Figure 7.

Mutations in the Smc Hinge Domain Cause Unstable Association of Cohesin with Chromosomes

Stability of cohesin's association with chromosomes was measured mainly in pericentromeric regions by means of fluorescence recovery after photobleaching (FRAP) in diploid yeast cells. A portion of the barrel-shaped fluorescent signal was bleached by exposure to an argon laser for 200 ms. Images were captured every 15 s for 8 min postbleaching to measure the recovery rate. Red circles in the upper panels of (A)–(D) depict the region of interest that was bleached and analyzed for recovery. Relative fluorescence intensities (RFI) of unbleached (blue) and bleached (red) signals are plotted over time in the lower panels. Wild-type Smc1-GFP (A; K16252: SMC1-GFP::URA3) and wild-type Smc3-GFP (C; K16113: SMC3-GFP) had no recovery in any of the cells analyzed. Smc1(M665R)-GFP (B; K16253) recovered with t_1/2 = 45.2 ± 12.84 s (n = 6). Smc3(M577K)-GFP (D; K16114) recovered with t_1/2 = 28.57 ± 7.2 s (n = 6). Error bars represent SD.