RSC and ISW2 Diffusion on Lambda DNA and Impacts of Ionic Strength.

(A) Schematic: RSC and ISW2 bind nucleosome-free DNA at yeast promoters. (B) Optical tweezers and confocal scanning microscopy assay. (C) Aligned trajectories for RSC, ISW2, and dCas9. (D) Rolling window analysis assigns diffusion coefficients (Dcoef) and percentages for non-diffusive, low, and high diffusion. (E) Heterogeneous 1D diffusion of RSC and ISW2 on lambda DNA (50 mM KCl, no ATP). Scatter violin plots show mean Dcoef per diffusion type. Mann-Whitney tests compare RSC vs ISW2 distributions. Median Dcoef values with 95% confidence intervals are shown. (F) Dwell times for RSC and ISW2 at varying KCl concentrations; single-exponential decay fit and half-lives shown. (G) Ionic strength impact on high-mobility diffusion at different KCl concentrations. Scatter violin plots depict median values and 95% confidence intervals. (H) Model: RSC primarily undergoes 1D hopping, while ISW2 performs helically coupled 1D sliding.

Bimolecular Remodeler-Remodeler Interactions During 1D Encounters on DNA.

(A-B) Two-color kymographs of RSC-JFX554 and ISW2-JFX650 (A) or RSC-JFX554 and RSC-JFX650 (B) diffusing together on naked DNA. (C-E) Three types of encounter events are observed: short colocalizations (C), long colocalizations (D), and bypass events (E). Representative kymograph sections and corresponding cartoons illustrate each interaction. (F-G) Dwell times of short colocalization events for RSC-ISW2 (F) and RSC-RSC (G). (H-I) Dwell times of long colocalization events for RSC-ISW2 (H) and RSC-RSC (I). Interaction lifetimes are determined by measuring the time remodelers spend in close proximity. Interaction half-lives (τ) are calculated from single exponential fits to PDF plots of lifetimes. (J-K) Proportions of each event type quantified as pie charts for RSC-ISW2 (J) and RSC-RSC (K).

Nucleosomes Constrain 1D Diffusion of RSC and ISW2.

(A) Schematic: RSC and ISW2 interact with promoters and flanking nucleosomes through 3D and 1D search processes. (B) Optical tweezers and confocal microscopy assay for measuring remodeler diffusion on lambda DNA nucleosome arrays. (C) Electrophoretic mobility shift assay (EMSA) of lambda nucleosome arrays with increasing octamer concentration. The asterisk indicates a condition that produced ∼10 nucleosomes per array. (D) Force distance plots are used to assess the number of nucleosomes on arrays, with ∼25 nm lengthening per nucleosome at 15 pN (inset). (E) Histograms show nucleosome numbers per array for three arrays used, with mean values and standard deviations. (F-G) Kymograph segments of RSC (F) and ISW2 (G) encountering a nucleosome on the array with 1 mM ATP. Diffusion levels are binned on time trace plots. (H-I) Survival plots of dwell times in the ‘non-diffusive’ state for RSC (H) and ISW2 (I) on naked DNA or nucleosome array. (J-M) Half-lives are determined using double exponential fits and are indicated for the slow and fast percentages (pie charts). Values are given for various ATP conditions and for diffusion on naked DNA (RSC; J-ISW2; K) and nucleosome array (RSC; L-ISW2; M).

ATP-Dependent Processive Nucleosome Translocation by RSC and ISW2.

(A-B) Kymographs of RSC (A) and ISW2 (B) showing static colocalization in 1 mM ATPγS. (C-D) Kymographs of RSC (C) and ISW2 (D) moving directionally with nucleosome signals in 1 mM ATP. The green laser is either blinked or continuous, and the initial position of the nucleosome is marked with a dashed green line. (E-F) Examples of translocation events following 1D encounter for RSC (E) and ISW2 (F). (G-H) Trace fragmentation plots illustrate translocating (blue), non-translocating (red), and 1D-search (green) segments for RSC (G) and ISW2 (H). (I) Linear fit to all translocation events, highlighting speed changes. (J) Linear fits to constant-speed segments (>300 bp, >5 s, r2 >0.5), with ATPγS static colocalization controls in gray. (K) Scatter plots of translocation speeds for RSC (cyan) and ISW2 (magenta), including mean values and standard deviation error bars; ATPγS control shown in gray. (L) Summary schematic.

crRNA sequences for dCas9 binding oligonucleotide sequences used for lambda DNA preparation