Fig. 1.
Nearest-neighbor sequence context affects the mismatch-dependent steady-state ATPase activity of hMSH2-hMSH6. (A) Michaelis–Menten analysis of hMSH2-hMSH6 in the presence of 240 nM DNA substrate and increasing concentrations of ATP (see Table S1, Table S2, Table S3, Table S4, Table S5, and Table S6 (47). Average ATP turnover (kcat) of the 2 × 3′ purine sequence context (red bar); average kcat of the 1 × 3′ purine plus 1 × 3′ pyrimidine sequence context (green bar); average kcat of the 2 × 3′ pyrimidine sequence context (blue bar). Error bars indicate the ± standard deviation for kcat. The gray box shows ATPase activation by a homoduplex DNA containing an A/T or G/C base pair at the mismatch site (± standard deviation; see text). The dashed line indicates ATPase activity of biotin-streptavidin blocked-end mismatched or homoduplex DNA (29). (B) Nearest-neighbor sequence context affects dissociation equilibrium binding constants (KD) for hMSH2-hMSH6. IAsys total internal reflectance (TIR) real-time binding studies using 41-mer single 3′-biotinylated mismatched DNA substrates immobilized on the TIR surface via biotin–streptavidin linkage (47). Model 2 × 3′-purine (CxA; red bar) and 2 × 3′-pyrimidine (AxC; blue bar) sequence contexts extremes were examined. Association rate constants (kON) and dissociation rate constants (kOFF) were derived from binding isotherms (Table 1). Error bars (some within the colored bar) represent the standard deviation calculated from multiple experiments. (C) Nearest-neighbor sequence context affects thermal denaturation of mismatched DNA. Melting curves for mismatched 10-mer duplexes embedded in model 2 × 3′-purine (CxA; red bar) and 2 × 3′-pyrimidine (AxC; blue bar) sequence contexts extremes were examined. The G/T* mismatch sequence context was CxG (red bar) and GxC (blue bar). Error bars (some within the colored bar) represent the standard deviation calculated from multiple experiments.