Engineering Multiple Site-Specific Modifications in Supercoiled DNAs

New techniques are needed in order to study the effects of DNA supercoiling on loop-mediated regulation. The thermodynamics of DNA looping depend periodically on the helical phasing between recognition sites. A long-term goal is to provide enabling technologies for ensemble and single-molecule FRET studies of DNA looping mediated by lac repressor in vitro and in vivo. Other applications of the methodology include engineering of DNA substrates for studies of site-specific recombination and other enzyme systems.