DNA-BINDING MODE OF CLASS-IIS RESTRICTION-ENDONUCLEASE FOKI REVEALED BY DNA FOOTPRINTING ANALYSIS

We investigate the interaction of FokI with its DNA recognition sequen ce by several footprinting techniques. Methylation of three guanine ba ses in the recognition sequence 5/-GGATG-3' is strongly protected by F okI binding, whereas other guanine bases are not masked from the modif ication. In footprinting using the methidiumpropyl-EDTA-Fe(II) complex , binding of FokI strongly inhibits cleavage by the footprinting reage nt at and near the recognition sequence. In high-resolution footprinti ng 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 p hosphate 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 wi th its target DNA from one side of the DNA helix.