Figure 4

Geminin at low or high concentrations prevents TopoIIα activity. (A) TopoIIα immunoprecipitated from the chromatin of HME cells 72 hours after transfection with control, geminin, Cdc7 or TopoIIα small interfering RNA (siRNA) or 24 hours after doxorubicin (10 μM) or etoposide (10 μM) treatment were used to decatenate kinetoplast (k)-DNA as a substrate using a TopoGen assay as described in Materials and methods. NCi-kD indicates the position of nicked circular decatenated k-DNA minicircles and NNCi-kD indicates the position of non-nicked circular decatenated k-DNA minicircles, while the catenated DNA networks are retained in the wells of the gel (C-kD). The markers are 1-kb DNA ladder (lane 1) or linear and decatenated k-DNA (lanes 8 and 9). (B) Quantification of the reactions shown in (A). Values presented are means ± SD. **P ≤ 0.01. Inset shows the expression of TopoIIα on chromatin (upper) or whole cell (lower) extracts. (C) A decatenation assay was carried out using k-DNA as the substrate with 0, 1 or 2 U of purified TopoIIα in the presence of 150 ng of glutathione S-transferase (GST) alone or 10, 50, 100 or 150 ng of GST-geminin. The reactions on the right are decatenation reactions in the presence of 2 U of TopoIIα alone or with 150 ng of GST-geminin or 150 ng of GST-geminin that was incubated earlier with an excess anti-geminin antibody. (D) Relaxation of supercoiled pBR233 DNA assay. The plasmid pBR233 was incubated with 0, 1 or 2 U of purified TopoIIα in the presence of 50 ng of GST alone or 0, 10, 50 or 100 ng of GST-geminin as described in Materials and methods. SCi-D, RCi-D, L-D and NCi-D indicate the positions of supercoiled, relaxed, linear and nicked circular pBR233 DNA.