Single-Molecule Assessment of DNA Supercoiling and Relaxation by S. Typhimurium and E. Coli DNA Gyrases

Abstract

Type II topoisomerases maintain DNA topology by regulating the level of supercoiling of chromosomes. DNA gyrase is a unique and highly conserved bacterial Type II topoisomerase which is able to introduce negative supercoils into the genome. Using magnetic-tweezers, we assessed the supercoil generation and relaxation activities of Escherichia coli and Salmonella typhimurium DNA gyrases. Our results indicate that under single-enzyme conditions and 0.6-pN tension, both enzymes relax DNA at similar rates, but Salmonella gyrase pauses more often. At high enzyme concentration and lower tensions, Salmonella gyrase introduced negative supercoils faster and to a larger extent than E. coli gyrase. Sequence and structure analyses show that most of the differences between the two enzymes are in the C-terminal domain, involved in DNA wrapping. Our ongoing single-molecule experiments to assess the DNA wrapping activities of both enzymes may reveal differences in their mechanistic properties.View Large Image | View Hi-Res Image | Download PowerPoint Slide