DNA binding mode of class-IIS restriction endonuclease FokI revealed by DNA footprinting analysis

We investigate the interaction of FokI with its DNA recognition sequence by several footprinting techniques. Methylation of three guanine bases in the recognition sequence 5'-GGATG-3' is strongly protected by FokI binding, whereas other guanine bases are not masked from the modification. In footprinting using the methidiumpropyl-EDTA-Fe(II) complex, binding of FokI strongly inhibits cleavage by the footprinting reagent at and near the recognition sequence. In high-resolution footprinting techniques using hydroxyl radical and the bleomycin-Fe(II) complex, all footprints in each binding site clearly face one side of the DNA helix. Interference analysis with FokI digestion by preethylation of phosphate groups suggests that essential phosphates for FokI digestion are located at and near the recognition sequence and the cleavage site. Evidently, the results indicate that (i) the sequence-recognition of FokI occurs in the major groove and that (ii) the enzyme interacts with its target DNA from one side of the DNA helix.