title: Chimeric restriction enzymes: what is next?
abstract: Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of Fokl (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes (Carroll et al., 1999). The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy.
authors.affiliation: Department of Environmental Health Sciences, School of Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: S
authors.initials: S
authors.lastname: Chandrasegaran
authors.forename: J
authors.initials: J
authors.lastname: Smith
mesh_terms: D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D011995:Recombination, Genetic; D013379:Substrate Specificity
chemical_list: D011993:Recombinant Fusion Proteins; D004262:DNA Restriction Enzymes