Figure 2.

Biochemical studies of full length TDG, TDG^82-308 and TDG^111-308. (A) Equilibrium binding of a given TDG construct to DNA (10 nM) containing a G·T^F mismatch, where T^F is a non-cleavable Thd analog, monitored by electrophoretic mobility shift assays (EMSA). The concentration of enzyme (nM) is indicated. For TDG^111-308, the left lane marked ‘20*’ indicates binding to abasic DNA product (10 nM, 28 bp), to show the mobility of a tight 1:1 complex, given that such a complex is not clearly observed for G·T^F DNA in the gel. (B) Single-turnover kinetics for excision of T from a G·T DNA substrate by the three TDG constructs, at 37°C and 22°C (TDG^111-308 not stable at 37°C). Rate constants are k_max = 0.651 ± 0.044 min⁻¹ for TDG and k_max = 0.655 ± 0.020 min⁻¹ for TDG^82-308 at 37°C, and k_max = 0.126 ± 0.008 for TDG, k_max = 0.108 ± 0.006 min⁻¹ for TDG^82-308, and k_max = 0.022 ± 0.002 min⁻¹ for TDG^111-308 at 22°C.