Chimeric restriction endonuclease.
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 (FN) of Fok I restriction endonuclease. The hybrid enzyme, Ubx-FN, 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.
Kim
Y G
YG
Department of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205-2179.
Chandrasegaran
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000737:chemistry / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004330:Drosophila / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D005801:Genes, Homeobox; D017344:Genes, Insect; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific