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. 2019 Mar 19;8:e43008. doi: 10.7554/eLife.43008

Figure 6. Reb1ΔN binds and dissociates similarly to WT Reb1.

(A) Schematic comparison of WT Reb1 and the deletion variant Reb1-ΔN, which is comprised of residues 395–810. (B) Cy3 image of the EMSA of Reb1-ΔN binding to the 25-bp DNA molecule (S1/2 Reb1ΔN–DNA EMSA = 12.8 ± 1.2 nM). In addition, Reb1-ΔN titration with the smPIFE DNA results in a Cy3 emission increase of ~1.4-fold and fits to a binding isotherm with an S1/2 Reb1ΔN–DNA PIFE = 7.8 ± 0.5 nM. (C) Cy5 image of Reb1-ΔN binding to smFRET nucleosomes containing the P8 Reb1-binding site (S1/2 Reb1ΔN -Nuc P8 EMSA = 8.5 ± 0.5 nM). In addition, ensemble FRET measurements with these nucleosomes fit to a binding isotherm with an S1/2 Reb1ΔN–Nuc P8 FRET = 11.8 ± 0.9 nM. (D) Example time traces of single nucleosomes for two separate Reb1-ΔN concentrations, which are fitted to a two-state Hidden-Markov Model. As the Reb1 concentration increases, the immobilized nucleosome shifts to the low FRET state. (E) The primary Reb1-binding (red) and -dissociation (blue) rates for increasing Reb1-ΔN concentrations. The dissociation rates are constant with an average rate of koff Reb1ΔN–Nuc primary = 0.0044 ± 0.0008 s−1, whereas the binding rates fit to a line with a slope that equals the overall binding rate of kon Reb1ΔN–Nuc primary = 0.0003 ± 0.00001 s−1 nM−1.

Figure 6.

Figure 6—figure supplement 1. Strong nucleosome displacing factors are characterized by their overall negative charge.

Figure 6—figure supplement 1.

(A) The isoelectric points for budding yeast TFs that have strong (red), weak (blue), or no (green) nucleosome displacing activity (Yan et al., 2018). (B) Binding curves fit to the Reb-ΔN EMSAs shown in Figure 6B–C.
Figure 6—figure supplement 2. Characterizing Reb1ΔN interactions with nucleosomes.

Figure 6—figure supplement 2.

(A) Cumulative sums of high FRET (red) and low FRET (blue) dwell times for nucleosomes at each Reb1ΔN concentration. (B) Table of the log-likelihood ratio that was used to test the assumption that a cumulative sum fits to a double exponential. If the result was more than 0.01 for three of the Reb1ΔN concentrations, we rejected the assumption and fitted a single exponential. For Reb1ΔN binding to nucleosomes, the low FRET cumulative sums followed double-exponentials. (C) Plots of the Reb1ΔN binding rates (red) to nucleosomes, and of both the fast and slow dissociation rates (blue) from nucleosomes for four concentrations of Reb1ΔN. These rates were determined from the exponential fits of the cumulative sums in panel (A). (D) The fraction of fast dissociation rates of Reb1 from nucleosomes for increasing Reb1 concentrations. For Reb1ΔN dissociation from nucleosomes, the slower rate accounted for ~50% of all of the dissociation events.