FIGURE 4.
DNA-PKcs titration for Artemis activity on blunt ends. 20 nm 5′ 32P-radiolabeled, 34-bp dsDNA (*HC79/82) with four dAs was created by annealing the radiolabeled (asterisk) top strand with its complementary strand with a 5′ biotin (B). The substrate was incubated with 50 nm Ku, 50 nm Artemis, and various amounts of DNA-PKcs in the respective reactions for 30 min at 37 °C, followed by resolving the sample on a 14% denaturing PAGE. In addition, 0.5 mm ATP and 0.1 μm streptavidin (SA) were included in all reactions to suppress Artemis activity on the 3′ end. Reactions consisted of Ku (lane 2), DNA-PKcs (lane 3), Artemis (lane 4), Ku and Artemis (lane 5), Artemis with increasing amounts of DNA-PKcs (lanes 6–9), and Ku and Artemis with increasing amounts of DNA-PKcs (lanes 10–13). The quantified 5′ resection percentage is noted at the bottom of the gel. This is a representative gel of at least two identical experiments. (Several additional similar experiments gave results indistinguishable from those shown here.)