Figure 2.

SNF2h and ACF function as dimers of ATPases. (a) Schematic of nucleosome structure with dye attachment sites for (b) and (d). The DNA is end-labeled with Cy3 (blue) on the shorter flanking DNA. The octamer is labeled with Cy5 at H2A-120C (yellow). (b) Cy3 fluorescence intensity of the construct shown in (a) as a function of SNF2h concentration, using nucleosomes with 78bp of flanking DNA on one side. A representative replicate curve is shown. Data are fit to the general equation for cooperative binding (see Methods). Hill Coefficient (n) = 1.8 ±0.17; K_1/2 = 353±30nM. (c) Schematic of FRET-based nucleosome remodeling assay. Rate constant of remodeling is measured by following the decrease in FRET between Cy3 and Cy5 in the presence of ATP. (d) Left panel: Nucleosome remodeling rate constant as a function of SNF2h concentration for nucleosomes with 78bp of flanking DNA. Right panel: Nucleosome remodeling rate constant as a function of ACF concentration for nucleosomes with 20 bp of flanking DNA. Hill Coefficient (n) = 1.8±0.1; K’_1/2 = 281±32 nM for SNF2h and Hill Coefficient (n) = 1.9±.3; K’_1/2 = 26±3 nM for ACF. Each panel represents global fits to data obtained from three independent experiments. (e) SNF2h binds as a cooperative dimer to the nucleosome in the absence of nucleotide (black circles), and in the presence of ADP (blue squares). In the presence of ADP•BeF_x, SNF2h binds non-cooperatively (red triangles). These binding measurements were carried out with nucleosomes containing 40bp of flanking DNA on one side and a Cy3 label on the short DNA end. Binding of SNF2h to these nucleosomes is ~2-fold weaker relative to the nucleosomes used in (b)⁹,⁴⁶. A representative replicate curve with each nucleotide analogue is shown (left panel), and the average K_1/2 and Hill Coefficient from three replicates is shown (table). Errors represent s.e.m.