Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain

Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1156-60. doi: 10.1073/pnas.93.3.1156.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriophage lambda
  • Base Sequence
  • Cloning, Molecular
  • DNA Primers
  • DNA, Viral / metabolism
  • Deoxyribonucleases, Type II Site-Specific / isolation & purification
  • Deoxyribonucleases, Type II Site-Specific / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Polymerase Chain Reaction
  • Protein Multimerization*
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism*
  • Restriction Mapping
  • Substrate Specificity
  • Zinc Fingers*


  • DNA Primers
  • DNA, Viral
  • Recombinant Fusion Proteins
  • endodeoxyribonuclease FokI
  • Deoxyribonucleases, Type II Site-Specific