Figure 6. LS1 is a nonintercalating TOP2-α inhibitor. (A) Effect of LS1 on decatenation of C. fasciculata kinetoplastid DNA (kDNA) by purified human TOP2-α. Inset shows a negative image of the ethidium bromide stained agarose gel separating kDNA species. Quantitation of decatenated kDNA was done with GelQuant.NET 1.8.2 software and results were plotted using KaleidaGraph 4.1.0 software. (B) A DNA topoisomerase I (Top1) DNA unwinding assay was used to assess the ability of LS1 to intercalate as indicated in Methods. Known Top2 poisons that either intercalate (doxorubicin) or do not (etoposide) were included as controls. An E. coli-compatible plasmid (puc18) exhibiting both supercoiled (*) and nicked/relaxed (**) forms was used as the substrate (lane 1). In the absence of an intercalator (lane 2) Top1 converts the plasmid to fully nicked/relaxed (**) or intermediate relaxed forms (***). Intercalation was assessed for DOX (10µM or 50µM; lanes 3-4), ELLIP (100µM; lane 5), 100mM ETOP (lane 6) and LS1 (10µM, 50µM, or 100µM; lanes 7-9).