Alteration of the Cleavage Distance of Fok I Restriction Endonuclease by Insertion Mutagenesis

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 thi...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1993-04, Vol.90 (7), p.2764-2768
Main Authors: Li, Lin, Chandrasegaran, Srinivasan
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino acids
Analytical, structural and metabolic biochemistry
Bacteria
Base Sequence
Binding Sites
Biological and medical sciences
Codon - genetics
Codons
Connected regions
Deoxyribonucleases, Type II Site-Specific - genetics
Deoxyribonucleases, Type II Site-Specific - isolation & purification
Deoxyribonucleases, Type II Site-Specific - metabolism
DNA
DNA - genetics
DNA - metabolism
Enzymes
Enzymes and enzyme inhibitors
Flavobacterium okeanokoites
Fundamental and applied biological sciences. Psychology
Gels
Hydrolases
Molecular Sequence Data
Mutagenesis, Insertional
Mutation
Nucleotide sequences
Nucleotides
Oligodeoxyribonucleotides - metabolism
Polymerase chain reaction
Proteins
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Sequencing
Substrate Specificity
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Description
Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.90.7.2764