Figure 2.
Activity of immobilized FLAG-integrase and inhibition by L-731,988. FLAG-integrase was assembled on the SPA beads and incubated with 32P-labeled U5 viral ends in the absence or presence of L-731,988. The beads were pelleted and resuspended in gel loading buffer. Lane 1, no FLAG-integrase; lane 2, SPA beads with FLAG-integrase; lanes 3–14, FLAG-integrase with a titration of L-731,988; lane 3, 100 μM; lane 4, 33 μM; lane 5, 11 μM; lane 6, 3.7 μM; lane 7, 1.23 μM; lane 8, 0.41 μM; lane 9, 0.14 μM; lane 10, 0.045 μM; lane 11, 0.015 μM; lane 12, 0.005 μM; lane 13, 0.0016 μM; lane 14, 0.0005 μM. (A) Inhibition of strand transfer (1-h exposure). (B) Inhibition of 3′ processing. N-2 processing product is indicated (arrowhead) (15-min exposure). (C) Inhibition of disintegration. The cleaved (arrow) and uncleaved (arrowhead) disintegration products are indicated. Lane 1, dumbbell probe; lane 2, FLAG-integrase in solution; lane 3, FLAG-integrase associated with SPA beads; lanes 4–7, FLAG-integrase associated with SPA beads with a titration of L-731,988; lane 4, 100 μM; lane 5, 10 μM; lane 6, 1 μM; lane 7, 0.1 μM.