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. 2015 Jun 5;4:e06249. doi: 10.7554/eLife.06249

Figure 1. Single molecule unzipping technique detects Gal4DBD and nucleosome to near base-pair accuracy.

DNA molecules, each containing a nucleosome and a bound Gal4DBD, were unzipped. All unzipped DNA molecules used in this work were in the region of 600 bp to 1.2 kbp. For clarity, much smaller regions are shown in all figures, with the origin of a template sequence defined as center position (the dyad) of the 601NPE. Shaded regions indicate locations of the Gal4 binding sequence and the 601NPE. (top panel) Cartoon illustrating the unzipping template design using for this experiment. A Gal4 sequence was separated from a 601NPE by 10 bp. The orientation of the 601NPE sequence is indicated by a white arrow. (middle panel) Unzipping in the direction in which the bound Gal4DBD was encountered first. (bottom panel) Unzipping in the direction in which the nucleosome was encountered first.

DOI: http://dx.doi.org/10.7554/eLife.06249.003

Figure 1.

Figure 1—figure supplement 1. Unzipping experimental configuration.

Figure 1—figure supplement 1.

The DNA template was attached, at one end, to the surface of a glass coverslip via a digoxigenin–antidigoxigenin linkage, and at its other end to a microsphere via a biotin–streptavidin linkage. As the coverslip was moved away from the trapped microsphere, using a loading-rate clamp, the dsDNA was sequentially converted into ssDNA upon base pair separation. The presence of force peaks above the naked DNA baseline revealed the detected locations of protein–DNA interactions.
DOI: http://dx.doi.org/10.7554/eLife.06249.004
Figure 1—figure supplement 2. Characterization of the precision and accuracy of detection of the locations of Gal4DBD and nucleosome.

Figure 1—figure supplement 2.

Single molecule unzipping detected Gal4DBD and a nucleosome simultaneously. The histograms for detected locations of Gal4DBD (green) and nucleosome (red) were obtained by pooling data from multiple single molecule traces, with the expected bound locations represented by their respective dashed lines. For each histogram, the precision was determined by the standard deviation of each histogram, and the accuracy by the difference between the mean of the histogram and the expected value (the vertical dashed line). These data demonstrate both the precision and accuracy to be near base-pair.
DOI: http://dx.doi.org/10.7554/eLife.06249.005
Figure 1—figure supplement 3. Characterization of Gal4DBD binding.

Figure 1—figure supplement 3.

To characterize Gal4DBD binding to its binding sequence, DNA unzipping was carried out in the presence of known Gal4DBD concentrations. (A) A representative unzipping trace of a bound Gal4DBD. The location of the binding sequence is shown as a shaded region. Naked DNA unzipping baseline is shown in gray. (B) Fraction of bound Gal4DBD vs the concentration of Gal4DBD. For a given concentration of Gal4DBD, measurements were on multiple DNA molecules to obtain the fraction of Gal4DBD. Data points are represented as (mean ± s.e.m.). The relation for the fraction bound vs [Gal4DBD] was fit to:[Gal4DBD][Gal4DBD]+Kd (red smooth curve), which yielded the dissociation equilibrium constant Kd= 3.4 nM. (C) Fraction of bound Gal4DBD vs time. This relation shows no significant Gal4DBD dissociation from its binding sequence over a course of one hour. Data were fit to a straight line to guide the eye.
DOI: http://dx.doi.org/10.7554/eLife.06249.006
Figure 1—figure supplement 4. Detection of Gal4DBD binding.

Figure 1—figure supplement 4.

The presence of a bound Gal4DBD was determined by the magnitude of the force peak at the Gal4 binding sequence. In the presence of a bound Gal4DBD, the peak force increased substantially and was readily differentiable from the baseline DNA force.
DOI: http://dx.doi.org/10.7554/eLife.06249.007