Waugh D S, Sauer R T
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
J Biol Chem. 1994 Apr 22;269(16):12298-303.
A genetic screen was used to identify amino acid substitutions that enable the FokI restriction endonuclease to cleave DNA in cells that express the cognate methyltransferase activity. Missense mutations that give rise to this phenotype were isolated at eight different positions (G188K, P196S, T343I, S388N, S395F, E407K, E410K, D421N), clustered in two regions of the polypeptide sequence of FokI. Two of the mutant endonucleases (P196S and D421N) were purified to homogeneity and analyzed in detail. Both mutants cleave FokI target sites (5'-GGATG-3') in a manner similar to the wild-type enzyme. Neither mutant cleaved noncanonical sequences, but both efficiently cleaved DNA substrates containing hemi-methylated FokI sites. This class of mutations has not been observed with other restriction enzymes.
利用遗传筛选来鉴定氨基酸替换，这些替换能使FokI限制性内切核酸酶在表达同源甲基转移酶活性的细胞中切割DNA。在八个不同位置（G188K、P196S、T343I、S388N、S395F、E407K、E410K、D421N）分离出了导致这种表型的错义突变，这些突变聚集在FokI多肽序列的两个区域。将其中两个突变内切核酸酶（P196S和D421N）纯化至同质，并进行了详细分析。这两个突变体均以与野生型酶相似的方式切割FokI靶位点（5'-GGATG-3'）。这两个突变体均未切割非规范序列，但都能有效切割含有半甲基化FokI位点的DNA底物。其他限制性酶未观察到这类突变。
