Conversion of the FokI endonuclease to a universal restriction enzyme: cleavage of phage M13mp7 DNA at predetermined sites.
Endonuclease FokI belongs to class IIS of restriction enzymes, for which the DNA cut points lie outside the enzyme-recognition sites. This permitted conferring new cleavage specificities by combining FokI with tailored oligodeoxynucleotide adapters. Such adapters carry a single-stranded (ss) target-recognition domain, complementary to the selected ss target DNA, and a double-stranded (ds) enzyme-recognition site. Neither enzyme nor adapter alone has endonucleolytic activity toward phage M13mp7 ss DNA, whereas the enzyme-adapter complex cleaves this ss target DNA at the particular sites foreordained by the sequence of the ss domain of the adapter. Two kinds of adapters (32 and 34 nucleotides long), with opposing orientations of the asymmetric FokI recognition site, were constructed and shown to direct specific cleavage under a variety of conditions. In addition to FokI, other class IIS enzymes, HphI, MboII and BbvI, which alone do not cleave ss DNA, are suitable for construction of tailored enzyme-adapter complexes with predictable cleavage specificities. This report provides a preliminary experimental confirmation for the proposal of Szybalski [Gene 40 (1985) 169-173] for the design of adapter-enzyme complexes with novel and predictable specificities. Theoretically, using this approach any sequence could be precisely cleaved at a predetermined point.
D001483:Base Sequence; D003090:Coliphages; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli; D005822:Genetic Vectors; D007700:Kinetics; D009994:Osmolar Concentration; D013379:Substrate Specificity
D004279:DNA, Viral; D004262:DNA Restriction Enzymes; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific