Figure 4.

Inhibition of UDGs by uracil and AP-DNA. (A) Inhibition by uracil: reactions were carried out using SSU9 with appropriately diluted UDGs, and the total product formed in the reactions with the wild-type, Y66F, Y66L, Y66H and Y66W UDGs was 2570, 2310, 3610, 2890 and 600 fmol, respectively, in the absence of externally added uracil, which was used as a reference for 100% activities. The products formed with the same dilutions of UDGs in the presence of uracil (2–12 mM) were calculated with respect to the reference values of the respective UDGs and plotted against uracil concentration (0–12 mM): wild-type (triangles), Y66F (squares), Y66H (crosses), Y66L (diamonds) and Y66W (circles) UDGs. (B) Inhibition by uracil and AP-DNA: UDG reactions were carried out in the presence of 5 mM uracil along with 0.062, 0.125 or 0.25 µM AP-DNA. The total amounts of product formed in the reactions with the wild-type, Y66F, Y66L, Y66H and Y66W UDGs were 1194, 999, 985, 1070 and 1870 fmol, respectively, in the absence of both the AP-DNA and uracil, which were used as reference for 100% activities. Activities in the presence of 5 mM uracil and the indicated amounts of AP-DNA were calculated with respect to these reference values for the respective UDGs and plotted against the AP-DNA concentration: wild-type (triangles), Y66F (squares), Y66H (crosses), Y66L (diamonds) and Y66W (circles) UDGs. (C) Same as (B) except that the experiment was carried in the absence of externally added uracil and in the presence of AP-DNA as inhibitor. The activities in the presence of AP-DNA were calculated with respect to the reference activities (100%) given in (B) and plotted as a histogram.