Chimeric restriction enzyme: Gal4 fusion to FokI 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).
Kim
Y G
YG
Department of Environmental Health Sciences, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205-2179, USA.
Smith
J
J
Durgesha
M
M
Chandrasegaran
S
S
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific