Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.
A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.
Kim
Y G
YG
Department of Environmental Health Sciences, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205-2179, USA.
Cha
J
J
Chandrasegaran
S
S
D010582:Bacteriophage lambda; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004279:DNA, Viral / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity; D016335:Zinc Fingers
D017931:DNA Primers; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific