| Recent developments in single-molecule manipulation of DNA have allowed researchers to test fundamental theories of polymer elasticity and directly measure mechanical properties of DNA while providing the conceptual and experimental basis for studying molecular motors that act on DNA. In this thesis, I report on the development and application of the rotor bead tracking technique, a novel single-molecule assay designed to study the twist and torque of DNA. This technique involves the attachment of a small "rotor" bead just below a single strand nick in the middle of a stretched DNA molecule. The rotor bead spins in response to torque generated in the lower DNA segment.;By measuring the magnitude of the angular fluctuations in the rotor bead we have been able to determine the twist rigidity of DNA in a variety of different buffers and over a range of tensions. We conclude that the torsional rigidity of DNA is C = 460 +/- 20 pNnm2, about 50% larger than most previous estimates. Although DNA can be modeled well as an isotropic rod, the double helical structure of DNA suggests that some chiral mechanical properties should exist. In particular, simple physical intuition predicts that stretching DNA should pull the molecule towards a denatured structure, which is unwound with respect to canonical B-form DNA. We have directly measured this twist-stretch coupling and have found that DNA overwinds under tension, with a 1% stretching of DNA leading to an increase in the twist of ∼0.1%. A simple physical model explains this anamolous behavior.;We also employed the rotor bead tracking technique to study the mechanochemical cycle of DNA gyrase, a molecular machine that uses the energy of ATP hydrolysis to introduce essential negative supercoils into DNA. In the presence of gyrase and ATP, we observe bursts of rotation of the rotor bead corresponding to the processive, stepwise introduction of negative supercoils in strict multiples of two. In a high-resolution variant of our assay, we directly detect rotational pauses corresponding to two kinetic substeps: an ATP-independent step at the end of the reaction cycle and an ATP-binding step in the middle of the cycle, subsequent to DNA wrapping.;The rotor bead tracking technique has proven to be a general and powerful probe with which to study DNA and its interaction with proteins. In the final chapter, I briefly discuss several other systems that we have begun to study, including the binding/unbinding of small molecules to DNA and translocation by motors such as RNA polymerase. |
