Single-molecule methods that allow for the precise application of forces and torques on individual DNA molecules have transformed our understanding of topoisomerase dynamics. These methods provide unparalleled information about the sequence of mechanical events that take place within topoisomerases, how and when these machines apply controlled torques to reconfigure DNA, and how these events are temporally correlated with supercoiling dynamics. These approaches have been applied across a wide range of experimental conditions with a diverse collection of drugs with important therapeutic implications. However, limited throughput remains a major challenge that often prevents detailed comparisons of different enzyme types, mutants, and activity rates in a reasonable amount of time. Moreover, isolating and characterizing rare intermediates has remained exceedingly difficult. Flow Magnetic Tweezers (FMTs) overcome these challenges by the introduction of a controlled laminar flow to traditional magnetic tweezers. This provides a 100-fold improvement in throughput by allowing for DNA length changes to be tracked as lateral bead displacements at lower magnification. In studies with gyrase, this approach has revealed rare breaks in DNA correlated with DNA relaxation events. Here, we present ongoing work adapting this technique to study human topoisomerase dynamics. Our data reveal significant differences in break frequencies among human topoisomerase subtypes and specific mutations, highlighting their impact on enzymatic function and genomic stability. The high-throughput and single-molecule resolution of FMT promise to uncover previously inaccessible mechanistic insights into topoisomerase dysfunction, enabling the identification of novel drug targets and therapeutic strategies for diseases linked to topological defects, such as cancer and neurodegenerative disorders. Overall, FMT provide an unprecedented combination of throughput and precision, establishing them as a powerful tool for studying topoisomerases, detecting rare enzymatic events, and accelerating the development of precision therapeutics.
Filipovic et al. (Sun,) studied this question.