Chimeric restriction enzymes: What is next?

Research output: Contribution to journalArticle

Abstract

Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of FokI (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. 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.

Original languageEnglish (US)
Pages (from-to)841-848
Number of pages8
JournalBiological Chemistry
Volume380
Issue number7-8
DOIs
StatePublished - Jul 1999

Fingerprint

Zinc Fingers
Nucleotide Motifs
DNA
Enzymes
Zinc
Helix-Turn-Helix Motifs
Genome
Leucine Zippers
DNA Cleavage
Genes
Homologous Recombination
DNA Restriction Enzymes
DNA-Binding Proteins
Microinjections
Gene therapy
Genetic Therapy
Anura
Chromatin
Oocytes
Availability

Keywords

  • Chimeric restriction endonuclease
  • Flavobacterium okeanokoites
  • Gene therapy
  • Genome engineering
  • Hybrid restrictions enzymes
  • Recognition and cleavage domains

ASJC Scopus subject areas

  • Biochemistry

Cite this

Chimeric restriction enzymes : What is next? / Chandrasegaran, Srinivasan; Smith, Jeff.

In: Biological Chemistry, Vol. 380, No. 7-8, 07.1999, p. 841-848.

Research output: Contribution to journalArticle

Chandrasegaran, Srinivasan ; Smith, Jeff. / Chimeric restriction enzymes : What is next?. In: Biological Chemistry. 1999 ; Vol. 380, No. 7-8. pp. 841-848.
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