A kinetic clutch governs religation by type IB topoisomerases and determines camptothecin sensitivity

摘要

Type IB topoisomerases (TopIBs) relax excessive DNA supercoiling associated with replication and transcription by catalyzing a transient nick in one strand to permit controlled rotation of the DNA about the intact strand. The natural compound camptothecin (CPT) and the cancer chemotherapeutics derived from it, irinotecan and topotecan, are highly specific inhibitors of human nuclear Top1B (nTopi). Previous work on vaccinia Top1B led to an elegant model that describes a straightforward dependence of rotation and religation on the torque caused by supercoiling. Here, we used a single-molecule DNA supercoil relaxation assay to measure the torque dependence of nTopi and its inhibition by CPT. For comparison, we also examined mitochondrial Top1B and an N-terminal deletion mutant of nTopi. Despite substantial sequence homology in their core domains, nTopi and mitochondrial Top1B exhibit dramatic differences in sensitivity to torque and CPT, with the N-terminal deletion mutant of nTopi showing intermediate characteristics. In particular, nTopi displays nearly torque-independent religation probability, distinguishing it from other Top1B enzymes studied to date. Kinetic modeling reveals a hitherto unobserved torque-independent transition linking the DNA rotation and religation phases of the enzymatic cycle. The parameters of this transition determine the torque sensitivity of religation and the efficiency of CPT binding. This "kinetic clutch" mechanism explains the molecular basis of CPT sensitivity and more generally provides a framework with which to interpret Top1B activity and inhibition.