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. Author manuscript; available in PMC: 2016 Feb 25.
Published in final edited form as: Mol Cell. 2010 Sep 10;39(5):689–699. doi: 10.1016/j.molcel.2010.08.008

Figure 5. Smc3 Needs to be Acetylated Specifically in S Phase to be Competent for Establishment of Cohesion.

Figure 5

Exponential phase cells of strains K17515 (WT, PMET3CDC20, SMC3-PK6), K17516 (hos1 Δ, PMET3CDC20, SMC3-PK6), and K17376 (td-Eco1, PMET3CDC20, SMC3-PK6) and K17382 (td-Eco1 hos1 Δ, PMET3CDC20, SMC3-PK6) were grown to log phase in synthetic medium lacking methionine and arrested in metaphase for 2 hr in YEP medium supplemented with 2 mM methionine, 2% raffinose, and 2% galactose (M1). Eco1 was degraded by shifting cultures to degron-mediated proteolysis conditions (YEP raff/gal, 20 μg/ml doxycycline, at 37°C) for 90 min (M2). Subsequently, the cells were released from G2/M arrest to raff/gal media (lacking methionine) containing 20 μg/ml doxycycline. Thirty minutes after release at 37°C, nocodazole was added to arrest the cells again in G2/M phase.

(A) Strains 16354 (WT) and 16353 (Eco1-deg) were grown at 25°C for 2 days on YP plates containing 2% raffinose and 2% galactose, then transferred to 37°C on plates containing 2% raffinose, 2% galactose, and doxycycline (5 μg/ml).

(B) Western blots show degradation of endogenous HA-tagged td-Eco1 proteins

(C) Fluorescence-activated cell sorting analysis shows that the release from Cdc20 arrest was complete and that all strains had completed DNA replication after 90 min.

(D) Smc3-PK6 was immunopurified, and the level of acetylated Smc3 was evaluated by western blotting using α-acetyl lysine antibody (ST1027, Calbiochem).

(E) Cohesion was measured by scoring the percentages of separated GFP dots at the TRP1 locus (12.6 kb from CEN4).

(F) K17987 (hos1 Δ, 256LacO::TRP1, PMET3CDC20, YEp-SMC1/SMC3) K17988 (hos1 Δ, 256LacO::TRP1, PMET3CDC20, YEp), K17989 (256LacO::TRP1, PMET3CDC20, YEp-SMC1/SMC3), and K17990 (256LacO::TRP1, PMET3CDC20) were grown to log phase in synthetic medium lacking methionine and uracil and arrested in metaphase for 2 hr in YEP medium supplemented with 2 mM methionine. Cohesion defect was measured by scoring the percentages of separated GFP dots at the TRP1 locus (12.6 kb from CEN4).