Insertion and deletion mutants of FokI restriction endonuclease

Details Insertion and deletion mutants of FokI restriction endonuclease Insertion and deletion mutants of FokI restriction endonuclease

1994-11-23

1996-01-30

Patterson, Jr., Charles L.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

435193, C12N 922

Patent

active

054879949

ABSTRACT:
The present invention reveals the construction of several insertion (4, 8, 12, 18, 19 or 23 amino acid residues) and deletion (4 or 7 amino acid residues) mutants of the linker region of FokI endonuclease in Flavobacterium okeanokoites. 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, the insertion mutants cleave predominantly one nucleotide further away from the recognition site on both strands of the DNA substrate. The four codon deletion mutant shows relaxed specificity at the cut site while the seven codon deletion appears to inactivate the enzyme. The DNA-binding and cleavage domains of FokI appear to be linked by a relatively malleable linker. No simple linear relationship exists between the linker length and the distance of the cut site from the recognition site. Furthermore, the four codon insertion mutants cleave DNA substrates containing hemi-methylated FokI sites; they do not cleave fully-methylated substrates.

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