Article Figures & Data
Figures
- Figure 1. Purification of budding yeast cohesin and its loader.
(A) Purified budding yeast cohesin and cohesin loader were analyzed by SDS–PAGE, followed by Coomassie Blue staining (CBB) and immunoblotting with the indicated antibodies. (B) Time course analysis of ATP hydrolysis by cohesin in the presence of DNA, with or without the cohesin loader. (C) Purified cohesin and Walker B motif mutant EQ-cohesin were analyzed by SDS–PAGE, followed by Coomassie Blue staining and immunoblotting. (D) Comparison of the ATP-hydrolysis rates of wild type and EQ-cohesin, in the presence or absence of the cohesin loader. A reaction with the cohesin loader (Scc2–Scc4) but without cohesin served as a negative control. The mean values and standard deviations from three independent experiments are shown. Hydrolysis rates calculated per cohesin complex are listed.
- Figure S1. Purification and characterization of budding yeast cohesin and its loader.
(A) Purification scheme for the cohesin tetramer complex. Size exclusion chromatography was the final purification step. Fractions from the column were analyzed by SDS–PAGE, followed by Coomassie Blue staining. Peak fractions corresponding to the cohesin tetramer complex were pooled and used for biochemical experiments described in this article. The cohesin tetramer complex contained a substoichiometric contamination from chaperones of the Hsp70 family (Ssa1-4, Ssb1-2, identified by mass spectrometry), indicated by “*”. The UV absorbance elution profile from the size exclusion column is also shown, together with the profiles of size markers used to calibrate the column. (B) As (A), but the purification scheme and final size exclusion chromatography step of the Scc2–Scc4 cohesin loader complex purification are shown.
- Figure S2. Characterization of budding yeast cohesin and its loader.
(A) DNA binding of cohesin to covalently closed circular DNA (CCC) and to linear DNA (L) was measured using an electrophoretic mobility shift assay. (B) DNA binding of the Scc2–Scc4 cohesin loader complex, next to the Scc2C fragment, was analyzed in an electrophoretic mobility shift assay. (C) Dose-dependent stimulation of cohesin loading by the cohesin loader. Gel image and quantification of the recovered DNA from cohesin loading assays performed with the indicated concentrations of the Scc2–Scc4 complex. The mean values and standard deviations from three independent experiments are shown. (D) Stimulation of ATP hydrolysis by the Scc2C fragment. Rates of ATP hydrolysis were determined in reactions containing cohesin and DNA, without or with the cohesin loader or with the Scc2C fragment. The mean values and standard deviations from three independent experiments are shown.
- Figure 2. Cohesin-loader−stimulated cohesin loading.
(A) Schematic of the cohesin loading assay. Circular DNA and cohesin, with or without the cohesin loader, are incubated in the presence of ATP. Cohesin-DNA complex are retrieved by immunoprecipitation using an antibody against the Pk epitope tag on the Smc1 subunit. The recovered DNA is analyzed by agarose gel electrophoresis. (B) Gel image and quantification of a cohesin loading time course experiment in the presence or absence of the cohesin loader. (C) The Scc2–Scc4 complex was analyzed by SDS–PAGE and Coomassie Blue staining next to the Scc2C fragment. (D) Gel image and quantification of recovered DNA from the cohesin loading assay performed with the indicated concentration of Scc2C in comparison with the Scc2/Scc4 complex. Mean values and standard deviations from three independent experiments are shown.
- Figure S3. Cohesin retains its oligomeric state during incubation at low ionic strength.
15 pmol of tetrameric cohesin was diluted to 150 nM in cohesin loading buffer (35 mM Tris–HCl pH 7.0, 20 mM NaCl, 0.5 mM MgCl2, 13.3% glycerol, 0.5 mM ATP, 0.003% Tween, 1 mM TCEP) to mimic the conditions of a cohesin loading reaction. The final cohesin concentration was somewhat higher than in a typical loading reaction, which was required to detect the protein in the following analysis. The sample was separated on a Superose 6 Increase 10/300 GL column equilibrated in R buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 10% glycerol, 0.5 mM TCEP) or was first incubated at 29°C for 120 min and then separated. In both samples, cohesin eluted at the expected size of the tetrameric complex (compare Fig S1A). At later elution volumes, the absorptions of detergent and ATP become apparent. The positions of the separation peaks of blue dextran (>2 MD), thyroglobulin (660 kD), aldolase (158 kD), carbonic anhydrase (29 kD) and aprotinin (6.5 kD), under the same conditions, are shown as a reference.
- Figure S4. Topological loading of the budding yeast cohesin ring onto DNA.
(A) Gel image of input DNA of various topologies and of recovered DNA following cohesin loading. (B) Topological DNA loading promoted by Scc2C was assessed by DNA release following linearization with PstI. Supernatant (S) and bead-bound fractions (B) were retrieved following PstI or mock treatment. DNA in both fractions was analyzed by agarose gel electrophoresis. CCC, covalently closed circular DNA; L, linear DNA; NC, nicked circular DNA; RC, relaxed circular DNA.
- Figure 3. Topological DNA embrace by the budding yeast cohesin ring.
(A) Schematic of DNA release by DNA linearization. Immobilized cohesin-DNA complexes were incubated in the presence or absence of PstI. The supernatant fraction (S) and bead-bound fraction (B) were collected, and DNA in each fraction was analyzed by agarose gel electrophoresis. (B) Gel image of an experiment as outlined in (A). Cohesin loading was performed with or without Scc2–Scc4. Covalently closed circular (CCC) and linear (L) forms of the input DNA were included as a comparison. (C) Schematic of DNA release by cohesin cleavage. (D) Wild type and TEV protease (TEV)-cleavable cohesin were loaded onto DNA, then TEV protease was added to half of the reaction. Cohesin was retrieved and recovered DNA analyzed by agarose gel electrophoresis. Scc1 cleavage was monitored by immunoblotting. Note that TEV-cleavable cohesin was partially cleaved even without TEV addition. This could be due to similarities between the TEV and PreScission protease recognitions sites, the latter was used during the cohesin purification.
- Figure 4. ATP binding, but not hydrolysis, is required for cohesin loading.
(A) Gel image and quantification of recovered DNA from cohesin loading reactions performed with or without added ATP. (B) Gel image and quantification of recovered DNA from cohesin-loading reactions performed with wild-type or Walker B motif mutant EQ-cohesin. (C) An assay in which ATP and the indicated nucleotide derivatives were compared for their ability to support cohesin loading. The mean values and standard deviations from three independent experiments are shown in panels (A–C). (D) The topological nature of cohesin loading, supported by ADP·AlFx, was analyzed following DNA linearization. (E) Fission yeast cohesin loading onto DNA was measured in the presence of ATP or the indicated nucleotide derivatives.
- Figure S5. Topological loading onto DNA without added ATP.
(A) A DNA release experiment by restriction enzyme cleavage was performed following a cohesin loading reaction in the presence or absence of added ATP. The immobilized cohesin-DNA complex was incubated with or without PstI. Supernatant (S) and bead-bound (B) fractions were collected and DNA in each fraction was analyzed by agarose gel electrophoresis. (B) Gel image and quantification of the recovered DNA from cohesin loading reactions performed with or without added ATP using Scc2C as the cohesin loader. (C) Topological DNA loading by Walker B motif mutant EQ-cohesin, in the absence or presence of the cohesin loader, was analyzed by DNA release following linearization with PstI. Supernatant (S) and bead-bound fractions (B) were retrieved following PstI or mock treatment. DNA in both fractions was analyzed by agarose gel electrophoresis.
