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. 2017 Sep 25;45(21):12413–12424. doi: 10.1093/nar/gkx848

Figure 5.

Figure 5.

Dependence of the translocation efficiency (Ptrans) on site spacing. (A) The uracil-containing probe DNAs are transfected into Hap1 cells for 4 h and hUNG2 induction was initiated by addition of doxycycline (Dox). At 6 h post-induction, cells were harvested and assessed for uracil excision (see Materials and Methods). The electrophoretically resolved band fragments from the site transfer measurements with substrates U10, U40 and U80 are shown. Each experiment was performed at least three times. The in vitro treatments that were performed with each DNA prior to electrophoresis are described in the legend to Figure 3C. The reaction extents (in percent) for each sample are shown at the bottom of the gel. The asterisk in the marker lane for U80 is a low level (<2%) of single-strand contaminant that was carried over from the native gel purification of the U80 probe. The Ptrans for the U40 substrate obtained from measurments in Hap1wt cells is 0.5 ± 0.2, which overlaps the value obtained with the inducible line. This measurement has a large error due to the weak band intensities arising from the low percentage reaction. (B) Comparison of cellular Ptrans measurements with previous in vitro measurements performed in the presence and absence of the polymer crowding agent PEG8K (25). The in vitro measurements used buffers containing 150 mM K+. All experiments were performed at least three times, and the standard errors of measurements are indicated. The values for the cellular measurements that are shown were corrected for small differences in extent of reaction for the three substrates (see Supplementary Information). (C) Determination of the mean translocation distance (λ) of hUNG2 in human cells and in vitro. The solid lines are best fits to a single Gaussian distribution (Equation 2) and the mean translocation length was determined using Equation (3). The fitted curve for the in vitro data set in the presence of PEG8K excluded the data point for the 10 bp spacing because it showed a negative deviation from the theoretical curve (see text).