Single molecule studies of DNA twisting and the mechanism of type II topoisomerases.

摘要

The elastic and topological properties of DNA are vital to its biological function. In order to access the information of the genetic code, cellular enzymes must bend and twist DNA to promote unwinding. A specialized class of enzymes called topoisomerases must untwist and unlink DNA at various stages of the cell cycle, facilitating DNA replication, transcription, and repair. The research within this thesis focuses upon the study of the elastic and topological properties of DNA, with particular emphasis on the catalytic mechanism of topoisomerases.;The first chapter is a general review of the basic principles of DNA topology, DNA replication and unlinking, topoisomerases, and emerging single molecule biophysical methods. In the second chapter, I describe an optical tweezers based DNA twisting apparatus, capable of applying specific amounts of tension and twist to single DNA molecules. With this system, I investigated the mechanism underlying the chiral substrate preference of Escherichia coli topoisomerase IV. I found that topo IV recognizes the chiral crossings imposed by the left-handed superhelix of a (+) supercoiled DNA, rather than global topology, twist deformation, or local writhe. Single-enzyme experiments also provided a direct measure of the processivity of the enzyme and offered insight into its mechanochemical cycle.;In chapter III, I describe a system in which torsional strain in over- or underwound molecules was used to power the rotation of submicron beads serving as calibrated loads. These experiments tested the linearity of DNA's twist elasticity, directly measured the torsional modulus (finding a value 40% higher than generally accepted), characterized torque-induced structural transitions, and established a framework for future assays of torque and twist generation by DNA-dependent enzymes.;Chapter IV describes the design, construction, and implementation of a magnetic tweezers system for low force single molecule DNA twisting experiments. I present data reproducing the well-established elastic properties of relaxed and supercoiled DNA, serving as a demonstration of the magnetic tweezers functionality. Finally, I present data from a novel magnetic tweezers assay designed to study the mechanism of Escherichia coli DNA gyrase.