Structural Requirements forFokI-DNA Interaction and Oligodeoxyribonucleotide-instructed Cleavage

Abstract The Fok I restriction endonuclease recognizes the double-stranded (ds) 5′-GGATG-3′ site and cuts at the 9th and 13th nucleotides downstream from the 5′-3′ and 3′-5′ strands, respectively. To elucidate the interaction between Fok I and DNA, and the effect of Mg 2+ on this interaction, we used Fok I with various combinations of dsDNA, single-stranded (ss) DNA and oligodeoxyribonucleotides (oligos) containing a double-stranded hairpin carrying the Fok I recognition site. Oligo- and dsDNA- Fok I interactions showed that for fully effective recognition, two or more base-pairs were required outside the 5′-GGATG-3′ site. When using Fok I with ssDNA and oligos, precise cutting with no observable byproducts was observed at the 9th or 13th nucleotide. This was independent of whether the region between the recognition and cut sites was perfectly complementary or whether there were up to four mismatches in this region, or a single mismatch within the cut site. Moreover, Fok I cleavage, when followed by step-wise filling-in of Fok I cohesive ends in the dsDNA, allowed Fok I to recleave such sites when two or more nucleotides were added, releasing 2-mer, 3-mer, or 4-mer single-stranded chains. Electrophoretic mobility shift assays showed that the DNA helix was bent when complexed with Fok I (without Mg 2+ ). Such a complex, when formed in the absence of Mg 2+ , did not accept the subsequently added Mg 2+ for several minutes. This suggests a tight, diffusion-resistant contact between the enzyme and the cognate DNA sequence. In the presence of Mg 2+ , the half-life of the complex Fok I and dsDNA was 12 minutes at 22°C. In the absence of Mg 2+ , such a complex, possessing a terminally located 5′-GGATG-3′ site, had a half-life of 1.5 to 2 minutes. However, if magnesium ions were present, this complex had a stability similar to that of a complex formed with dsDNA containing a centrally located 5′-GGATG-3′ site.