Wah D A, Hirsch J A, Dorner L F, Schildkraut I, Aggarwal A K
Department of Biochemistry and Molecular Biophysics, Columbia University, New York 10032, USA.
Nature. 1997 Jul 3;388(6637):97-100. doi: 10.1038/40446.
FokI is a member of an unusual class of bipartite restriction enzymes that recognize a specific DNA sequence and cleave DNA nonspecifically a short distance away from that sequence. Because of its unusual bipartite nature, FokI has been used to create artificial enzymes with new specificities. We have determined the crystal structure at 2.8A resolution of the complete FokI enzyme bound to DNA. As anticipated, the enzyme contains amino- and carboxy-terminal domains corresponding to the DNA-recognition and cleavage functions, respectively. The recognition domain is made of three smaller subdomains (D1, D2 and D3) which are evolutionarily related to the helix-turn-helix-containing DNA-binding domain of the catabolite gene activator protein CAP. The CAP core has been extensively embellished in the first two subdomains, whereas in the third subdomain it has been co-opted for protein-protein interactions. Surprisingly, the cleavage domain contains only a single catalytic centre, raising the question of how monomeric FokI manages to cleave both DNA strands. Unexpectedly, the cleavage domain is sequestered in a 'piggyback' fashion by the recognition domain. The structure suggests a new mechanism for nuclease activation and provides a framework for the design of chimaeric enzymes with altered specificities.
FokI是一类特殊的双功能限制酶的成员，这类酶识别特定的DNA序列，并在距该序列一段短距离处非特异性地切割DNA。由于其独特的双功能性质，FokI已被用于创建具有新特异性的人工酶。我们已经确定了与DNA结合的完整FokI酶在2.8埃分辨率下的晶体结构。正如预期的那样，该酶分别包含对应于DNA识别和切割功能的氨基末端和羧基末端结构域。识别结构域由三个较小的亚结构域（D1、D2和D3）组成，它们在进化上与分解代谢基因激活蛋白CAP的含螺旋-转角-螺旋的DNA结合结构域相关。CAP核心在前两个亚结构域中得到了广泛修饰，而在第三个亚结构域中它被用于蛋白质-蛋白质相互作用。令人惊讶的是，切割结构域仅包含一个催化中心，这就提出了单体FokI如何切割两条DNA链的问题。出乎意料的是，切割结构域以“搭便车”的方式被识别结构域隔离。该结构揭示了一种核酸酶激活的新机制，并为设计具有改变特异性的嵌合酶提供了框架。
