FIGURE 2:

Effect of Tbf1 depletion on localization of MRX and Tel1 to DNA ends with telomeric or subtelomeric sequences. (A) Effect of tbf1-degron mutation on colony formation on galactose medium at 37°C. Wild-type or tbf1-degron mutant (tbf1-d; KSC2853) cells were transformed with YCpU-TBF1 or the control vector. Cells were serially diluted and spotted on sucrose or galactose plates selectable for the plasmids. Plates were incubated at the indicated temperatures. (B) UBR1-induced Tbf1 protein degradation at 37°C. tbf1-degron mutant (tbf1-d) (KSC2853) cells were initially grown in sucrose at 30°C and were then incubated with galactose and concomitantly shifted to 37°C. Aliquots of cells were collected at the indicated times after incubation with galactose at 37°C and subjected to immunoblotting analysis with anti-myc antibodies. The degron cassette is fused to a myc epitope tag. (C, D) Proliferation of tbf1-degron mutants after incubation with galactose at 37°C. tbf1-degron mutant (tbf1-d) (KSC2853) cells were initially grown in sucrose at 30°C and were then incubated with galactose and concomitantly shifted to 37°C. Aliquots of cells were collected at the indicated times after incubation with galactose at 37°C to determine the cell division rate (C) and DNA content of cells (D). DNA content was determined by flow cytometric analysis. (E, F) Effect of Tbf1 depletion on Mre11 or Tel1 localization to XIII_R-TG₈₁ ends. XIII_R-TG₈₁-HO tbf1-d cells expressing Mre11-HA (KSC2854) or Tel1-HA (KSC2855) were transformed with the YCpU-TBF1 plasmid or the control vector, together with the GAL-HO plasmid. Transformed cells were treated as in A and analyzed by ChIP assay as in Figure 1B to monitor the accumulation of Mre11 (E) or Tel1 (F) at DNA ends. HO cutting efficiency of cells carrying each plasmid is shown in square brackets.