Chimeric restriction enzyme: Gal4 fusion to FokI cleavage domain

Biol Chem. Apr-May 1998;379(4-5):489-95. doi: 10.1515/bchm.1998.379.4-5.489.

Abstract

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).

Publication types

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

MeSH terms

  • Base Sequence
  • Binding Sites
  • Cloning, Molecular
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Deoxyribonucleases, Type II Site-Specific / genetics
  • Deoxyribonucleases, Type II Site-Specific / metabolism*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae Proteins*
  • Structure-Activity Relationship
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*

Substances

  • DNA-Binding Proteins
  • Fungal Proteins
  • GAL4 protein, S cerevisiae
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • endodeoxyribonuclease FokI
  • Deoxyribonucleases, Type II Site-Specific