Figure 5

Effect of BTBD2 on TOP1 activity in vitro.A. Supercoil relaxation activity. HeLa cell nuclear extract containing TOP1 was mixed with GST-BTBD2 or GST alone for 15 min on ice before the addition of supercoiled DNA. The reactions were then incubated for 0–24 min at 30°C before stopping with SDS. The location of relaxed and supercoiled topoisomers are indicated (R and SC). B. DNA nicking at a strong TOP1 cleavage site. GST or GST-BTBD2 was incubated with TOP1 for 20 min at room temperature before the addition of a 36 bp 3' end-labeled DNA [24]. Cleavage reactions were continued for another 20 min before stopping with SDS. Lane 1: DNA alone; lane 2: + TOP1 (1.2 ng/reaction); lane 3: same + 10 μM camptothecin; lanes 4–8: reactions contained 0.25 μ g GST and 10 μ M camptothecin; lanes 9–13 contained 0.25 μg GST-BTBD2 + 10 μM camptothecin; lanes 4 & 9: no TOP1 added; lanes 5 & 10: 1.2 ng TOP1; lanes 6 & 11: 0.4 ng TOP1; lanes 7 & 12: 0.3 ng TOP1; lanes 8 & 13: 0.1 ng TOP1). C. As in B, but using a 161 bp substrate DNA [25]. Numbering of cleavage sites according to Pommier et al. [25] is indicated on the right side.