Fig. 6. In vitro activity of Topo II in control and DmSMC4-depleted extracts. (A) Determination of Topo II activity of chromatin-associated protein extracts from control cells using kDNA as substrate. For each assay 10 µg of total extracts with (+) and without (-) 10 µM of Topo II inhibitor ICRF-187 were used and the reaction was allowed to proceed for different times. The DNA was deproteinised and analysed on a 0.8% agarose gel. In control extracts the kDNA was rapidly decatenated to produce relaxed minicircles after 5 minutes of the incubation. The addition of ICRF-187 for 10 minutes completely inhibited decatenation. (B) Decatenation of kDNA was used to measure Topo II activity in control or DmSMC4-depleted extracts after 96 hours of treatment without (-) or with (+) the addition of exogenous Topo II (2U, Amersham). Different levels of protein extracts were used: 0 µg (-), 5 µg (+) or 10 µg (++). DNA was deproteinised and run on a 0.8% agarose gel containing 0.5 µg/ml ethidium bromide. Decatenation of kDNA is observed in control extracts with or without addition of exogenous Topo II. DmSMC4-depleted extracts showed no decatenation activity in the absence of exogenously added Topo II.

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