Siksnys V, Pleckaityte M
Institute of Biotechnology, Fermentas, Vilnius, Lithuania.
Eur J Biochem. 1993 Oct 1;217(1):411-9. doi: 10.1111/j.1432-1033.1993.tb18260.x.
The Cfr9I restriction endonuclease recognizes and cleaves duplex DNA sequence C decreases CCGGG. The binding of restriction endonuclease Cfr9I to DNA was examined in the absence of Mg2+ using gel-mobility-shift and nitrocellulose-filter-binding assays. It was shown that restriction endonuclease Cfr9I bound DNA fragments either containing or lacking the canonical recognition sequence with equal affinity. These results suggest that the specificity of restriction endonuclease Cfr9I is expressed during the catalytic step. The cleavage of supercoiled pUC18 DNA by restriction endonuclease Cfr9I showed that at low concentrations of MgCl2, only with open-circular DNA, nicks appeared in one strand at the recognition sequence, while the cleavage of the second strand was very slow. At higher concentrations of MgCl2 the enzyme cleaves either one or both strands of the DNA. Under these conditions the supercoiled DNA was converted to open-circular and linear forms simultaneously rather than consecutively. It was shown that open-circular DNA was a poor substrate for restriction endonuclease Cfr9I. These results suggested that both Mg2+ and intact recognition sequence are required to drive the enzyme into correct conformation to ensure DNA cleavage.
Cfr9I 限制性内切核酸酶识别并切割双链 DNA 序列 C↓CCGGG。在不存在 Mg2+ 的情况下，使用凝胶迁移率变动分析和硝酸纤维素滤膜结合分析检测了限制性内切核酸酶 Cfr9I 与 DNA 的结合情况。结果表明，限制性内切核酸酶 Cfr9I 以相同的亲和力结合含有或缺乏典型识别序列的 DNA 片段。这些结果表明，限制性内切核酸酶 Cfr9I 的特异性在催化步骤中表现出来。限制性内切核酸酶 Cfr9I 对超螺旋 pUC18 DNA 的切割表明，在低浓度的 MgCl2 条件下，只有开环 DNA 在识别序列处的一条链上出现切口，而第二条链的切割非常缓慢。在较高浓度的 MgCl2 条件下，该酶切割 DNA 的一条链或两条链。在这些条件下，超螺旋 DNA 同时而非连续地转化为开环和线性形式。结果表明，开环 DNA 是限制性内切核酸酶 Cfr9I 的不良底物。这些结果表明，Mg2+ 和完整的识别序列都是驱动该酶进入正确构象以确保 DNA 切割所必需的。
