Van Ravenstein Sabrina X, Dewar James M
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
Methods Mol Biol. 2025;2928:151-172. doi: 10.1007/978-1-0716-4550-5_13.
Topoisomerases unlink chromosomes and relieve topological stress. Topoisomerase "poisons" are widely used chemotherapeutics that stabilize topoisomerase complexes on DNA, leading to cytotoxic DNA breaks and cancer cell killing. It is well established that topoisomerase poisons interfere with DNA replication, which is thought to be a major physiological target of these drugs. However, many questions remain about the mechanisms by which topoisomerase poisons impact DNA replication and the downstream consequences. Here, we describe assays to study topoisomerase poisons during vertebrate DNA replication using Xenopus egg extracts. These approaches allow for replication intermediates formed following poison treatment to be carefully monitored with high temporal resolution.
拓扑异构酶解开染色体并缓解拓扑压力。拓扑异构酶“毒药”是广泛使用的化疗药物，可使拓扑异构酶复合物稳定在DNA上，导致细胞毒性DNA断裂并杀死癌细胞。众所周知，拓扑异构酶毒药会干扰DNA复制，这被认为是这些药物的主要生理靶点。然而，关于拓扑异构酶毒药影响DNA复制的机制及其下游后果仍有许多问题。在这里，我们描述了使用非洲爪蟾卵提取物在脊椎动物DNA复制过程中研究拓扑异构酶毒药的实验方法。这些方法能够以高时间分辨率仔细监测毒药处理后形成的复制中间体。
