INSERTION AND DELETION MUTANTS OF FOKI RESTRICTION-ENDONUCLEASE

FokI restriction endonuclease recognizes the nonpalindromic pentadeoxy ribonucleotide, 5'-GGATG-3':5'CATCC-3' in duplex DNA and cleaves 9 and 13 nucleotides away from the recognition site, We have reported the p resence of two distinct and separable protein domains within this enzy me: one for the sequence-specific recognition of DNA (the DNA binding domain) and the other for the endonucleases activity (the cleavage dom ain), Our studies have suggested that the two domains are connected by a linker region, which appears to be amenable for repositioning of th e DNA-sequence recognition domain with respect to the catalytic domain , Here, we report the construction of several insertion (4-, 8-, 12-, 18-, 19-, or 23-amino acid residues) and deletion (4- or 7-amino acid residues) mutants of the linker region of FokI endonuclease. The mutan t enzymes were purified, and their cleavage properties were characteri zed, The mutants have the same DNA sequence specificity as the wild-ty pe enzyme, However, compared with the wild-type enzyme, the insertion mutants cleave predominantly one nucleotide further away from the reco gnition site on both strands of the DNA substrate, The four-codon dele tion mutant shows relaxed specificity at the cut site while the seven- codon deletion appears to inactivate the enzyme, The DNA binding and c leavage domains of FokI appear to be Linked by a relatively malleable linker, No simple linear relationship exists between the linker length and the distance of the cut site from the recognition site, Furthermo re, the four-codon insertion mutants cleave DNA substrates containing hemi-methylated FokI sites; they do not cleave fully methylated substr ates, These results are best explained as a consequence of protein-pro tein interactions between the domains.