Figure 4 |. The APB motif is essential for ACF remodeling activity, and yeast viability.

(a) Remodeling activity of wild-type ACF complex (left) and the mutant ACF complex containing SNF2h 2A-APB (right) as read out by EMSA. ACF complexes (20 nM) were incubated with an end-positioned mono-nucleosome (10 nM) containing a 90 bp overhang in the presence of ATP. Aliquots of the reaction mixtures were analyzed at various time-points by native gel with SYBR gold staining. (n=3 independent experiments). Uncropped data are shown in Supplementary Fig. 17. (b,c) S. cerevisiae viability assay. Genomic copies of ISW1 and ISW2 were replaced by plasmid encoded copies of either ISW1 or mutant (panel b) or ISW2 or mutant (panel c). Yeast were then grown at indicated temperatures. Empty vector = pRS416 vector. wt ISW1 = pRS416 vector carrying wild-type ISW1 gene. isw1-APB = pRS416 vector carrying isw1-⁷⁶⁸AAEAA mutant gene (basic motif mutant). isw1-K227A = pRS416 vector carrying isw1-K227A mutant gene (known to abolish ATPase activity). isw1-AAA = pRS416 vector carrying isw1-⁷⁵¹AAA mutant gene. wt ISW1-ISW2 = RS416 vector carrying wild-type ISW1-ISW2 genes. Wt ISW2: pRS416 vector carrying wild-type ISW2 gene. isw2-APB = pRS416 vector carrying isw2-⁷⁴³AAEAAA mutant gene (basic motif mutant). isw2-AAA = pRS416 vector carrying isw2-⁷²⁸AAA mutant gene. For b,c: two biologically independent samples, n=6 independent experiments.