Hairpin-loop formation by inverted repeats in supercoiled DNA is a local and transmissible property

Short inverted repeat sequences adopt hairpin stem-loop type structures in supercoiled closed circular DNA molecules, demonstrated by S1 nuclease cleavage. Fine mapping of cleavage frequencies is in good agreement with expected cleavage patterns based upon the interaction between an unpaired loop and a sterically bulky enzyme molecule. Whilst the topological properties of underwound DNA circles depend ultimately upon reduced linkage, necessarily a global molecular property, hairpin loop formation is an essentially local property. Thus molecular size is unimportant for the S1 hypersensitivity of the Co1E1 inverted repeat. Furthermore, a 440 bp Sau3AI, EcoRI fragment of Co1E1 which contains the inverted repeat has been cloned into pBR322 whereupon it exhibits S1 cleavage similar to Co1E1 in the supercoiled recombinant molecule. The effect is therefore both local and transmissible. Direct competition, between inverted repeats in the recombinant, coupled with close examination of flanking sequences, enables some simple 'rules' for base pairing in hairpin loops to be formulated. Whilst limited G-T and A-C base pairing appears not to be destabilising, A-G, T-C or loop outs are highly destabilising.