Fig. 7. The effect of depletion of topo II on centromeric topo II cleavage activity. The HTETOP-minichromosome hybrid cells expressing topo II or depleted (72 hours exposure to dox) for topo II and/or topo IIβ (72 hours exposure to siRNA) were exposed in culture to etoposide [0 (DMSO only), 100 or 500 µM] for 60 minutes at 37° C and embedded in agarose. (A) Undigested HMW DNA was resolved by PFGE and stained using ethidium bromide. (B) After transfer, the Southern blot was probed using the X -satellite DNA DXZ1 to detect the 2.7 Mb X centromere-based minichromosome (position indicated by black arrowhead). An etoposide-specific DXZ1-hybridising fragment of 1.85 Mb (position indicated by `<') could be detected, in addition to a more general smear of hybridisation (this was more noticeable in DNA from cells expressing topo II). At the lower etoposide concentration (100 µM), a signal in the 1.85 Mb range could only be detected in cells expressing topo II (either alone, or together with the β isoform); at the higher etoposide concentration (500 µM), the 1.85 Mb signal was barely detectable after depletion of both isoforms, but could still be detected following depletion of topo II alone (although the signal was reduced relative to that of the intact minichromosome in the same sample). This suggests that both isoforms of topo II contribute to this cleavage site within the centromeric DNA. LM, limit mobility.