Figure 6.

The contribution of Eso1 to cohesin stabilization on chromosomes. (A) Eso1 is not required for sustained Rad21 binding to chromatin in HU-arrested cells. HU was added to cycling cells at 25°C and the culture was immediately shifted to 32°C to induce the early S-phase arrest at 32°C, a restrictive temperature for eso1-H17. Cells were then shifted to 37°C for 2 h to inactivate mis4–367 in the presence of HU. Rad21-HA association with chromatin was monitored on chromosome spreads by immunofluorescence before and after the shift to 37°C. Rad21-HA fluorescence was measured in 50–100 nuclei. The error bars show the confidence interval of the mean with α=0.05. DNA content analysis and septation index (%) show that cells remained arrested in early S-phase throughout the experiment. (B) Cohesin stability on chromosomes is compromised after passage through an unperturbed S-phase without Eso1. Cells were arrested in G1 by nitrogen starvation (25°C−N) and released to pass through S-phase at 32°C, a restrictive temperature for eso1-H17. At 3.75 h after release, when cells had completed S-phase (32°C+N), the temperature was raised to 37°C for 1.5 h to inactivate Mis4 and probe the stability of cohesin on chromosomes in G2 (37°C+N). Samples were analyzed before and after temperature shift to 37°C as in (A). Scale bar, 5 μm. The quantification is based on two independent experiments with the error bars indicating the s.d. of the means. The fluorescence for each sample is normalized to the value of the mis4–367 sample before the 37°C shift.