Figure 2.

Kinetics and sequence context dependences of TDG-catalyzed excision of mismatched T. (A) Graphic presentation of pre-steady-state single turnover kinetic of TDG-catalyzed cleavage of various oligonucleotide duplexes. Time kinetics were performed using 500 nM TDG and 50 nM 5′-[³²P]-labeled 40-mer oligonucleotide duplex (⋄) T•G, (•) T•Hx, (▪) T•ϵA, (▾) T•THF and (Δ) T•8oxoA. Each bar represents the mean values of TDG activity ± SD of three independent experiments. (B) Separation of TDG-cleavage products on denaturing PAGE. 5 nM 5′-[³²P]-labeled 40-mer T•Hx oligonucleotide duplexes, where T-containing strand is labeled and Hx placed in different sequence context: CXC, CXG, CXA, CXT, GXC, AXC, TXC, GXG, AXG and TXG, where X is Hx, were incubated with 50 nM TDG for 30 min at 37°C. Arrows 40 mer and 20 mer indicate substrate and cleavage products, respectively. The reaction products were analyzed as described in the Materials and Methods section.