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

Figure 1. A Wapl-independent activity releases cohesin from chromosomes in G1 cells.

(A) Cohesin’s association with DNA is regulated by two opposing activities: Scc2-Scc4 complex loads cohesin onto DNA, while Pds5, Scc3 and Wapl constitute the releasing activity that releases cohesin from DNA by opening the Smc3-Scc1 interface. Mutations in Scc3 (scc3K404E) Pds5 (pds5S81R) and deletion of the WAPL gene abrogate Wapl mediated releasing activity and lead to cohesin’s stable association with DNA. (B) Schematic of the mini-chromosome IP assay: 6C strain (K23889) with cysteine pairs at all three ring subunit interfaces (2C Smc3: E570C S1043C, 2C Smc1: G22C K639C and 2C Scc1 C56 A547C) and carrying a 2.3 kb circular mini-chromosome was subjected to in vivo crosslinking with BMOE. DNAs associated with cohesin immune-precipitates (Scc1-PK6) were denatured with SDS and separated by agarose gel electrophoresis. Southern blotting reveals two forms of DNA unique to cells treated with BMOE: CMs (cohesin entrapping individual mini-chromosomes) and CDs (cohesin entrapping a pair of sister mini-chromosomes). (C) WT (K23972) and scc2-45 (K25238) 6C strains were arrested in late G1 by overexpression of nondegradable Sic1 at 25°C as described in Materials and Methods. The cultures were shifted to 37°C for 20 min, aliquots drawn before (0) and after (20) temperature shift (to inactivate Scc2) were subjected to mini-chromosome IP. (D) scc3K404E (K25313) and scc3K404E scc2-45 (K25316) 6C strains were arrested in late G1. The cultures were shifted to 37°C for 20 min, aliquots drawn before (0) and after (20) temperature shift (to inactivate Scc2) were subjected to mini-chromosome IP. Also see S1B. (E) pds5S81R (K25311) and pds5S81R scc2-45 (K25312) 6C strains were arrested in late G1. The cultures were shifted to 37°C for 20 min, aliquots drawn before (0) and after (20) temperature shift (to inactivate Scc2) were subjected to mini-chromosome IP. (F) scc3K404E (K25313) and scc3K404E scc2-45 (K25316) strains were arrested in late G1. The cultures were shifted to 37°C for 20 min, aliquots drawn before (0) and after (20) temperature shift (to inactivate Scc2) were analysed by Calibrated-ChIP- sequencing (Scc1-PK). Cohesin profile along chromosome four is shown. Also see Figure 1—figure supplement 1. (G) Even in cells that lack Wapl mediated releasing activity, inactivation of Scc2 in G1 cells leads to release of DNA entrapped within cohesin rings. This suggests that an activity that is Wapl-independent is capable of releasing cohesin from DNA.

Figure 1.

Figure 1—figure supplement 1. A Wapl-independent activity releases cohesin from chromosomes in G1 cells.

Figure 1—figure supplement 1.

(A) Data from Figure 1F is plotted to show the percentage of cohesin that remains on DNA upon Scc2 inactivation (after temperature shift) along the entire chromosome 4. The median cohesin level along the entire chromosome 4 (dotted line) is marked with arrowheads. (B) FACS analysis of the cultures described in Figure 1D (bottom two) and Figure 1—figure supplement 1F (top two). (C) Strain containing smc3R1008I mutation was crossed with an eco1Δ waplΔ strain and the resultant diploid analysed by tetrad dissection. The spores with eco1Δ waplΔ and eco1Δ waplΔ smc3R1008I are highlighted. (D) FACS analysis of strains grown in Figure 2A. (E) FACS analysis of strains grown in Figure 2B. (F) scc3K404E (K25313) and scc3K404E scc2-45 (K25316) 6C strains were arrested in G2 with nocadazole as described in Methods. The cultures were shifted to 37°C for 20 min, aliquots drawn before (0) and after (20) temperature shift (to inactivate Scc2) were subjected to mini-chromosome IP. Also see Figure 1—figure supplement 1B. (G) The cultures from experiment described in Figure 2A were analysed by western blotting with the indicated antibodies. (H) The Scc2-3XmAID strain described in Figure 2G was analysed by western blotting with anti-mAID antibody and anti PK antibody before and after auxin addition. (I) The Scc2-3XmAID strain described in Figure 2H was analysed by western blotting with anti-mAID antibody and anti PK antibody before and after auxin addition.