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. 2014 Apr 21;289(22):15810–15819. doi: 10.1074/jbc.M114.572081

FIGURE 5.

FIGURE 5.

Single-turnover assays of TDG modification by preloaded E2∼SUMO-1. A, immunoblot analysis of TDG modification by preformed E2∼SUMO-1 in the presence or absence of DNA. Preformed E2∼SUMO-1 conjugate was generated by adding ATP (10 μm) to buffer containing E1 (0.1 μm), E2 (1 μm), and SUMO-1 (1 μm). Reactions were incubated for 15 min at 37 °C. Then, TDG modification reactions were initiated by adding 3 μl of the E2∼SUMO-1 reaction to 27 μl of buffer containing TDG (2 μm) and EDTA (5 mm). Samples were extracted from the reaction and quenched at the indicated times (given in seconds), and the formation of TDG∼SUMO-1 was analyzed by SDS-PAGE under nonreducing conditions. Detection of SUMO-1 conjugates was performed via immunoblotting with anti-SUMO-1 primary antibody, followed by blotting with a fluorescent secondary antibody. B, kinetics for modification of TDG by preformed E2∼SUMO-1 in the presence or absence of DNA. Linear regression analysis provides initial rate constants of kobs = 1.1 min−1 (no DNA), 0.78 min−1 (AP-DNA), and 0.53 min−1 (NS-DNA). Error bars represent 1 S.D. The asterisk indicates the 60-s time point for free TDG, which was not used in data fitting.