The elimination of TopoIIα SUMOylation at Lys660 blocks SUMOylation-dependent inhibition of TopoIIα activity. (a) Unmodified TopoIIα WT and K660R proteins were incubated with kDNA to determine relative activity. K660R had ∼20 times less activity than WT. (b) Electrophoretic mobility shift assay. Unmodified TopoIIα WT and K660R were incubated with plasmid DNA to determine relative DNA binding affinity. Both WT and K660R displayed similar binding affinity to DNA. oc and cc stand for open and closed circle, respectively. (c and e) The TopoIIα WT and K660R were SUMO2-modified in vitro with 60 nM of Ubc9 and 30 nM of PIASy. Control reactions (Cont.) using the same condition except for SUMO2-G were also performed. Non-SUMOylated or SUMOylated TopoIIα samples were assayed for decatenation activity. (d and f) Representative results of decatenation activity assays with TopoIIα WT (d) and K660R (f) are shown. The mean decatenation activity from five independent experiments with TopoIIα WT (g) and four independent experiments with TopoIIα K660R (h) are displayed as the percentage of catenated kDNA remaining, with standard error (error bars). The strong inhibition of TopoIIα decatenation activity by SUMOylation was abolished in reactions using TopoIIα K660R. Positions of molecular mass standards (kD) are indicated on the sides of the gel blots.