拓扑异构酶（TOP1、TOP2α和β）是核酶，在DNA代谢的几乎所有方面都起着关键作用。它们也是重要的抗肿瘤化疗药物的靶点，这些药物通过困住可逆拓扑异构酶-DNA共价复合物中间体（TOPccs）来发挥作用，在催化反应期间形成长寿命拓扑异构酶DNA蛋白交联物（TOP-DPCs），从而干扰DNA交互作用。泛素连接酶ADP核糖基多聚合酶（PARP）家族蛋白PARP1在DNA损伤后被激活，以招募DNA修复蛋白，而PARP抑制剂是另一类常用化疗药物，可以与DNA上的PARP分子结合和困住。到目前为止，TOPccs和PARP由它们各自的抑制剂困住只能通过细胞内的免疫生化方法来测量。在这里，我们开发了一种基于成像的方法，能够实时监测药物诱导的TOPccs和PARP1在活细胞中的单分子水平的困住。利用这种方法，我们计算了药物在实时中困住标记了自荧光标签的拓扑异构酶和PARP单分子的比例。这种新颖的技术应有助于阐明修复TOPcc和PARP困住的分子过程，并促进新型拓扑异构酶和PARP抑制剂为基础的治疗方法的开发。由牛津大学出版社代表核酸研究出版于2023年。

Topoisomerases (TOP1, TOP2α, and β) are nuclear enzymes crucial for virtually all aspects of DNA metabolisms. They also are the targets of important anti-tumor chemotherapeutics that act by trapping the otherwise reversible topoisomerase-DNA covalent complex intermediates (TOPccs) that are formed during their catalytic reactions, resulting in long-lived topoisomerase DNA-protein crosslinks (TOP-DPCs) that interfere with DNA transactions. The Poly(ADP-ribose) polymerase (PARP) family protein PARP1 is activated by DNA damage to recruit DNA repair proteins, and PARP inhibitors are another class of commonly used chemotherapeutics, which bind and trap PARP molecules on DNA. To date, the trapping of TOPccs and PARP by their respective inhibitors can only be measured by immune-biochemical methods in cells. Here, we developed an imaging-based approach enabling real-time monitoring of drug-induced trapping of TOPccs and PARP1 in live cells at the single-molecule level. Capitalizing on this approach, we calculated the fraction of self-fluorescence tag-labeled topoisomerases and PARP single-molecules that are trapped by their respective inhibitors in real time. This novel technique should help elucidate the molecular processes that repair TOPcc and PARP trapping and facilitate the development of novel topoisomerase and PARP inhibitor-based therapies.Published by Oxford University Press on behalf of Nucleic Acids Research 2023.