FIGURE 3.

Sumoylation of TDG at 37 °C in the presence and absence of DNA. A–C, in vitro sumoylation reactions for modification of TDG by SUMO-1 or SUMO-2 performed at 37 °C in the absence of DNA (A) or in the presence of abasic DNA (B) or nonspecific (undamaged) DNA (C), and monitored by electrophoresis. The first lane of each gel contains pure TDG, and the second lane contains pure SUMO-1-modified TDG (TDG∼SUMO-1 or TDG-S). The sumoylation reactions were initiated by adding ATP (2.5 mm) to buffer containing E1 (0.1 μm), E2 (1 μm), SUMO-1 or SUMO-2 (10 μm), TDG (2 μm), with no DNA or with abasic DNA (2.5 μm) or nonspecific DNA (4.1 μm). D, in vitro sumoylation of RanGAP1-NΔ419 at 37 °C. Samples were extracted from the reaction and quenched at the indicated times (given in minutes), and the reaction progress was analyzed by SDS-PAGE under reducing conditions.