Figure 7. Disruptions in the nucleosome acid patch only moderately decrease sliding by Chd1.

(A) Overview of disruptions introduced on H2A/H2B. Nucleosome crystal structure shown is PDB code 1KX5 (Davey et al., 2002). (B) Stopped flow sliding reactions using asymmetric nucleosomes containing H2A or H2B disruptions on the entry side H2A or H2B. Asymmetric nucleosomes were generated by incubating 10 nM 0-601-80 hexasomes with 12 nM H2A/H2B containing one of the following sequence variants: Wt (blue); FACT/SWR1 binding surface disruption (H2B-Y39A/M56A) (cyan); H2A C-terminal tail truncation (∆110–129) (green); acid patch single mutant (H2A-E64R) (brown); acid patch double mutant (H2A-D90R/E92A) (orange); acid patch quadruple mutant (H2A-E61A/E64A/D90A/E92A) (magenta). Reactions were performed with 400 nM Chd1 and 25 μM ATP and followed by Cy3B-Dabcyl SQOF. Each progress curve is an average of 3–6 technical replicates. (C) Summary of observed rates (k₁, k₂) obtained from double exponential fits to stopped flow data as shown in (A). In every case the observed fast rate (k₁) contributes >90% of the amplitude of the progress curve. Error bars represent standard deviation from three (six for Wt) independent experiments. Statistics compare k₁ rates for indicated constructs: *** p-value <0.00001; **** p-value <0.0000001; n.s., not significant. See also Figure 7—figure supplements 1 and 2.

DOI: http://dx.doi.org/10.7554/eLife.21356.013

Figure 7—figure supplement 1. With subsaturating H2A/H2B dimer addition, rates of nucleosome sliding by Chd1 are not sensitive to nucleosome:hexasome ratios.

(A) Stopped flow nucleosome sliding reactions monitored by Cy3-Cy3 fluorescence quenching . The legend indicates the amount of H2A/H2B dimer in each reaction, which contained 10 nM 0-601-80 hexasome, 50 nM Chd1, and 25 μM ATP. Each trace is an average of 4 or more injections from the same stopped flow experiment. The black curves represent double exponential fits to the data. (B) Graph of overall intensity changes at each H2A/H2B dimer concentration added to hexasomes, with higher intensity reflecting a greater proportion of nucleosomes that were shifted. Error bars indicate the range from two independent sets of experiments. (C) Graph of sliding rates for stopped flow H2A/H2B dimer addition. The observed rates (given as k₁ and k₂) were determined from double exponential fits to the data. Error bars indicate the range from two independent sets of experiments. (D) Native PAGE visualization of nucleosomes generated by addition of H2A/H2B dimer to hexasomes. Shown is a representative of ten similar titrations performed using wild-type or modified H2A/H2B dimers.

DOI: http://dx.doi.org/10.7554/eLife.21356.014

Figure 7—figure supplement 2. With limiting ATP, remodeling saturates at 400 nM Chd1.

Nucleosomes formed by adding 12 nM dimer to 10 nM 0-601-80 hexasomes were titrated with 10, 25, 50, 100, 200, 400, and 800 nM Chd1 and 25 μM ATP. Reactions were monitored by Cy3-Cy3 SQOF via fluorometer, and show that remodeling plateaued at 400 nM Chd1. The progress curves shown are representative of two independent Chd1 titrations using unmodified H2B (Wt-Wt). Similar results were observed for duplicate titrations using Ub-Wt, Wt-Ub, and Ub-Ub nucleosomes.

DOI: http://dx.doi.org/10.7554/eLife.21356.015