Type IB topoisomerases (Top1Bs) 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 (nTop1). 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 nTop1 and its inhibition by
CPT. For comparison, we also examined mitochondrial Top1B and an N-terminal deletion mutant of nTop1. Despite substantial sequence homology in their core domains, nTop1 and mitochondrial Top1B exhibit dramatic differences in sensitivity to torque and
CPT, with the N-terminal deletion mutant of nTop1 showing intermediate characteristics. In particular, nTop1 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.