|dc.description.abstract||The rotation of DNA is an intrinsic part of many protein activities. Many DNA motor proteins, including helicases, translocases, and polymerases, rotate their DNA substrate due to DNA’s helical structure. Others induce DNA rotation by distorting the DNA upon binding or introduce turns as their primary enzymatic activity. Additionally, in the constrained environment of the cell, DNA rotation affects the buildup of torsional strain, which can influence the activity of many proteins. The direct measurement of single-molecule DNA rotation can serve as a readout of these protein activities, creating opportunities to study mechanism and regulation. However, existing methods are limited in resolution, throughput, and by certain experimental requirements. In this dissertation, I describe the development of Origami-Rotor-Based Imaging and Tracking (ORBIT), a new approach for direct, single-molecule measurement of DNA rotation at high resolution and throughput.
ORBIT uses structural amplification to turn the very small movement associated with DNA rotation into a signal measurable using fluorescence tracking. Specifically, we use fluorescently labeled DNA origami rotors to report on the rotation of double-stranded DNA that extends perpendicular to the structure. High resolution is achieved by minimizing Brownian noise with a small origami rotor and short double-stranded DNA. ORBIT does not require applying a stretching force or a biasing torque to the DNA. We demonstrate that ORBIT is capable of resolving rotation equivalent to the twist between adjacent base pairs with an integration time of only ~20 ms.
We have applied ORBIT for three biological applications. First, by measuring the Brownian dynamics of origami rotors tethered to a coverslip surface by double-stranded DNA, we determined the torsional rigidity of DNA in the absence of applied force. This value determines how twist and torque are transmitted through DNA and influences how the activity of one protein can affect another. Second, we applied ORBIT to the homologous recombination enzyme RecBCD during initiation and processive unwinding of DNA. We characterize the pausing and backtracking behaviors of the enzyme, finding evidence of two distinct pausing states during processive unwinding. Furthermore, with ORBIT, we are able to track activity starting from DNA binding, allowing us to observe the series of events during initiation on double-stranded breaks with variable end geometries. We find reversible, ATP-independent 5 bp transitions between wound and unwound states during initiation, and that the engagement of RecB with the 3' strand is important for initiation. Third, we track the rotation of DNA induced by RNA polymerase (RNAP) during transcription, and directly detect single base-pair steps in the rotational movement of the DNA, indicating that RNAP tracks the double helix at this short length scale. We anticipate that ORBIT will be useful for studying DNA rotation in many contexts.||