Enhancer-origin interaction in plasmid R6K involves a DNA loop mediated by initiator protein

Abstract

Initiation of DNA replication from on β of plasmid R6K requires the presence of the on γ sequence in cis. We demonstrate that binding of initiator protein to the seven strong, tandem binding sites in γ increases binding of the protein at the very weak binding site present in on β by cooperativity at a distance. The γ-β interaction via the initiator results in a DNA loop, as revealed by the novel technique of cyclization enhancement and as confirmed by exonuclease III protection, electron microscopy, and chemical footprinting. The protein-mediated γ-β interaction in vitro suggests that the cooperative interaction of γ-bound protein with the β sequence by DNA looping is an early step in the initiation of DNA replication at the β origin of R6K.