STRUCTURAL REQUIREMENTS FOR FOKI-DNA INTERACTION AND OLIGODEOXYRIBONUCLEOTIDE-INSTRUCTED CLEAVAGE

The FokI restriction endonuclease recognizes the double-stranded (ds) 5'-GGATG-3' site and cuts at the 9th and 13th nucleotides downstream f rom the 5'-3' and 3'-5' strands, respectively. To elucidate the intera ction between FokI and DNA, and the effect of Mg2+ on this interaction , we used FokI with various combinations of dsDNA, single-stranded (ss ) DNA and oligodeoxyribonucleotides (oligos) containing a double-stran ded hairpin carrying the FokI recognition site. Oligo- and dsDNA-FokI interactions showed that for fully effective recognition, two or more base-pairs were required outside the 5'-GGATG-3' site. When using FokI with ssDNA and oligos, precise cutting with no observable byproducts was observed at the 9th or 13th nucleotide. This was independent of wh ether the region between the recognition and cut sites was perfectly c omplementary or whether there were up to four mismatches in this regio n, or a single mismatch within the cut site. Moreover, FokI cleavage, when followed by step-wise filling-in of FokI cohesive ends in the dsD NA, allowed FokI to recleave such sites when two or more nucleotides w ere added, releasing 2-mer, 3-mer, or 4-mer single-stranded chains. El ectrophoretic mobility shift assays showed that the DNA helix was bent when complexed with FokI (without Mg2+). Such a complex, when formed in the absence of Mg2+ did not accept the subsequently added Mg2+ for several minutes. This suggests a tight, diffusion-resistant contact be tween the enzyme and the cognate DNA sequence. in the presence of Mg2, the half-life of the complex FokI and dsDNA was 12 minutes at 22 deg rees C. In the absence of Mg2+, such a complex, possessing a terminall y located 5'-GGATG-3' site, had a half-life of 1.5 to 2 minutes. Howev er, if magnesium ions were present, this complex had a stability simil ar to that of a complex formed with dsDNA containing a centrally locat ed 5'-GGATG-3' site. (C) 1996 Academic Press Limited