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. 2021 Jul 14;10:e67268. doi: 10.7554/eLife.67268

Figure 2. smc1D588Y restores cohesin occupancy on chromosome arms in the absence of Scc4.

(A) Average calibrated ChIP-seq profiles of Scc1-PK6 in smc1D588Y, scc4-4, and smc1D588Y scc4-4 cells 60 kb either side of CDEIII plotted as a percentage of the average number of reads obtained for wild-type (WT) cells. Cells were pheromone arrested in G1 at 25°C before release at 37°C into medium containing nocodazole. Samples were taken 75 min after release (K22005, K22009, K21999, K22001). (B) Average calibrated ChIP-seq profiles of Scc1-PK6 in smc1D588Y, and smc1D588Y scc4Δ cells 60 kb either side of CDEIII plotted as a percentage of the average number of reads obtained for WT cells. Cells were pheromone arrested in G1 at 25°C before release at 25°C into medium containing nocodazole. Samples were taken 60 min after release (K22005, K22009, K19624). (C) Average calibrated ChIP-seq profiles of Scc2-PK6 2 kb either side of CDEIII in cycling WT, smc1D588Y, and smc1D588Y scc4Δ cells at 25°C (K21388, K24680, K24678). (D) Average calibrated ChIP-seq profiles of ectopically expressed Smc3E1155Q-PK6 2 kb either side of CDEIII in cycling WT, smc1D588Y, and smc1D588Y scc4Δ cells at 25°C (K24562, K24689, K24564). (E) ATPase activity of WT or mutant tetramers on addition of ATP and Scc2 in the presence and absence of DNA.

Figure 2.

Figure 2—figure supplement 1. smc1D588Y restores cohesin occupancy on chromosome arms in the absence of Scc4.

Figure 2—figure supplement 1.

(A) Calibrated ChIP-seq profiles of Scc1-PK6 in wild-type (WT), smc1D588Y, scc4-4, and smc1D588Y scc4-4 cells across chromosome I. Cells were pheromone arrested in G1 at 25°C before release at 37°C into medium containing nocodazole. Samples were taken 75 min after release (K22005, K22009, K21999, K22001). (B) Calibrated ChIP-seq profiles of Scc1-PK6 in WT, smc1D588Y, and smc1D588Y scc4Δ cells across chromosome I. Cells were pheromone arrested in G1 before release into medium containing nocodazole at 25°C. Samples were taken 60 min after release (K22005, K22009, K19624). (C) A fraction of the ATPase reaction stained with Coomassie after SDS-PAGE to confirm protein levels. (D) ATPase activity of WT or mutant trimers on addition of ATP and Scc2 in the presence or absence of DNA.