt and mutant cells (mean ± SD N = 3 clones). (A–D) ***p < 0.001.DOI:http://dx.doi.org/10.7554/eLife.03790.007
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fig3: The SAGA complex mediates retention of DNA circles including ERCs.(A) Representative images and quantifications of plasmid propagation frequencies (pf) in wt and different mutant cells (mean ± SD N ≥ 3 clones). (B) Mother-bud distribution of the rDNA containing plasmid pDS163 in wt, gcn5∆ and sgf73∆ mutant cells (mean ± SD N = 10 independent experiments). (C) Detection of ERC levels in wt, sgf73∆ and gcn5∆ mutant cells by Southern blotting (« depict ERCs) in 11 and 14 generations old cells. (D) Quantifications of half-sectors representing recombination events within the rDNA repeats in wt and mutant cells (mean ± SD N = 3 clones). (A–D) ***p < 0.001.DOI:http://dx.doi.org/10.7554/eLife.03790.007

Mentions: Next, we wondered what caused the interaction of the circles with the nuclear periphery and if this contributed to circle retention. Therefore, we asked whether any of the protein complexes already reported to mediate chromatin interaction with the nuclear periphery facilitate the retention of DNA circles. We screened a collection of knockout strains for defects in circle partitioning, using the labeled plasmid described above. Three hours after centromere excision, individual plasmid dots were visualized and the frequency at which they segregated to the bud was determined (propagation frequency, pf, see ‘Materials and methods’). In wild-type cells, non-centromeric circles rarely passed to the daughter cells (pf = 3.9 ± 1.1; Figure 3A), that is 2.5- to 4-fold less frequently than predicted by the passive model (pf = 10–15; (Gehlen et al., 2011)). Circle propagation into the bud was significantly increased in yku70∆ and bud6∆ cells, as previously reported ((Shcheprova et al., 2008; Gehlen et al., 2011); pf = 10.3 ± 1.3 and 11.8 ± 0.8, respectively, N = 3 clones, >50 cells each, p < 0.001; Figure 3A), validating our approach.10.7554/eLife.03790.007Figure 3.The SAGA complex mediates retention of DNA circles including ERCs.


Role of SAGA in the asymmetric segregation of DNA circles during yeast ageing.

Denoth-Lippuner A, Krzyzanowski MK, Stober C, Barral Y - Elife (2014)

The SAGA complex mediates retention of DNA circles including ERCs.(A) Representative images and quantifications of plasmid propagation frequencies (pf) in wt and different mutant cells (mean ± SD N ≥ 3 clones). (B) Mother-bud distribution of the rDNA containing plasmid pDS163 in wt, gcn5∆ and sgf73∆ mutant cells (mean ± SD N = 10 independent experiments). (C) Detection of ERC levels in wt, sgf73∆ and gcn5∆ mutant cells by Southern blotting (« depict ERCs) in 11 and 14 generations old cells. (D) Quantifications of half-sectors representing recombination events within the rDNA repeats in wt and mutant cells (mean ± SD N = 3 clones). (A–D) ***p < 0.001.DOI:http://dx.doi.org/10.7554/eLife.03790.007
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232608&req=5

fig3: The SAGA complex mediates retention of DNA circles including ERCs.(A) Representative images and quantifications of plasmid propagation frequencies (pf) in wt and different mutant cells (mean ± SD N ≥ 3 clones). (B) Mother-bud distribution of the rDNA containing plasmid pDS163 in wt, gcn5∆ and sgf73∆ mutant cells (mean ± SD N = 10 independent experiments). (C) Detection of ERC levels in wt, sgf73∆ and gcn5∆ mutant cells by Southern blotting (« depict ERCs) in 11 and 14 generations old cells. (D) Quantifications of half-sectors representing recombination events within the rDNA repeats in wt and mutant cells (mean ± SD N = 3 clones). (A–D) ***p < 0.001.DOI:http://dx.doi.org/10.7554/eLife.03790.007
Mentions: Next, we wondered what caused the interaction of the circles with the nuclear periphery and if this contributed to circle retention. Therefore, we asked whether any of the protein complexes already reported to mediate chromatin interaction with the nuclear periphery facilitate the retention of DNA circles. We screened a collection of knockout strains for defects in circle partitioning, using the labeled plasmid described above. Three hours after centromere excision, individual plasmid dots were visualized and the frequency at which they segregated to the bud was determined (propagation frequency, pf, see ‘Materials and methods’). In wild-type cells, non-centromeric circles rarely passed to the daughter cells (pf = 3.9 ± 1.1; Figure 3A), that is 2.5- to 4-fold less frequently than predicted by the passive model (pf = 10–15; (Gehlen et al., 2011)). Circle propagation into the bud was significantly increased in yku70∆ and bud6∆ cells, as previously reported ((Shcheprova et al., 2008; Gehlen et al., 2011); pf = 10.3 ± 1.3 and 11.8 ± 0.8, respectively, N = 3 clones, >50 cells each, p < 0.001; Figure 3A), validating our approach.10.7554/eLife.03790.007Figure 3.The SAGA complex mediates retention of DNA circles including ERCs.

Bottom Line: Reciprocally, this causes retention and accumulation of NPCs, which affects the organization of ageing nuclei.Thus, SAGA prevents the spreading of DNA circles by linking them to NPCs, but unavoidably causes accumulation of circles and NPCs in the mother cell, and thereby promotes ageing.Together, our data provide a unifying model for the asymmetric segregation of DNA circles and how age affects nuclear organization.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Department of Biology, ETH Zürich, Zürich, Switzerland.

ABSTRACT
In eukaryotes, intra-chromosomal recombination generates DNA circles, but little is known about how cells react to them. In yeast, partitioning of such circles to the mother cell at mitosis ensures their loss from the population but promotes replicative ageing. Nevertheless, the mechanisms of partitioning are debated. In this study, we show that the SAGA complex mediates the interaction of non-chromosomal DNA circles with nuclear pore complexes (NPCs) and thereby promotes their confinement in the mother cell. Reciprocally, this causes retention and accumulation of NPCs, which affects the organization of ageing nuclei. Thus, SAGA prevents the spreading of DNA circles by linking them to NPCs, but unavoidably causes accumulation of circles and NPCs in the mother cell, and thereby promotes ageing. Together, our data provide a unifying model for the asymmetric segregation of DNA circles and how age affects nuclear organization.

Show MeSH
Related in: MedlinePlus