Figure 6. AcidicN is a strong negative regulator of the ATPase.
(A) Design of AcidicN derivatives of ISWI (see also Figure 6—figure supplement 1A). (B) Effects of AcidicN mutation on ATP hydrolysis in absence or presence of saturating concentrations of DNA and chromatin. In absence of DNA, ATPase activities of ISWIWT (#) and ISWI+3 (§) were ≤0.06 s−1. Errors are s.d. (n ≥ 4). (C) Effects of AcidicN mutation on the remodeling activities. Nucleosomal arrays containing wild-type H4 were used. Errors are s.d. (n ≥ 3) except for ISWI+3 for which minimal and maximal values of two independent measurements are shown. Color code as in panel (B). Raw data of the remodeling assay can be found in Figure 8—figure supplement 1. Results for ISWIWT (*) are replotted for comparison from Figure 3B,C.
Figure 6—figure supplement 1. AcidicN and AutoN mutants.
(A) Construct design. Only the NTR region of ISWI including affinity and solubility tags are shown (not to scale). Blue arrowheads indicate a TEV cleavage site. Tags were removed by protease cleavage as indicated. (B) Coomassie-stained SDS-PAGE of purified recombinant proteins.
Figure 6—figure supplement 2. Comparison of ATPase and remodeling activities of ISWI control variants used in this study.
(A) The Z2 solubility tag did not interfere with DNA- and chromatin-stimulated ATPase activities. Saturating amounts of nucleic acid ligands (0.2 mg/ml of linearized pT7blue and 0.1 mg/ml of chromatin assembled on the same DNA, respectively) and ATP (1 mM) were used. The unstimulated basal activity was ≤0.05 s−1. Errors are s.d. (n ≥ 3). (B) The Z2 solubility tag did not interfere with remodeling rates on wild-type H4 containing chromatin and tail-less H4 chromatin. Z2-tagged ISWI+6; E257Q, which contained a point mutation in the ATPase domain rendering it catalytically inactive, was included as a control. Its activity on tail-less H4 arrays was undetectable (§). Errors are s.d. (n ≥ 3) except for the ATPase-dead construct (ISWI+6; E257Q), which was tested once. (C) DNA- and chromatin-stimulated ATP hydrolysis rates of the ATPase dead double mutant ISWI+6; E257Q were negligible (≤0.04 s−1). Errors are s.d. (n ≥ 3) for ISWIWT and minimal and maximal values of two independent measurements for ISWI+6; E257Q. The asterisks (*) mark data that were replotted for comparison from Figure 3B,C.
Figure 6—figure supplement 3. Saturation controls for ISWI+6 in ATPase assays.
(A) Linearized pT7blue DNA was titrated over a 16-fold range. 0.2 mg/ml were saturating. (B) Titration of chromatin assembled on linearized pT7blue DNA. 0.1 mg/ml were close to saturation.
Figure 6—figure supplement 4. AcidicN mutations upregulate the ATPase activity of ISWIH483B.
Relative to ISWIWT, ISWIH483B had a ~fourfold diminished DNA- and chromatin-stimulated ATPase activity. Additional mutation of AcidicN (+3; +6) strongly activated both DNA- and chromatin-stimulated ATP turnover. Errors are s.d. for ISWIWT and minimal and maximal values of two independent measurements for all other constructs. Data for ISWIWT (*) were replotted for comparison from Figure 3B.