Cowell Ian G, Ling Elise M, Austin Caroline A
Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK.
Methods Mol Biol. 2025;2928:97-108. doi: 10.1007/978-1-0716-4550-5_9.
DNA topoisomerase II (TOP2) regulates DNA topological states including supercoiling and knotting via a strand-passage reaction that involves transiently breaking and rejoining both strands of the DNA. These enzymes are the targets of a category of drugs termed topoisomerase poisons, which block the rejoining reaction, leading to DNA cleavage. In vitro DNA cleavage assays have been very useful in studying various properties of TOP2, including cleavage site preferences and the effects of TOP2 amino acid substitutions or reaction conditions on DNA cleavage. These assays employ purified or recombinant TOP2 in the presence or absence of TOP2 poisons, followed by electrophoretic separation of DNA cleavage products. The substrates for these assays have typically been radioactively end-labelled DNA fragments. Here, we describe an alternative method employing fluorescently labelled oligonucleotide substrates, combined with convenient mini-gel electrophoretic separation. This methodology combines stable, long-lived substrates with an easily used gel system and convenient imaging and quantification.
DNA拓扑异构酶II（TOP2）通过链通过反应调节DNA拓扑状态，包括超螺旋和打结，该反应涉及DNA两条链的瞬时断裂和重新连接。这些酶是一类称为拓扑异构酶毒物的药物的作用靶点，这些药物会阻断重新连接反应，导致DNA裂解。体外DNA裂解试验在研究TOP2的各种特性方面非常有用，包括裂解位点偏好以及TOP2氨基酸取代或反应条件对DNA裂解的影响。这些试验在有或没有TOP2毒物的情况下使用纯化的或重组的TOP2，然后对DNA裂解产物进行电泳分离。这些试验的底物通常是放射性末端标记的DNA片段。在这里，我们描述了一种替代方法，该方法使用荧光标记的寡核苷酸底物，并结合方便的微型凝胶电泳分离。这种方法将稳定、寿命长的底物与易于使用的凝胶系统以及方便的成像和定量相结合。
