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 language | English (US) |
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Pages (from-to) | 883-887 |
Number of pages | 5 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 91 |
Issue number | 3 |
DOIs | |
State | Published - Feb 1 1994 |
Keywords
- Escherichia coli
- Flavobacterium okeanokoites
- hybrid restriction endonuclease
- protein engineering
- recognition and cleavage domains
ASJC Scopus subject areas
- General