Division of Oncology, Department of Medicine, Washington University in St. Louis, St. Louis, MO, United States.
Division of Oncology, Department of Medicine, Washington University in St. Louis, St. Louis, MO, United States.
Methods Enzymol. 2022;672:369-381. doi: 10.1016/bs.mie.2022.03.036. Epub 2022 Apr 11.
DNA topoisomerases resolve topological stress by introducing transient single- or double-strand breaks into the DNA duplex. This reaction requires the covalent binding of topoisomerases to DNA while the topological stress is being released. This transient intermediate is known as topoisomerase-covalent complex and represents the target of many anti-cancer drugs. Here, we describe a protocol to quantitatively detect topoisomerase-covalent complexes in vivo, called RADAR (rapid approach to DNA adduct recovery). DNA and protein-DNA covalent complexes are rapidly isolated from cells through chaotropic extraction. After normalization, samples are loaded on a slot blot, and the covalent complexes are detected using specific topoisomerase antibodies. In addition to being fast and robust, this assay produces quantitative results. Consequently, the RADAR assay can be applied to investigate the topoisomerase-covalent complex biology, including the effect of specific topoisomerase inhibitors. Finally, the same assay can be more generally applied to study covalent complexes of other enzymes with DNA.
DNA 拓扑异构酶通过在 DNA 双链中引入瞬时单链或双链断裂来解决拓扑结构应力。该反应需要拓扑异构酶与 DNA 的共价结合，同时释放拓扑结构应力。这种瞬时中间产物称为拓扑异构酶共价复合物，是许多抗癌药物的作用靶点。在这里，我们描述了一种在体内定量检测拓扑异构酶共价复合物的方法，称为 RADAR（快速 DNA 加合物回收方法）。通过剧烈提取，快速从细胞中分离 DNA 和蛋白-DNA 共价复合物。归一化后，将样品加载到狭缝印迹上，并用特定的拓扑异构酶抗体检测共价复合物。除了快速和稳健之外，该测定还产生定量结果。因此，RADAR 测定法可用于研究拓扑异构酶共价复合物生物学，包括特定拓扑异构酶抑制剂的作用。最后，相同的测定法可更一般地应用于研究其他与 DNA 结合的酶的共价复合物。
