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. 2015 Oct 1;26(19):3480–3488. doi: 10.1091/mbc.E15-05-0259

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© 2015 Ogi, Goto, Ghosh, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — Effect of tel1-21 or tel1-ΔC mutation on association with DNA ends. (A) Effect of tel1-21 or tel1-ΔC mutation on Tel1 localization to an HO-induced DSB. Wild-type (KSC1709), tel1-21 (KSC3087) or tel1-ΔC (KSC3549) cells expressing Tel1-HA were transformed with the YCpA-GAL-HO plasmid. Transformed cells were grown in sucrose and treated with nocodazole. After arrest at G2/M, the culture was incubated with galactose for 3 h to induce HO expression. Top, the strains contain an HO cleavage site, marked with HIS2, at the ADH4 locus on chromosome (Chr.) VII. The HO1 primer pair amplifies a region 1 kb away from the HO cleavage site. An arrow represents the telomere. Bottom, cells were subjected to chromatin immunoprecipitation with anti-HA antibodies. Association of Tel1 with an HO-induced DSB was analyzed by real-time PCR. Relative enrichment was determined from three independent experiments. (B) Effect of tel1-ΔC mutation on the intracellular distribution. TEL1-HA (KSC1709) or tel1-ΔC-HA (KSC3549) cells were grown to mid log phase, harvested, and spheroplasted. Spheroplasts were homogenized to prepare whole-cell extracts (W) and then separated into the cytoplasmic (C) and nuclear (N) fractions. Samples from each fraction were separated by SDS–PAGE and immunoblotted with anti-HA, anti-Zwf1 (glucose-6-phosphate dehydrogenase; G6PDH) or anti-nuclear pore complex (NPC) antibodies. (C) Effect of tel1-ΔC mutation on Tel1–Tel2 interaction. Whole-cell extracts (WCE) prepared from cells expressing Tel1-HA (KSC1709), Tel2-FLAG Tel1-HA (KSC3633), or Tel2-FLAG tel1-ΔC-HA (KSC3634) were subjected to immunoprecipitation (IP) with anti-FLAG antibodies. Samples were immunoblotted with anti-HA or anti-FLAG antibodies. (D) Effect of tel1-ΔC mutation on Tel1–Xrs2 interaction. FLAG-Tel1 or FLAG-Tel1-ΔC was incubated with GST-Xrs2C–bound glutathione beads. Proteins bound to glutathione beads were subjected to immunoblotting analysis with anti-FLAG antibodies. The binding assay used 0.5 pmol of FLAG-Tel1 proteins and 70 pmol of GST-Xrs2C.