Dynamics of cruciform extrusion in supercoiled DNA: use of a synthetic inverted repeat to study conformational...

An inverted repeat has been created in a plasmid by ligation of two 13 nucleotide synthetic oligonucleotides into the cloning vector pAT153. The resulting recombinant plasmid, pIRbke8, is hypersensitive to cleavage by the single-strand-specific nuclease S1, and to modification by the single-strand-selective reagent bromoacetaldehyde, when the plasmid is negatively supercoiled. The new inverted repeat is a stronger S1 site than those derived from pBR322, but, in contrast to the ColE1 and phi X174 RF inverted repeats, these repeats share a similar temperature dependence. The kinetics of EcoRI cleavage at the centre of the synthetic inverted repeat have been studied in supercoiled and linear molecules. It is found that in the supercoiled molecule this target is not refractory to EcoRI cleavage to an extent which is greater than the resolution of the experiment. We conclude that in this molecule the cruciform is in a dynamic equilibrium with the regular duplex, in which the cruciform constitutes a relatively small subpopulation of conformational species.