Figure 5.

Direct competition of target DNA for the L-731,988 binding site. (A) Competition of target DNA for L-731,988. L-731,988 was incubated with NL4–3 integrase assembled on SPA beads coated with 2′,3′-dideoxy donor DNA. The ability of two nonspecific target DNA sequences to compete for inhibitor binding was determined. A representative experiment done with duplicate samples is shown with bars indicating standard error. ●, ▴, target DNAs 1 and 2 as described; ■, unlabeled L-731,988; ○ and ×, single-stranded G-rich and T-rich 20-mer. (B) Exonuclease protection of donor DNA in the presence of target DNA. NL4–3 integrase was assembled on SPA beads coated with ³²P-labeled, 2′,3′- dideoxy donor DNA. The assembled beads were incubated overnight with target DNA, washed, and treated with exonuclease III. Lane 1, no integrase; lanes 2–9, beads with assembled integrase incubated with a titration of target DNA; lane 2, 1,000 nM; lane 3, 333 nM; lane 4, 111 nM; lane 5, 37 nM; lane 6, 12.3 nM; lane 7, 4.1 nM; lane 8, 1.4 nM; lane 9, 0.45 nM; lane 10, no target DNA added; lane 11, no exonuclease III treatment.