Rotation tracking of genome-processing enzymes using DNA origami rotors

• Published in: Nature (London) 2019-08, Vol.572 (7767), p.136-140
• Main Authors: Kosuri, Pallav, Altheimer, Benjamin D., Dai, Mingjie, Yin, Peng, Zhuang, Xiaowei
• Format: Article
• Language: English
• Subjects: 631/1647/2204; 631/337/1644; 631/337/1645; 631/57/2265; 631/57/2272; Base Pairing; Base pairs; Deoxyribonucleic acid; DNA; DNA - analysis; DNA - chemistry; DNA - metabolism; DNA biosynthesis; DNA Breaks, Double-Stranded; DNA helicase; DNA Helicases - metabolism; DNA repair; DNA-directed RNA polymerase; DNA-Directed RNA Polymerases - metabolism; E coli; Enzymes; Exodeoxyribonuclease V - metabolism; Genome - genetics; Genomes; Humanities and Social Sciences; Letter; Measurement methods; Mechanical properties; Molecular rotation; multidisciplinary; Nanotechnology; Nucleic Acid Conformation; Observations; Optical trapping; RecBCD; Recombination; Repair; RNA polymerase; RNA processing; Rotation; Rotors; Science; Science (multidisciplinary); Structure; Tracking; Transcription; Transcription, Genetic; Translocation; Unwinding
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/s41586-019-1397-7

Many genome-processing reactions, including transcription, replication and repair, generate DNA rotation. Methods that directly measure DNA rotation, such as rotor bead tracking 1 – 3 , angular optical trapping 4 and magnetic tweezers 5 , have helped to unravel the action mechanisms of a range of genome-processing enzymes that includes RNA polymerase (RNAP) 6 , gyrase 2 , a viral DNA packaging motor 7 and DNA recombination enzymes 8 . 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 for tracking the 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 labelled DNA origami rotors to track DNA rotation at the single-molecule level with a time resolution of milliseconds. We used ORBIT to track the DNA rotations that result from unwinding by the RecBCD complex, a helicase that is involved in DNA repair 9 , as well as from transcription by RNAP. We characterized a series of events that occur 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 that correspond to the unwinding of single base pairs. We envisage that ORBIT will enable studies of a wide range of interactions between proteins and DNA. ORBIT (origami-rotor-based imaging and tracking) is used to track the DNA rotation that results from DNA unwinding by RecBCD helicase and transcription by RNAP at a single-molecule scale and millisecond time resolution.