Figure 1. SWR1 binds DNA in short- and long-lived states and prefers longer DNAs.

(A) Proposed facilitated search mechanism for how SWR1 locates the +1 nucleosome. (B) A denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of reconstituted Cy3-SWR1 imaged for Coomassie (left) and Cy3 fluorescence (right). Cy3-Swc7 is faint when stained with Coomassie but is a prominent band in the Cy3 scan. The two diffuse bands that run at higher molecular weight and appear in the Cy3 scan are carry over from the ladder loaded in the adjacent lane. (C) A schematic of the single-molecule colocalization experiment used for kinetic measurements of Cy3-SWR1 binding to Cy5-labeled DNA of different lengths. Representative traces of Cy3-SWR1 binding to (D) 20 bp Cy5-DNA and to (E) 150 bp Cy5-DNA. A second Cy3-SWR1 can be seen binding at approximately 100 s. (F) The on-rate binding constant for the initial binding event, (k_bind) for SWR1 to DNA of different lengths, error bars are standard deviation. N values: 20 bp (40), 40 bp (67), 60 bp (267), 80 bp (129), 100 bp (221), 150 bp (409). The red line is a linear fit to the data, where R² = 0.99 [two technical replicates represented, statistical differences determine via Student’s t-test, where asterisks indicate the level of significance as conventionally defined (* = p < 0.05; ** = p < 0.01, *** = p < 0.001, n.s. = not significant)]. (G) The lifetime (t_bound) of Cy3-SWR1 bound to DNAs of different lengths, error bars are standard deviation. N values: 20 bp (48), 40 bp (118), 60 bp (291), 80 bp (382), 100 bp (339), 150 bp (363) [two technical replicates represented, statistical differences determine via Student’s t-test, where asterisks indicate the level of significance as conventionally defined].

Figure 1—figure supplement 1. Cy3-SWR1 purification and DNA-binding kinetics.

(A) A glycerol gradient purification of the Cy3–SWR1 complex after Cy3-Swc7 reincorporation. A silver stain (top image) shows that the SWR1 complex eluted in fractions 6 and 7 of the gradient. A Cy3 scan of the same gel shows that Cy3-Swc7 is also found in fractions 6 and 7 (confirmed by a Cy3-Swc7 only control at the end of the gel). This demonstrates that the Cy3-Swc7 is incorporated into the SWR1ΔSwc7 complex. (B) A histone exchange assay that shows Cy3-SWR1 (right lanes) is as active as the unlabeled wild-type SWR1 complex. The gel shift is caused by the incorporation of triple flag-tagged ZB dimers (denoted Z^f) into the nucleosome (AB dimer denoted with a letter A). (C) Normalized plot shown in Figure 1F of k_bind. Values and errors are normalized to the longest DNA tested; values are divided by the quotient of the length of DNA divided by 150 bp. No statistical difference between DNA lengths as assessed using a Student’s t-test. (D) t_bound for Cy3-SWR1 bound to 150 bp DNA measured at different laser powers. N values: 7 W (154), 3.5 W (139), 1 W (88) [one technical replicate represented]. (E) 1-CDF for Cy3-SWR1 bound to 150 bp DNA in histone exchange reaction buffer without ATP, with 1 mM ATP, with 200 mM NaCl, or with 0.2 µg/ml of salmon sperm DNA [three technical replicates]. (F) Quantification of panel E; t_bound for Cy3-SWR1 bound to 150 bp DNA with error bars representing standard deviation for the different conditions. N values: buffer + ATP (588), buffer only (563), high salt (538), salmon sperm DNA (750) [three technical replicates represented, statistical differences determine via Student’s t-test, where asterisks indicate the level of significance as conventionally defined (* = p < 0.05; ** = p < 0.01)]. (G) 1-CDF for Cy3-Swc2 bound to 150 bp DNA in histone exchange reaction buffer without ATP, with 200 mM NaCl or with 0.2 µg/m of salmon sperm DNA [three technical replicates per condition represented; all data are shown] (H) Quantification of panel G; t_bound for Cy3-Swc2 bound to 150 bp DNA with error bars representing standard deviation for the different conditions. N values: buffer only (1027), high salt (605), salmon sperm DNA (699) [three technical replicates; statistical differences determine via Student’s t-test]. (E, G) 1-CDF plots for both SWR1 and Swc2 binding to 150 bp DNA under all conditions tested were best fit to double exponential decay functions. (F, H) t_bound values are amplitude averages which normalize the reported t_bound based on the relative contribution of the short- versus long-lived tau to the fit data. While the short tau values are reported as part of the amplitude averages, for C-Trap experiments, the long tau is the more relevant value as very short-lived binding events are not easily observed in our C-Trap setup. The overall trends in tau-slow are consistent with the reported amplitude averages.