Human DNA topoisomerase II beta binds and cleaves four-way junction DNA invitro

We have used gel retardation analysis to show that human DNA topoisomerase II beta can bind a 40 bp linear duplex containing a single DNA topoisomeras e II beta cleavage site. Furthermore, we demonstrate for the first time tha t human DNA topoisomerase II beta binds to four-way junction DNA, This supp orts previous suggestions that topoisomerase II may be targeted to supercoi led DNA through the recognition of DNA cruciforms, helix-helix crossovers a nd hairpins, DNA topoisomerase II beta had a 4-fold higher affinity for the four-way junction than for the linear duplex, as demonstrated by protein t itration and competition analysis. Furthermore, the DNA topoisomerase II be ta:four-way junction complex was significantly more salt stable than the co mplex with linear DNA. The four-way junction contained potential topoisomer ase II beta cleavage sites straddling the points of strand exchange, and in deed, topoisomerase II beta was able to cleave three of these four predicte d sites. This indicates that topoisomerase II beta can bind to the centre o f the junction, Topoisomerase II has to bind both the transported and the g ated DNA helices prior to strand passage, and it is possible that both heli ces are provided by the four-way junction in this case. The stable complex of DNA topoisomerase II beta with four-way junction DNA may provide an idea l substrate for further studies into the mechanism of substrate recognition and binding by DNA topoisomerase II.