FIGURE 5.

The Time course of 5′ endonuclease activity on blunt-ended DNA reveals that Artemis resects up to 6 nt, requires DNA-PKcs, and is modulated by Ku to preferentially cut 2 nt into the duplex. 20 nm 5′ 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 3′ end. The samples were resolved on a 14% denaturing PAGE. M, marker lane; asterisk, [³²P]phosphate. The quantified 5′ 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, 5′ dA substrate (*HC79/82). B, 5′ dT substrate (*HC57/76). C, 5′ dG substrate (*HC80/83). D, 5′ dC substrate (*HC81/84).