Supplementary Figure 7: Characterization of Brn1 yeast mutants.
From: Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits
(a) Expression of wild type or mutant Brn1-PK6 constructs from one endogenous allele in diploid yeast strains (strains C3632, C3665, C3651, C3641, C3635, C3658, C3649 and C3634) was tested by western blotting of cell extracts against the PK epitope and tubulin as loading control. (b) Co-purification of Smc2-HA6 with wild type or mutant Brn1-PK6 from whole cell yeast extracts (strains C3632, C3665, C3651, C3641, C3635, C3658, C3649 and C3634) was probed by western blotting of input (IN), unbound (U), and bound (B, 10× concentrated compared to input) fractions with antibodies against the HA and PK epitopes. (c) Haploid yeast cells expressing wild type or mutant (M1 or M4) Brn1-PK6 from the endogenous BRN1 locus (strains C3890, C3892 and C3893) were arrested in G1 phase with mating pheromone and cell cycle progression after release at 37°C was monitored by FACScan analysis of DNA content. Segregation of chromosome V marked by the binding of TetR-GFP to tandem arrays of TetO sequences integrated proximal to the telomere of the right chromosome arm was monitored by live cell microscopy at the indicated time points. The fractions of cells from n > 200 cells per time point (sum from 3 independent experiments) are plotted. (d) Genomic binding sites of budding yeast condensin holocomplexes and condensin complexes lacking Ycg1. Relative enrichment of Brn1-PK6 wild type and M1 M2 M4 mutant at rRNA genes, tRNA genes, centromeres and coding regions of RNA pol II-transcribed genes (ORFs) is plotted. Horizontal lines define the median, boxes the 25th and 75th percentiles and whiskers the maximum and minimum values, extending up to 1.5-times the interquartile range. Outlier values are represented by circles.
