Journal of Biological Chemistry

A series of biochemical assays were developed and performed to monitor the molecular events that occur during the Hin-mediated DNA inversion reaction. These events can be divided into five different stages: 1) binding of proteins (Hin, Fis, and HU) to DNA; 2) pairing of Hin-binding sites; 3) invertasome formation; 4) DNA strand cleavage; 6) strand rotation and reli-gation. A series of topoisomers of the wild type DNA substrate plasmid (ranging from fully relaxed mole-cules to those with more than the physiological super-helical density (the physiological superhelical density of pKH336 from Escherichia coli DHlOB ” is-0.072 in this study)) was generated, and the role of negative supercoiling in each step of the inversion reaction was investigated. We found differences in the dependence