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. 2012 Jan 15;23(2):347–359. doi: 10.1091/mbc.E11-06-0568

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© 2012 Fukunaga 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 8: — Effect of Tbf1 binding on checkpoint activation at adjacent DNA ends with the 22–base pair TG₂₂ sequence. (A) Effect of the XIII_R and 22–base pair TG₂₂ sequence on Rad53 phosphorylation after HO expression. TG₂₂-HO-CA (KSC2230), XIII_R-HO-CA (KSC2894), or XIII_R-TG₂₂-HO-CA (KSC2834) cells were transformed with the GAL-HO plasmid. Transformed cells were grown in sucrose and synchronized at G2/M with nocodazole. After arrest, the culture was incubated with galactose to induce HO expression. Aliquots of cells were collected at the indicated times after HO expression, and cell extracts were analyzed by immunoblotting analysis with anti-Rad53 antibodies. (B) Effect of the XIII_R and 22–base pair TG₂₂ sequence on Mre11 accumulation at DNA ends. XIII_R-TG₂₂-HO-CA MRE11-myc (KSC2892), TG₂₂-HO-CA MRE11-myc (KSC2901), or XIII_R-HO-CA MRE11-myc (KSC2905) cells carrying the GAL-HO plasmid were analyzed by ChIP assay as in Figure 1B. (C) Effect of the XIII_R and 22–base pair TG₂₂ sequence on Tel1 accumulation at DNA ends. XIII_R-TG₂₂-HO-CA TEL1-HA (KSC2896), TG₂₂-HO-CA TEL1-HA (KSC2895), or XIII_R-HO-CA TEL1-HA (KSC2897) cells carrying the GAL-HO plasmid were analyzed by ChIP assay as B. (D) Effect of the XIII_R and 22–base pair TG₂₂ sequence on Mec1 accumulation at DNA ends. XIII_R-TG₂₂-HO-CA MEC1-HA (KSC2899), TG₂₂-HO-CA MEC1-HA (KSC2898), or XIII_R-HO-CA MEC1-HA (KSC2900) cells carrying the GAL-HO plasmid were analyzed by ChIP assay as in B. (E) Effect of Tbf1 depletion on checkpoint signaling from XIII_R-TG₂₂ ends. TG₂₂-HO-CA (KSC2230), XIII_R-TG₂₂-HO-CA (KSC2839), or XIII_R-TG₂₂-HO-CA tbf1-d (KSC2836) cells were transformed with the GAL-HO plasmid. Transformants were treated as in Figure 6C to induce HO expression and Tbf1 degradation. Cells were subjected to immunoblotting analysis as in A.