FIGURE 7.

The Time course of 3′ endonuclease activity on blunt-ended DNA reveals that Artemis resects predominantly 1–3 nt, requires DNA-PKcs, and is stimulated by Ku. 20 nm 3′ ³²P-radiolabeled, 34-bp biotinylated dsDNA (B) was incubated with 50 nm Ku, 50 nm DNA-PKcs, and 50 nm Artemis for 0, 15, 30, and 60 min at 37 °C in the lanes as indicated. In addition, 0.5 mm ATP and 0.1 μm streptavidin (SA) were included in all reactions to suppress Artemis activity on the 5′ end. The P on the bottom strand denotes a cold phosphate group on the 5′ end. The samples were resolved on a 14% denaturing PAGE. M, marker lane; asterisk, [³²P]phosphate. The quantified 3′ resection percentage is noted at the bottom of the gel. This is a representative gel of at least three identical experiments and at least two preparations of Artemis and DNA-PKcs. (Several additional similar time courses gave results indistinguishable from those shown here.). A, 3′ dA substrate (HC86*/89). B, 3′ dT substrate (HC74*/73). C, 3′ dG substrate (HC87*/90). D, 3′ dC substrate (HC88*/91).