Figure 7. In the absence of all forms of cohesin release, Scc2 becomes dispensable for cohesion establishment.

(A–C) Transient inhibition of CDK1 in mitotic cells induces re-replication: (A) Growth conditions to induce re-replication: scc3K404E cdc28-as1 (K25437) containing 2C Smc1 and 2C Smc3 along with galactose inducible 2C Scc1^NC was arrested in G2 with nocadazole. Scc1^NC expression induced by galactose addition for 60 min. After this, glucose and 1NMPP1 were added to the cultures. After 60 min, the culture was shifted to 37°C for 20 min. Subsequently, the culture was filtered, washed with YEP medium and resuspended into YEPD medium containing either DMSO or 1NMPP1at 37°C. 90 min later, the culture was analysed by western blotting with indicated antibodies (B) and FACS (C). (D and E) in the absence of release, Scc2 is dispensable for cohesion establishment: re-replication was induced in scc3K404E cdc28-as1 (K25437) and scc3K404E scc2-45 cdc28-as1 (K25440) expressing galactose inducible 6C non-cleavable cohesin using the growth regime described in (A). Samples drawn before (D) and after (E) induction of re-replication were analysed by in vivo crosslinking and minichromosome IP. (F) Inactivation of releasing activity suppresses the cohesion defect caused by Scc2 inactivation: Sister chromatid cohesion was measured as described in Materials and Methods in pds5S81R (K27443) scc2-4 (K15028) and scc2-4 pds5S81R (K27575) strains that were arrested in G1 or S (HU) and released into G2 arrest (by Cdc20 depletion) at non-permissive temperature (35.5°C).

Figure 7—figure supplement 1. In the absence of all forms of cohesin release, Scc2 becomes dispensable for cohesion establishment.

(A) CMs and CDs in WT (K23889) and scc2-45 (K24267) 6C strains arrested in G1 with α factor at 25°C in YPD medium and released into nocodazole at 37°C. (B) FACS profiles of the strains in the experiment described in Figure 7D (1NMPP1) and E (DMSO). (C) Two independent repetitions of the experiment described in Figure 7D and E. (D) CDs formed after re-replication are dependent on cohesin circularisation with BMOE: re-replication was induced in scc3K404E cdc28-as1 (K25437) and scc3K404E scc2-45 cdc28-as1 (K25440) expressing galactose inducible 6C non-cleavable cohesin using the growth regime described in Figure 7A. Samples that were crosslinled with BMOE were analysed along with those that were not crosslinked with BMOE by mini-chromosome IP. (E) Wild type strain (K699) was released from a G1 arrest into nocadazole or HU containing medium for indicated times and subjected to calibrated whole genome deep sequencing as described in Materials and Methods. The reads from 45 min HU, 90 min HU and G2 were normalised with the reads from G1 arrested culture (un-replicated, 1 copy of the genome). A portion of chromosome V is shown with the positions of ARS elements and URA3 gene marked. (F) Sister chromatid cohesion was measured in scc2-4 pds5S81R strain (K27575) that was arrested in S (HU) and released into G2 arrest (by Cdc20 depletion) at either permissive (blue curve) or non-permissive temperature (red curve).