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Published in final edited form as: J Cell Sci. 2007 Oct 23;120(Pt 22):3952–3964. doi: 10.1242/jcs.013730

Fig. 7 — The effect of topo II depletion on centromeric topo II cleavage activity. The HTETOP: minichromosome hybrid cells expressing, or depleted (72 hours dox) for, topo IIα and/or topo IIβ (72 hours 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 α-satellite DNA DXZ1 to detect the 2.7 Mb X centromere-based minichromosome (). An etoposide-specific DXZ1-hybridising fragment of ~1.85 Mb (<) could be detected, in addition to a more general smear of hybridisation (this was more noticeable in DNA from cells expressing topo IIα). At the lower etoposide concentration (100 μM) a signal in the 1.85 Mb range could only be detected in cells expressing topo IIα (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 IIα alone (although the signal was reduced relative to that of the intact minichromosome in the same sample). This suggests that both isoforms contribute to this cleavage site within the centromeric DNA.

Inline graphic — The effect of topo II depletion on centromeric topo II cleavage activity. The HTETOP: minichromosome hybrid cells expressing, or depleted (72 hours dox) for, topo IIα and/or topo IIβ (72 hours 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 α-satellite DNA DXZ1 to detect the 2.7 Mb X centromere-based minichromosome (). An etoposide-specific DXZ1-hybridising fragment of ~1.85 Mb (<) could be detected, in addition to a more general smear of hybridisation (this was more noticeable in DNA from cells expressing topo IIα). At the lower etoposide concentration (100 μM) a signal in the 1.85 Mb range could only be detected in cells expressing topo IIα (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 IIα alone (although the signal was reduced relative to that of the intact minichromosome in the same sample). This suggests that both isoforms contribute to this cleavage site within the centromeric DNA.