Equilibrium binding assays

Equilibrium binding of enzyme (TDG, TDG^82-308 or TDG^111-308) to a G·T^F substrate analog was analyzed using electrophoretic mobility shift assays (EMSAs), performed essentially as described (20), where T^F is the 2′-fluoroarabino analogue of dT (described above). Samples contained a 10 nM concentration of G·T^F DNA and varying concentrations of enzyme. Binding reactions were incubated at room temperature for 30 min, loaded onto a 6% native denaturing polyacrylamide gel (Invitrogen) and run at 4°C for 2 h at 50 V. Gels were imaged using a Typhoon 9400 variable mode imager (GE Healthcare) as described (56).