Topological approaches to studies of protein-mediated looping of DNA in vivo

This chapter presents topological approaches for studying the protein-mediated looping of DNA in vivo. It describes methods for studying protein-mediated looping of DNA in vivo using the lac repressor–operator complex as a model system. The tetrameric lac repressor has been shown to bind simultaneously to two operators both in vitro and in vivo. The simultaneous binding of the lac repressor to two operators on the same DNA molecule causes looping of the intervening DNA. Such lac repressor-mediated looping of DNA interferes with template supercoiling during RNA transcription and can cause changes in the supercoiled state of plasmid DNAs. The looping of DNA due to lac repressor binding requires the presence of at least two lac repressor-binding sites on the same molecule. For plasmid DNAs containing only a single lac repressor binding site, only the multimeric forms of the plasmid DNAs can be efficiently looped by the tetrameric lac repressor complex. The preferential supercoiling of the multimeric forms of the plasmid DNAs can, therefore, be a good indication of protein-mediated looping of DNA in vivo.