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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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SAGA preferentially interacts with non-centromeric DNA circles (A).Fluorescent images (deconvolved max Z-projections) of Gcn5-GFP and Sgf73-GFP (green) in cells with and without accumulated plasmids (red). (B) ChIP-qPCR analysis to test the binding of the SAGA components Gcn5 and Spt20 to three sequences on pYB1670 (ARS, non-coding sequences (NCS) 1 and 2; see scheme of plasmid) and a control sequence on chromosome V (mean ± SD, N = 5 independent experiments, ***p < 0.001, **p < 0.01, *p < 0.05). (C) pf in cells expressing Gcn5 or Sir2 fused to TetR (mean ± SD, N = 3 clones, ***p < 0.001).DOI:http://dx.doi.org/10.7554/eLife.03790.010
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fig7: SAGA preferentially interacts with non-centromeric DNA circles (A).Fluorescent images (deconvolved max Z-projections) of Gcn5-GFP and Sgf73-GFP (green) in cells with and without accumulated plasmids (red). (B) ChIP-qPCR analysis to test the binding of the SAGA components Gcn5 and Spt20 to three sequences on pYB1670 (ARS, non-coding sequences (NCS) 1 and 2; see scheme of plasmid) and a control sequence on chromosome V (mean ± SD, N = 5 independent experiments, ***p < 0.001, **p < 0.01, *p < 0.05). (C) pf in cells expressing Gcn5 or Sir2 fused to TetR (mean ± SD, N = 3 clones, ***p < 0.001).DOI:http://dx.doi.org/10.7554/eLife.03790.010

Mentions: To address whether SAGA interacts with non-centromeric DNA circles, we then analyzed the localization of Gcn5-GFP and Sgf73-GFP fusion proteins in cells accumulating non-centromeric DNA circles labeled with TetR-mCherry. Both SAGA components were strongly enriched in the plasmid area, indicating that SAGA interacts more readily with non-chromosomal DNA than with the rest of the genome (Figure 7A). Accordingly, chromatin immunoprecipitation (ChIP) experiments (N = 5 independent experiments) reproducibly showed a clear enrichment of the SAGA proteins Gcn5 and Spt20 on a non-centromeric reporter plasmid (pYB1670/CEN-), as judged by probing for the autonomous replication sequence (ARS) and two non-coding sequences of bacterial origin. Strikingly, this enrichment was nearly lost (p < 0.05) when the same plasmid contained a centromere (CEN+; Figure 7B). Thus, SAGA is recruited to higher levels on non-centromeric than on centromeric DNA circles. Together, these data indicate that SAGA more stably associates with non-chromosomal than with chromosomal DNA.10.7554/eLife.03790.010Figure 7.SAGA preferentially interacts with non-centromeric DNA circles (A).


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

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

SAGA preferentially interacts with non-centromeric DNA circles (A).Fluorescent images (deconvolved max Z-projections) of Gcn5-GFP and Sgf73-GFP (green) in cells with and without accumulated plasmids (red). (B) ChIP-qPCR analysis to test the binding of the SAGA components Gcn5 and Spt20 to three sequences on pYB1670 (ARS, non-coding sequences (NCS) 1 and 2; see scheme of plasmid) and a control sequence on chromosome V (mean ± SD, N = 5 independent experiments, ***p < 0.001, **p < 0.01, *p < 0.05). (C) pf in cells expressing Gcn5 or Sir2 fused to TetR (mean ± SD, N = 3 clones, ***p < 0.001).DOI:http://dx.doi.org/10.7554/eLife.03790.010
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: SAGA preferentially interacts with non-centromeric DNA circles (A).Fluorescent images (deconvolved max Z-projections) of Gcn5-GFP and Sgf73-GFP (green) in cells with and without accumulated plasmids (red). (B) ChIP-qPCR analysis to test the binding of the SAGA components Gcn5 and Spt20 to three sequences on pYB1670 (ARS, non-coding sequences (NCS) 1 and 2; see scheme of plasmid) and a control sequence on chromosome V (mean ± SD, N = 5 independent experiments, ***p < 0.001, **p < 0.01, *p < 0.05). (C) pf in cells expressing Gcn5 or Sir2 fused to TetR (mean ± SD, N = 3 clones, ***p < 0.001).DOI:http://dx.doi.org/10.7554/eLife.03790.010
Mentions: To address whether SAGA interacts with non-centromeric DNA circles, we then analyzed the localization of Gcn5-GFP and Sgf73-GFP fusion proteins in cells accumulating non-centromeric DNA circles labeled with TetR-mCherry. Both SAGA components were strongly enriched in the plasmid area, indicating that SAGA interacts more readily with non-chromosomal DNA than with the rest of the genome (Figure 7A). Accordingly, chromatin immunoprecipitation (ChIP) experiments (N = 5 independent experiments) reproducibly showed a clear enrichment of the SAGA proteins Gcn5 and Spt20 on a non-centromeric reporter plasmid (pYB1670/CEN-), as judged by probing for the autonomous replication sequence (ARS) and two non-coding sequences of bacterial origin. Strikingly, this enrichment was nearly lost (p < 0.05) when the same plasmid contained a centromere (CEN+; Figure 7B). Thus, SAGA is recruited to higher levels on non-centromeric than on centromeric DNA circles. Together, these data indicate that SAGA more stably associates with non-chromosomal than with chromosomal DNA.10.7554/eLife.03790.010Figure 7.SAGA preferentially interacts with non-centromeric DNA circles (A).

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