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. 2011 Feb 7;192(3):417–431. doi: 10.1083/jcb.201008007

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© 2011 Ruault et al.

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Figure 6. — Nonacetylable Sir3 promotes telomere clustering in the absence of Sir2 and Sir4 but requires Rap1 C terminus. (A) Fluorescent images of Rap1-GFP in GAL1p-SIR3 (yAT208), GAL1p-SIR3 sir2Δ (yAT772), GAL1p-Sir3-A2Q (yAT1205), and GAL1p-Sir3-A2Q sir2Δ (yAT1334) cells grown in synthetic complete galactose medium overnight, diluted to OD_600nm = 0.2, and imaged at OD_600nm = 1. (B) ChIP analysis with an anti-Sir3 to study the spreading of Sir3 on telomere VIR: GAL1p-SIR3 (yAT208), GAL1p-Sir3-A2Q (yAT1205), GAL1p-SIR3 sir2Δ (yAT772), GAL1p-Sir3-A2Q sir2Δ (yAT1334), GAL1p-SIR3-GFP rap1–17 (yAT1357), and GAL1p-Sir3-A2Q-GFP rap1–17 (yAT1358) strains were grown in YPGal for 48 h. The bar graph represents the enrichment over the mitochondrial locus OLI1 for amplicons at different distances from the TG repeats as indicated. Enrichment at the internal chromosomal locus OGG1 is shown for comparison. The experiment was repeated three times. Error bars represent means ± SEM. (C) Fluorescent images of the overexpressed Sir3 or Sir3-A2Q proteins tagged with GFP in their C terminus: the GAL1p-SIR3-GFP (yAT780), GAL1p-SIR3-GFP sir2Δ (yAT782), GAL1p-Sir3-A2Q-GFP (yAT1337), GAL1p-Sir3-A2Q-GFP sir2Δ (yAT1338), and GAL1p-Sir3-A2Q-GFP rap1–17 (yAT1358) strains were grown as in A. (D) Fluorescent images of the GAL1p-SIR3-GFP sir2Δ sir4Δ (yAT1342) and GAL1p-Sir3-A2Q-GFP sir2Δ sir4Δ (yAT1340) strains grown as in A. (E) Fluorescent images of Rap1-GFP in GAL1p-Sir3-A2Q (yAT1205) and in GAL1p-Sir3-A2Q sir2Δ sir4Δ (yAT1336) cells grown as in A. WT, wild type. Bars, 2 µm.