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. 2019 Jun 21;8:e44736. doi: 10.7554/eLife.44736

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© 2019, Srinivasan et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 5. — (A) scc3K404E scc2-45 (K24709) and smc3K404 eco1Δ scc2-45 (K25947) strains arrested in G2 at 25°C and subjected to temperature shift to 37°C for 20 min. Ratio of average cohesin levels before and 20 min after the temperature shift is plotted. (B) Strains grown in (A) were analysed by western blotting against the indicated antibodies. (C) scc3K404E (K24697) and scc3K404E scc2-45 (K24738) strains containing 2C Smc1, 2C Smc3 and galactose inducible 2C Scc1^NC were arrested in G2 in YEP raffinose and Scc1^NC expression induced by addition of galactose. 60 min after galactose addition, glucose was added to the cultures and temperature shifted to 37°C. 0- and 20 min samples were subjected to mini-chromosomeIP (Scc1-PK6) following in vivo cohesin crosslinking with BMOE. (D) 0- and 20 min samples from (C) were subjected to Cal-ChIP-Seq. ratio of average cohesin levels before and temperature shift is plotted. Also see Figure 5—figure supplement 2B. (E) Cdc45 was depleted from wapl−AID cdc45-AID (K27169) and wapl-AID cdc45-AID scc2-45 (K27168) strains like described in Materials and Methods. Following temperature shift, 0- and 20 min samples of the cdc45 depleted strains were subjected to Cal-ChIP-Seq. ratio of average cohesin levels before and temperature shift from −100 kb to +60 KB relative to all 16 centromeres is plotted. Also see Figure 5—figure supplement 2C and D. (F) The percentage of cohesin that remains on DNA upon Scc2 inactivation (after temperature shift) along the entire chromosome four is shown. The median cohesin level along the entire chromosome 4 (dotted line) is marked with arrowheads. Data from SCC2 wild type cells arrested in lateG1 (Figure 2A) is shown in grey. Data from scc2-45 cells arrested in late G1 (Figure 2A) is shown in yellow. Data from Cdc45 depleted scc2-45 cells (Figure 5E) is shown in turquoise. Data from scc2-45 cells arrested in G2 (Figure 2B) is shown in green.

Figure 5—figure supplement 1. — (A) scc3K404E scc2-45 (K24709) and smc3K404 eco1Δ scc2-45 (K25947) strains arrested in G2 at 25°C and subjected to temperature shift to 37°C for 20 min. Ratio of average cohesin levels before and 20 min after the temperature shift is plotted. (B) Strains grown in (A) were analysed by western blotting against the indicated antibodies. (C) scc3K404E (K24697) and scc3K404E scc2-45 (K24738) strains containing 2C Smc1, 2C Smc3 and galactose inducible 2C Scc1^NC were arrested in G2 in YEP raffinose and Scc1^NC expression induced by addition of galactose. 60 min after galactose addition, glucose was added to the cultures and temperature shifted to 37°C. 0- and 20 min samples were subjected to mini-chromosomeIP (Scc1-PK6) following in vivo cohesin crosslinking with BMOE. (D) 0- and 20 min samples from (C) were subjected to Cal-ChIP-Seq. ratio of average cohesin levels before and temperature shift is plotted. Also see Figure 5—figure supplement 2B. (E) Cdc45 was depleted from wapl−AID cdc45-AID (K27169) and wapl-AID cdc45-AID scc2-45 (K27168) strains like described in Materials and Methods. Following temperature shift, 0- and 20 min samples of the cdc45 depleted strains were subjected to Cal-ChIP-Seq. ratio of average cohesin levels before and temperature shift from −100 kb to +60 KB relative to all 16 centromeres is plotted. Also see Figure 5—figure supplement 2C and D. (F) The percentage of cohesin that remains on DNA upon Scc2 inactivation (after temperature shift) along the entire chromosome four is shown. The median cohesin level along the entire chromosome 4 (dotted line) is marked with arrowheads. Data from SCC2 wild type cells arrested in lateG1 (Figure 2A) is shown in grey. Data from scc2-45 cells arrested in late G1 (Figure 2A) is shown in yellow. Data from Cdc45 depleted scc2-45 cells (Figure 5E) is shown in turquoise. Data from scc2-45 cells arrested in G2 (Figure 2B) is shown in green.