Supplementary Figure 3: Characterization of DNA- and chromosome-binding specificities of the non-SMC subcomplex.
From: Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits
(a) Coomassie-stained SDS PAGE of purified S. cerevisiae the Smc2–Smc4 hinge dimer and the Brn1–Ycs4–Ycg1 subcomplex used for DNA binding experiments. (b) Electrophoretic mobility shift and fluorescence anisotropy binding assays of 6-FAM-labeled 30-bp dsDNA and ssDNA substrates at increasing Smc2–Smc4 hinge concentrations. Unbound (*) and slower migrating species (**) are indicated. Points and error bars indicate mean and s.d. of 3 independent experiments. (c) Increasing concentrations of Brn1–Ycs4–Ycg1 (0.1–1.6 μM; lanes 1–6) or of the Smc2–Smc4 hinge (0.8–25.6 μM; lanes 7–13) were incubated with a fixed concentration of 30- bp 6-FAM-labeled dsDNA (0.2 μM) and binding was analyzed by agarose gel electrophoresis. Addition of the non-SMC subcomplex to pre-formed Smc2–Smc4 hinge–DNA complexes was analyzed at the same time (lanes 14–15). (d) Purified non-SMC subcomplex and reconstituted nucleosomes (Nuc-167) used in binding assays were analyzed by SDS PAGE and Coomassie staining. (e) The budding yeast genome was sub-divided into regions enriched or depleted of histone H2A.Z (S. cerevisiae Htz1; see Online Methods) and the relative enrichment of Brn1-PK6 in each of the two categories was determined for each chromosome in a ChIP-seq experiment. Horizontal lines define the median and boxes the 25th and 75th percentiles, whiskers represent the maximum and minimum values; P < 4×10–9 by Wilcoxon two-sided test. (f) The levels of condensin and histone H2A.Z bound in every 500-bp window of the budding yeast genome were expressed as relative enrichment of Brn1-PK6 or Htz1-TAP in ChIP-seq experiments were compared; the non-parametric correlation index (Spearman) is indicated. Panels b and c show one representative experiment of n = 3 independent replicates.
