Figure 2.

Chd1 tolerates increased length and flexibility in the DBD linker, but requires a minimal length linker for coordinated action of DBD and ATPase domains. (A) Nucleosome sliding as assessed by native PAGE. Data shown are representative experiments (n≥4). (B) Quantification of nucleosome sliding reactions shown in (A). Error bars represent standard deviations (s.d.) (n≥4). (C) Relative rate constants for nucleosome sliding. Rate constants were obtained from single exponential fits to the data shown in (B), with the relative rates shown as k_rel=k_{obs, Chd1 variant}/k_{obs, Chd1ΔNC}. For Chd1_ΔNC, k_obs was 0.33±0.02 min⁻¹. Error bars represent the calculated errors of the fits. (D) Deletion of the DBD linker does not disrupt nucleosome binding, as shown by native PAGE. Super-shifted bands indicate Chd1–nucleosome complexes. (E) Deletion of the DBD linker still allows the Chd1 ATPase motor to be stimulated by nucleosomes. Shown are the averages and s.d. (n≥3) of nucleosome-stimulated ATPase activities with increasing concentrations of nucleosome substrate. Nucleosome-free rates were subtracted from all values, and the resultant activities were fit to the Michaelis–Menton equation (Chd1_ΔNC and Chd1_ΔNC^{Δ961–1,005}, solid lines) or linearly (Chd1_ΔDBD, broken line). Chd1_ΔDBD contained residues 118–1,005. DBD, DNA-binding domain; PAGE, polyacrylamide gel electrophoresis.