Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain
Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain
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DOI：
10.1073/pnas.93.3.1156
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发表时间：
1996-02-06
影响因子：
11.1
通讯作者：
Chandrasegaran, S
中科院分区：
文献类型：
--
作者：
Kim, YG;Cha, J;Chandrasegaran, S
限制性修饰酶领域的长期目标是通过突变或工程改造现有酶来产生具有新序列特异性的限制性内切核酸酶。这将避免广泛筛选细菌和其他微生物以获得新酶的日益艰巨的任务。在这里，我们报告故意创建新的位点特异性核酸内切酶，通过连接两个不同的锌指蛋白的Fok I核酸内切酶的切割结构域。两种融合蛋白都是有活性的，并且在最佳条件下以序列特异性方式切割DNA。因此，Fok I内切核酸酶和锌指基序的模块化结构使得有可能产生将在预定位点附近切割DNA的“人工"核酸酶。这开辟了产生许多新酶的途径，这些酶具有各种应用所需的定制序列特异性。
A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create ''artificial'' nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.