(
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.