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Human DNA topoisomerase IIβ binds and cleaves four-way junction DNA in vitro

机译:人类DNA拓扑异构酶IIβ在体外与四向连接DNA结合并裂解

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We have used gel retardation analysis to show that human DNA topoisomerase IIβ can bind a 40 bp linear duplex containing a single DNA topoisomerase IIβ cleavage site. Furthermore, we demonstrate for the first time that human DNA topoisomerase IIβ 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 IIβ 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 IIβ:four-way junction complex was significantly more salt stable than the complex with linear DNA. The four-way junction contained potential topoisomerase IIβ cleavage sites straddling the points of strand exchange, and indeed, topoisomerase IIβ was able to cleave three of these four predicted sites. This indicates that topoisomerase IIβ 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 IIβ 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.
机译:我们已经使用凝胶阻滞分析来显示人类DNA拓扑异构酶IIβ可以结合一个包含单个DNA拓扑异构酶IIβ切割位点的40 bp线性双链体。此外,我们首次证明了人类DNA拓扑异构酶IIβ与四向连接DNA结合。这支持先前的建议,即拓扑异构酶II可能通过识别DNA十字形,螺旋-螺旋交叉和发夹而靶向超螺旋DNA。 DNA拓扑异构酶IIβ对四向连接的亲和力比对线性双链体高4倍,如蛋白质滴定和竞争分析所证明。此外,DNA拓扑异构酶IIβ:四向连接复合物比线性DNA的复合物盐稳定性更高。四向连接包含跨链交换点的潜在拓扑异构酶IIβ裂解位点,实际上,拓扑异构酶IIβ能够裂解这四个预测位点中的三个。这表明拓扑异构酶IIβ可以结合到连接的中心。拓扑异构酶II必须在链通过之前结合转运的和门控的DNA螺旋,在这种情况下,两个螺旋可能由四向连接提供。 DNA拓扑异构酶IIβ与四向连接DNA的稳定复合物可为进一步研究DNA拓扑异构酶II的底物识别和结合机理提供理想的底物。

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