Figure 4. Mutations to the Chd1 N-terminus have positive and negative effects on activity.
(A) Alignment of Chd1 proteins from the indicated yeast species indicates that sequences in the N-terminal region are conserved. Below this mutations to the N-terminal region characterised in this study are indicated. Numbering is to the Saccharomyces cerevisiae sequence. (B) 100 nM nucleosomes assembled on a DNA fragment consisting of the 601 nucleosome positioning sequence flanked by 47 bp linker DNA on one side were incubated at 30°C with 2 nM enzyme, aliquots of the reaction were stopped at 0, 4, 8, 16, 32, and 64 min. Repositioning of the nucleosome to the DNA centre show an increase in activity for the △57–88 and a decrease for the △1–35 mutation. As mutations in the basic patch resulted in no measurable repositioning, the reaction was run for t = 0 and 64 min. (C) Initial reaction rates relative to wild type were determined from a non-linear fit, and are presented as mean +/- standard deviation for N = 3. (D) 25 nM nucleosomes were incubated with increasing concentrations of enzyme (50, 100, 150, 200, and 300 nM). Representative gel images are shown with bar graphs below showing the mean +/- standard deviation of percent bound from triplicate experiments. At high concentrations of Chd1 super-shifted complexes corresponding to two or more Chd1 molecules binding a nucleosome are observed. (E) 5 nM enzyme was reacted with increasing amounts of nucleosome (10, 20, 40, 80, and 160 nM) and phosphate release from ATP hydrolysis was monitored by fluorescence intensity. Non-linear regression of triplicate experiments was used to define Km as the nucleosome concentration at half maximal reaction rate and kcat as the enzyme turnover at maximum rate. R squared values for the fits were above 0.9 in all cases. Phosphate release with high nucleosome concentration was not above background signal in either basic patch mutant, making calculation of kinetic parameters unfeasible.