Chimeric restriction endonuclease

Research output: Contribution to journalArticle

Abstract

Fok I restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide 5'-GGATG-3'·5'-CATCC-3' in duplex DNA and cleaves 9 and 13 nt away from the recognition site. Recently, we reported the presence of two distinct and separable domains within this enzyme: one for the sequence-specific recognition of DNA (the DNA-binding domain) and the other for the endonuclease activity (the cleavage domain). Here, we report the construction of a chimeric restriction endonuclease by linking the Drosophila Ultrabithorax homeodomain to the cleavage domain (F(N)) of Fok I restriction endonuclease. The hybrid enzyme, Ubx-F(N), was purified, and its cleavage properties were characterized. The hybrid enzyme shows the same DNA sequence- binding preference as that of Ubx; as expected, it cleaves the DNA away from the recognition site. On the 5'-TTAATGGTT-3' strand the hybrid enzyme cleaves 3 nt away from the recognition site, whereas it cuts the complementary 5'- AACCATTAA-3' strand 8, 9, or 10 nt away from the binding site. Similarly engineered hybrid enzymes could be valuable tools in physical mapping and sequencing of large eukaryotic genomes.

Original languageEnglish (US)
Pages (from-to)883-887
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number3
DOIs
StatePublished - Feb 1 1994

Keywords

  • Escherichia coli
  • Flavobacterium okeanokoites
  • hybrid restriction endonuclease
  • protein engineering
  • recognition and cleavage domains

ASJC Scopus subject areas

  • General

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