Figure 6.
Smc3 Acetylation Blocks NScc1 Dissociation
(A) HOS1 or hos1Δ strains with galactose inducible WPL1, K22555 (MATa SMC3 (S1043C)-HA6 MYC3-SCC1(TEV268) pGAL1-10-WPL1) and K22810 (MATa hos1Δ SMC3(S1043C)-HA6 MYC3-SCC1(TEV268) pGAL1-10-WPL1) were grown in YP Raff medium at 25°C and arrested in nocodazole for 2 hr. Galactose was then added to induce Wapl. Samples were taken at the indicated time points to induce in vivo crosslinking with 5 mM BMOE. Crosslinking was analyzed by western blotting using anti HA antibodies. Uncrosslinked samples were also analyzed similarly (shown in Figure S1B).
(B) Exponentially growing strains K24217 (MATa SMC3 URA3::SMC3 S1043C-PK6 MYC3-SCC1(TEV268)) and K24218 (MATa, SMC3 URA3::smc3K112 113Q S1043C-PK6 MYC3-SCC1(TEV268)) in YPD medium at 25°C were subjected to in vivo thiol-specific crosslinking with 5 mM BMOE. Crosslinking was analyzed by western blotting using anti PK(V5) antibody.
(C) Strain K24090 containing a 2.3 kb circular minichromosome, six cysteines within the Smc1-Smc3-Scc1 interfaces, eco1ts(G211H), and galactose-inducible WPL1 gene was grown at 25°C, arrested in G1 by pheromone, and permitted to go through S phase at 37°C in the presence of nocodazole. After addition of either galactose or glucose to induce Wapl expression (or not) samples were taken at times 0 and 60 min for in vivo crosslinking with BMOE. Scc1-PK6-immunoprecipitated DNA denatured with SDS was detected by Southern blotting. Catenated monomers (CM), catenated dimers (CD).
(D) Calibrated ChIP-seq profiles of Smc3 E1155Q and Smc3 E1155Q K112Q K113Q showing the number of reads at each base pair away from the CDEIII element averaged over all 16 chromosomes.