Institut für Biochemie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 58, D-35392, Giessen, Germany.
Nucleic Acids Res. 2012 Jan;40(2):847-60. doi: 10.1093/nar/gkr788. Epub 2011 Sep 29.
Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.
锌指核酸酶和 TALE 核酸酶是通过将特定的 DNA 结合模块和非特异性的 DNA 切割模块组合而成的,从而产生能够在独特序列处切割 DNA 的核酸酶。在这里,通过将具有催化活性的同种型内切酶 I-SceI 的变体融合到 IIP 型限制酶 PvuII 作为切割模块,来创建高度特异性核酸酶的新方法。融合酶被设计用来识别一个复合位点,该复合位点由 PvuII 的识别位点侧翼的 I-SceI 的识别位点组成。为了降低缺乏侧翼 I-SceI 位点的 PvuII 位点的活性,对酶进行了优化,使得 I-SceI 与其位点的结合使 PvuII 定位以切割复合位点。这是通过优化连接子和在 PvuII 中引入氨基酸取代来实现的,这些取代降低了其活性或干扰了其二聚体界面。最特异的变体对目标复合位点的偏好性超过 1000 倍,而对未处理的 PvuII 位点则没有偏好性。这些结果表明,使用特定的限制酶(如 PvuII)作为切割模块,提供了一种替代通常使用的 FokI 催化结构域的方法,而 FokI 本身并不能为工程化核酸酶的特异性做出贡献。