Li L, Chandrasegaran S
Department of Environmental Health Sciences, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205-2179.
Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2764-8. doi: 10.1073/pnas.90.7.2764.
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.
福克I限制性内切核酸酶识别双链DNA中5'-GGATG-3'.5'-CATCC-3'这种非回文五脱氧核糖核苷酸，并在距识别位点9和13个核苷酸处切割。最近，我们报道了该酶中存在两个不同且可分离的蛋白质结构域——一个用于序列特异性识别，另一个用于内切核酸酶活性。在此，我们报道了福克I内切核酸酶两个插入突变体的构建。对突变酶进行了纯化，并对其切割特性进行了表征。这些突变体与野生型酶具有相同的DNA序列特异性。然而，与野生型酶相比，它们在DNA底物的两条链上距识别位点多切割一个核苷酸。因此，通过蛋白质工程有可能改变福克I的切割距离。
