Detection of DNA looping due to simultaneous interaction of a DNA-binding protein with two spatially separated binding sites on DNA.

We describe different and relatively rapid biochemical techniques to detect protein-mediated DNA looping. These techniques, based on enhancement of DNA knotting and that of ligase-catalyzed cyclization, were used to show that the replication initiator protein of plasmid R6K can bring together two intramolecular gamma origin of replication sequences located as far apart as 2 kilobases. The site-site interaction causes looping out of the intervening DNA sequence as visualized by electron microscopy. Because the autoregulatory sequence of the initiator cistron also binds initiator protein, we investigated whether the gamma origin-bound protein can participate in autoregulation by interaction of the two sites through a protein bridge. We discovered that the two sites do not interact in vitro at their natural locations when on opposite faces of the double helix. Moving the two sites to the same face of the double helix by introducing a half turn into the intervening sequence allows protein-mediated site-site interaction to occur.