FIGURE 7:

Effect of Tbf1 depletion on checkpoint response to endogenous telomeres. (A) Effect of Tbf1 depletion on checkpoint activation in cells carrying normal-length telomeres. Wild-type (KSC1602) or tbf1-d (KSC2756) cells were cultured as in Figure 6C to degrade Tbf1 protein. Cells were further treated with 0.1% methyl methanesulfonate (MMS) for 1 h at 37°C. Aliquots of cells were collected and subjected to immunoblotting analysis with anti-Rad53 antibodies. Tubulin was detected as a loading control. (B) Effect of Tbf1 depletion on checkpoint activation in stn1-13 mutant cells. tbf1-d (KSC2756) or tbf1-d stn1-13 (KS2891) cells carrying YCpT-TBF1 or the control vector were grown and treated with nocodazole at 25°C. After synchronization, the culture was 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. As a control, aliquots of cells were treated with 0.1% MMS for 1 h at 37°C (labeled as M). Cells were subjected to immunoblotting analysis as in A.