Alteration of the cleavage distance of Fok I restriction endonuclease by insertion mutagenesis

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Fok I restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide 5′-GGATG-3′·5′-CATCC-3′ in duplex DNA and cleaves 9 and 13 nucleotides away from the recognition site. Recently, we reported the presence of two distinct and separable protein domains within this enzyme - one for the sequence-specific recognition and the other for endonuclease activity. Here, we report the construction of two insertion mutants of Fok I endonuclease. The mutant enzymes were purified, and their cleavage properties were characterized. The mutants have the same DNA sequence specificity as the wild-type enzyme. However, compared with the wild-type enzyme, they cleave one nucleotide further away from the recognition site on both strands of the DNA substrates. Thus, it is possible to alter the cleavage distance of Fok I by protein engineering.

Original languageEnglish (US)
Pages (from-to)2764-2768
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number7
StatePublished - Apr 1 1993


  • Escherichia coli
  • Flavobacterium okeanokoities
  • Protein engineering
  • Recognition and cleavage domains

ASJC Scopus subject areas

  • General

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