Figure 2.

Facilitated site transfer by human uracil DNA glycosylase (hUNG). In all facilitated transfer assays the hUNG concentration is 5–20 pM and the DNA substrate concentration is 40 nM. (a) Facilitated site transfer of hUNG between two uracil sites on the same DNA strand separated by 10 bps (S10). Reactions in the absence and presence of 10 mM uracil are shown. Facilitated transfer is qualitatively indicated by an excess of double excision fragments (A and C) relative single site excision products (AB and BC). (b) Facilitated site transfer by hUNG between sites separated by 5 bp (S5) in the absence and presence of 10 mM uracil. (c) Observed probability of site transfer (P_trans^obs) as a function of time and uracil concentration for the substrate with a 10 bp site separation. Linear extrapolation to the y axis provides the true transfer probability at zero time (P_trans). (d) P_trans′ as a function of increasing uracil for substrates with 5, 10 and 20 bp site spacings. Each data point represents an individual experiment as in panels a and b and the prime notation indicates correction for the efficiency of excision (see text). The non-linear least squares fits in (d) use a kinetic partitioning model that relates the dependence of the total transfer probability (P_trans′ = P_slide′ + P_hop′) to the uracil trap concentration (Supplementary Methods). Uncut gel images are shown in Supplementary Fig. 6. Error bars represent the mean ± 1 s.d. of at least three independent trials.