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

TLDR

The field seeks to engineer restriction enzymes with novel sequence specificities by mutating existing enzymes. The authors aim to create new site‑specific endonucleases by fusing zinc finger proteins to the Fok I cleavage domain, thereby obviating extensive microbial screening. They link two distinct zinc finger proteins to the Fok I cleavage domain to generate the novel endonucleases. Both fusion proteins are active and cleave DNA in a sequence‑specific manner under optimal conditions, proving that the modular Fok I structure and zinc finger motifs can produce artificial nucleases that cut near predetermined sites and enabling the creation of many tailor‑made enzymes.

Abstract

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.