A NOVEL CLASS OF FOKI RESTRICTION-ENDONUCLEASE MUTANTS THAT CLEAVE HEMI-METHYLATED SUBSTRATES

A genetic screen was used to identify amino acid substitutions that en able the FokI restriction endonuclease to cleave DNA in cells that exp ress the cognate methyltransferase activity. Missense mutations that g ive rise to this phenotype were isolated at eight different positions (G188K, P196S, T343I, S388N, S395F, E407K, E410K, D421N), clustered in two regions of the polypeptide sequence of FokI. Two of the mutant en donucleases (P196S and D421N) were purified to homogeneity and analyze d in detail. Both mutants cleave FokI target sites (5'-GGATG-3') in a manner similar to the wild-type enzyme. Neither mutant cleaved noncano nical sequences, but both efficiently cleaved DNA substrates containin g hemi-methylated FokI sites. This class of mutations has not been obs erved with other restriction enzymes.