Skip to main content
. 2003 Jan-Feb;9(1-2):26–36.

Figure 2.

Figure 2

Mechanistic study of DHBA-mediated DNA topoisomerase inhibition. A: DHBA prevents topoisomerase I-DNA binary complex formation. pHOT1 DNA (lane 1) was incubated with 20 units (lane 2) and 40 units (lane 3) of topoisomerase I at 8 °C for 15 min and electrophoresed on a 7% nondenaturing polyacrylamide gel at 4 °C. Preincubation of 50 units of topoisomerase I with increasing concentration of DHBA prevented the formation of DNA-enzyme binary complex (lanes 5 to 7). B: DHBA prevents topoisomerase II-DNA binary complex formation. Preincubation of 50 units of topoisomerase II with increasing DHBA concentration (lanes 5 to 8). Lanes 2 and 3, incubation of DNA with 20 and 40 units of enzyme, respectively. C: DHBA does not induce topoisomerase I–mediated DNA cleavage. pHOT1 DNA (lane 1) was incubated with 50 units of enzyme alone (lane 2) and further treated with SDS/PK (lane 3). Lanes 4 to 6, with increasing concentrations of camptothecin, and lanes 7 to 9, with increasing concentration of DHBA. D: DHBA antagonizes camptothecin-mediated DNA cleavage. Reactions were as described in Materials and Methods. Lane 1, supercoiled pHOT1 DNA; lane 2, same amount of DNA plus 50 units of topoisomerase I; lane 3, same as lane 2 plus 20 μM camptothecin; lanes 4 to 6, same as lane 3 but in presence of 1, 5, and 10 μM of DHBA added simultaneously; lanes 7 to 9, same as lanes 4 to 6 but enzyme was preincubated with different concentrations of DHBA before addition of camptothecin and DNA. E: DHBA does not induce topoisomerase II–mediated DNA cleavage and antagonizes etoposide-induced cleavage. Reactions were as described in Materials and Methods. Lane 1, control pRYG DNA; lanes 2 and 3, DNA with 25 and 50 units of L. donovani topoisomerase II; lanes 4 and 5, same as lane 3 but in presence of 10 and 50 μM etoposide; lanes 6 to 8, same as lane 3, but in presence of 1, 5, and 10 μM DHBA added simultaneously with DNA and topoisomerase II; lanes 9 to 11, same as lane 5, but enzyme was preincubated with increasing concentration of DHBA before addition of etoposide. F: DHBA did not induce cleavage of kDNA minicircles in L. donovani AG83 promastigotes. Reactions were as described in Materials and Methods. Lane 1, DNA from control cells; lane 2, cells treated with 0.5% DMSO; lanes 3 and 4, cells treated with etoposide at 100 and 200 μM; lanes 5 to 8, from cells treated with increasing concentration of DHBA. I, covalently closed minicircles; II, minicircles containing nicks or gaps; III, linearized minicircles. G: DHBA inhibits etoposide-promoted minicircle cleavage. Lanes 1 and 2, same as lanes 1 and 2 in F; lane 3, DNA from cells treated with 50 μM etoposide; lanes 4 and 5, DNA from cells treated with 50 μM etoposide along with 5 and 20 μM DHBA added simultaneously; lanes 6 and 7, DNA from cells preincubated with 5 and 10 μM DHBA followed by treatment with etoposide.