Rotation tracking of genome-processing enzymes using DNA origami rotors

Many genome-processing reactions, such as transcription, replication and repair, generate DNA rotation. Methods that directly measure DNA rotation, including rotor bead tracking(1–3), angular optical trap(4), and magnetic tweezers(5) have helped unravel the action mechanisms of a range of genome-processing enzymes, such as RNA polymerase (RNAP)(6), gyrase(2), viral DNA packaging motor(7), and DNA recombination enzymes(8). However, despite the potential of rotation measurements to transform our understanding of genome-processing reactions, measuring DNA rotation remains a difficult task. The time resolution of existing methods is insufficient to track rotation induced by many enzymes under physiological conditions, and the measurement throughput is typically low. Here we introduce Origami-Rotor-Based Imaging and Tracking (ORBIT), a method that uses fluorescently labeled DNA origami rotors to track DNA rotation at the single-molecule level with millisecond time resolution. We used ORBIT to track DNA rotation resulted from unwinding by RecBCD, a helicase involved in DNA repair(9), and transcription by RNAP. We characterized a series of events during RecBCD-induced DNA unwinding, including initiation, processive translocation, pausing and backtracking, and revealed an initiation mechanism that involves reversible, ATP-independent DNA unwinding and engagement of the RecB motor. During transcription by RNAP, we directly observed rotational steps corresponding to single-base-pair unwinding. We envision ORBIT will enable studies of a wide range of protein-DNA interactions.