Figure 4.

ATBC inhibits plasmid incision by Mtb UvrA, UvrB, and UvrC without intercalating in DNA. (A) Compound structures of ATBC and related compounds 1 and 2. (B) Inhibition of NER activity by ATBC in a concentration-dependent manner. Irradiated plasmid DNA (25 nM) incubated with Uvr proteins (100 nM UvrA for 10 min, 300 nM UvrB for 20 min, and 150 nM UvrC for 30 min at 37 °C) in the presence of increasing concentrations of compound ATBC (0−5 × 10⁻⁵ M); lanes a and b show DNA plasmid substrate without or with UvrABC, respectively. (C) ATBC but not closely related congeners inhibit NER activity. Irradiated plasmid DNA (25 nM) was incubated with Uvr proteins as in (B) in the presence of ATBC or analogues 1−6 at 50 μM. (D) and (E) are plots of data from (B) and (C), respectively; means ± SD from triplicates. (F) Lack of intercalation. ATBC (1, 10, or 100 μM) was incubated with relaxed circular plasmid (25 nM) in the presence of 3 units of topoisomerase I for 45 min at 37 °C. The same concentrations of ethidium bromide were tested as a positive control. Samples were processed as described in Experimental Procedures. Compounds were omitted in lanes x (relaxed plasmid) and y (supercoiled plasmid). Topoisomerase was omitted in lane z, which contained only supercoiled plasmid. Lanes are from a single gel in one experiment representative of three. Intervening lanes with unrelated compounds were removed from the image.