Supplementary Figure 2: The non-SMC subcomplex binds DNA.
From: Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits
(a) The electrophoretic mobility of a 6.5-kb linear dsDNA at increasing S. cerevisiae Brn1–Ycs4–Ycg1 concentrations was resolved on a 0.8% native agarose gel and detected by ethidium bromide staining. Unbound (*), major (**) and minor (***) slower migrating species are indicated. (b) Sequences of the 15 to 60-bp dsDNA substrates; green asterisks indicate the positions of the 6-FAM labels. (c) Electrophoretic mobility shift assay of a 30-bp dsDNA substrate (lanes 1-6) after addition of unlabeled 30-bp competitor DNA (lanes 7-12) or an antibody against the His6 tag on Ycs4 (lanes 13-14). Unbound (*), shifted (**) and antibody super-shifted (***) species are indicated. (d) Electrophoretic mobility of the Brn1–Ycs4–Ycg1 subcomplex during native protein gel electrophoresis with or without addition of 30-bp dsDNA at 4:1 molar DNA/protein ratio. Unbound (*) and shifted (**) species detected by post-run Coomassie staining are indicated. (e) Electrophoretic mobility shift assay of a 6.5-kb linear dsDNA with Brn1ΔNC–Ycs4–Ycg1 as in a. (f) Binding affinities of Brn1–Ycs4–Ycg1 and Brn1ΔNC–Ycs4–Ycg1 to a 30-bp 6-FAM-labeled dsDNA substrate were compared by measuring fluorescence anisotropy changes upon addition of the indicated protein concentrations. Dissociation constants (Kd) were calculated by fitting mean ΔA values for each protein concentration assuming a single-site binding model. Points and error bars indicate mean and s.d. of n = 3 independent experiments. Panels a, c and e show one representative experiment of n = 3 independent replicates.
