Figure 2. Chromosomal cohesin is converted into cohesive structures in the absence of Scc2 activity.

(A) A schematic depiction of the cohesin conversion assay, see the results and Materials and methods sections for detailed description of the assay. Green rings denote endogenous cohesin and red rings denote non-cleavable 6C cohesin. Only the latter molecules are capable of generating SDS-resistant catenated monomers (CMs) and catenated dimers (CDs). (B) Wild type (K24697) and scc2-45 (temperature sensitive mutant of Scc2) (K24738) strains that contain genes coding for 6C non cleavable cohesin (2C SMC1 2C SMC3 and GALp-2C SCC1^NC) were arrested in G2 phase, the expression of 2C Scc1^NC was induced by addition of galactose for 45 min. Mini-chromosome IP of the cultures at this stage shows CMs formed in both strains. The cultures were released from the G2 arrest and arrested in the subsequent the G1 phase, mini-chromosome IP of the cultures at this stage shows CMs formed in the previous G2 phase being retained in the subsequent G1 in both strains. The cultures were released from the G1 arrest and allowed to undergo replication at 37°C (in order to inactivate Scc2 in the scc2-45 strain), mini-chromosome IP shows formation of CDs in both the wild type and scc2-45 strains, in the scc2-45 mutant strain this is accompanied by a reduction in the amount of CMs. The FACS profiles of the two cultures at different stages of the experiment is shown below the respective southern blots. The data shown is representative of three independent biological repeats.

Figure 2—figure supplement 1. Non cleavable cohesin expressed in the G2 phase survives mitosis and remains stably associated with the chromosomes in the subsequent G1 phase.

(A) 6C non cleavable cohesin was expressed for 45 min in Wild type (K24697) strain arrested in G2 phase. The culture was released from the G2 arrest and arrested in the subsequent the G1 phase, a sample of the culture was drawn at this stage and fixed with formaldehyde (time 0). The rest of the culture was maintained in a prolonged G1 arrest by regular addition of the mating pheromone α-factor for a further 60 min. A sample was drawn at 60 min (time 60) and fixed with formaldehyde. The 0 and 60 min samples were subjected to calibrated ChIP sequencing with anti-PK antibody. The occupancy of Scc1^NC along the entire chromosome IV is shown for both t = 0 and t = 60 min samples. (B) The difference in the level of Scc1^NC-PK (time 60/time0) between the two conditions is shown as % of cohesin that remains on DNA upon prolonged G1 arrest. The median cohesin levels across the entire chromosome IV (dotted line) is marked with an arrowhead.