Figure 1.

Cohesin Loading Requires RSC Catalytic Activity

(A) The ATPase mutant Sth1 fails to restore cell viability following Sth1 depletion. Wild-type Sth1^Degron cells and Sth1^Degron cells expressing wild-type Sth1 or Sth1^K501R were streaked onto rich yeast extract peptone dextrose (YPD) medium containing methionine to repress Sth1^Degron expression and indole-3-acetic acid (IAA) to promote its degradation.

(B) Ectopic Sth1 and Sth1^K501R are expressed at similar levels as endogenous Sth1. Cells of the indicated genotypes were synchronized in G1, endogenous Sth1 was depleted, and cells were released into nocodazole-imposed mitotic arrest. Levels of Sth1 and of the cohesin subunit Scc1 were monitored by immunoblot. Tubulin served as a loading control. End., endogenous Sth1; D, Sth1^Degron; wild-type (wt), Sth1^Degron + wild-type Sth1; KR, Sth1^Degron + Sth1^K501R.

(C) RSC catalytic activity is required for sister chromatid cohesion; as in (B), but sister chromatid cohesion at the GFP-marked URA3 locus was scored. Means and SEM of three independent experiments are shown. Sth1^Degron and Sth1^Degron + Sth1^K501R, p < 0.01; Sth1^Degron + Sth1 wt, p not significant; Student’s t test compared with the wild-type strain.

(D) RSC catalytic activity promotes cohesin loading; as in (B), but Scc1 levels at three chromosome arm cohesin binding sites (POA1, MRP10, and MET10), two centromeres (CEN3 and CEN9), and a negative control site (GLT1) were measured by ChIP, followed by real-time qPCR. Means and SEM of three independent experiments are shown. Sth1^Degron and Sth1^Degron + Sth1^K501R, p < 0.01; Sth1^Degron + Sth1 wt, p not significant; two-way ANOVA test compared with the wild-type strain.

See also Figure S1 for a schematic of cell synchronization and cell cycle progression analysis by fluorescence-activated cell sorting (FACS) analysis of DNA content as well as Figure S2 for additional ChIP microarray and quantitative analyses of the cohesin distribution.