Figure 3.

RAL and EVG inhibit concerted integration at low nM concentrations. (A) IN (40 nM) was pre-assembled with 5′-³²P end-labeled 1.6 kb U5 blunt-ended DNA substrate (1 nM) at 14°C for 15 min. Upon addition of varying concentrations of inhibitors and supercoiled DNA (1.5 nM), samples were incubated for 2 h at 37°C. Reactions were stopped with EDTA, deproteinized, and the strand transfer products were analyzed on 0.7% agarose gels. Individual products are identified on the right. Lane 1, marked C, contains DNA only without IN. Lanes 2 and 3 are control reactions without inhibitor. Increasing concentrations of RAL were added in lanes 4–13 and EVG in lanes 15–24. Lane 14, marked M, contain ³²P- labeled molecular weight markers (kb ladder). (B) Inhibition of concerted or FS, CHS, and D-D products by increasing concentrations of RAL. IN (20 nM) was pre-assembled with 5′-³²P end-labeled 1.6 kb U5 blunt-ended DNA substrate (0.5 nM) at 14°C for 15 min. Upon addition of varying concentrations of RAL and supercoiled DNA (1.5 nM), samples were incubated for 2 h at 37°C and processed as mentioned in A. Inhibition of each product (FS, D-D and CHS) was plotted against RAL concentration. The error bars indicate the SD from at least four independent experiments. (C) Inhibition of integration products with increasing concentrations of EVG. Experiments were done as described in panel B. The error bars indicate the SD from at least four independent experiments. The dotted horizontal line indicates 50% inhibition of FS product. The IC₅₀ values for inhibition of FS, D-D, and CHS integration products are summarized in Table 1.