Chimeric Restriction Enzyme: Gal4 Fusion to Fokl Cleavage Domain

Gal4, a yeast protein, activates transcription of genes required for metabolism of galactose and melibiose. It binds as a dimer to a consensus palindromic 17-base pair DNA sequence. It is a member of the third family of proteins that contain zinc-mediated peptide loops that interact specifically with nucleic acids. Gal4 has a very distinctive zinc coordination profile and mode of DNA-binding. Here, we report the creation of a novel site-specific endonuclease by linking the N-terminal 147 amino acids of Gal4 to the cleavage domain of FokI endonuclease. The fusion protein is active and under optimal conditions, binds to a 17 bp consensus DNA site and cleaves near this site. As expected, the cleavage occurs on either side of the consensus binding site(s).

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