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

摘要

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 topoisomerase II beta cleavage site. Furthermore, we demonstrate for the first time that human DNA topoisomerase II beta binds to four-way junction DNA, This supports previous suggestions that topoisomerase II may be targeted to supercoiled DNA through the recognition of DNA cruciforms, helix-helix crossovers and 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 titration and competition analysis. Furthermore, the DNA topoisomerase II beta:four-way junction complex was significantly more salt stable than the complex with linear DNA. The four-way junction contained potential topoisomerase II beta cleavage sites straddling the points of strand exchange, and indeed, topoisomerase II beta was able to cleave three of these four predicted sites. This indicates that topoisomerase II beta can bind to the centre of the junction, Topoisomerase II has to bind both the transported and the gated DNA helices prior to strand passage, and it is possible that both helices 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 ideal substrate for further studies into the mechanism of substrate recognition and binding by DNA topoisomerase II.