“归巢核酸内切酶”或“（兆碱基）大范围核酸酶”是一种切割得非常罕见的核酸内切酶。它们可以识别的DNA序列要比“经典”限制酶可以识别的大得多，所以它们在人类基因组中产生解理的频率低。这使得它们有可能用于基因疗法，作为具有高度针对性的分子刀来瞄准特定的基因。
在一项原理证明实验中，Redondo等人设计出的“归巢核酸内切酶”有可能修复在“着色性干皮病”中发生突变的基因。“着色性干皮病”影响核苷酸切除修复机制，削弱身体消除紫外线损伤的能力。“着色性干皮病”患者发生突变的频率高，因此患皮肤癌的可能性大。新设计的酶名叫Amel3-4 和 Ini3-4，是“归巢核酸内切酶” I-CreI的衍生物，这种内切酶在体外和体内都能解理XPC人基因。结构分析表明，它们的解理催化机制与野生型homodimeric I-CreI的相似，Amel3-4 和 Ini3-4在哺乳动物细胞中都能诱导高水平的基因定向。这项工作展示了一种基因修复技术，该技术有可能修复“着色性干皮病”中产生其他单亲遗传疾病的基因。（生物谷Bioon.com）
生物谷推荐原始出处：
Nature 456, 107-111 (6 November 2008) | doi:10.1038/nature07343
Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases
Pilar Redondo1,6, Jesús Prieto2,6, Inés G. Mu?oz1, Andreu Alibés3, Francois Stricher3, Luis Serrano3,4, Jean-Pierre Cabaniols5, Fayza Daboussi5, Sylvain Arnould5, Christophe Perez5, Philippe Duchateau5, Frédéric Paques5, Francisco J. Blanco2,7 & Guillermo Montoya1
1 Macromolecular Crystallography Group,
2 NMR group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), c/Melchor Fdez. Almagro 3, 28029 Madrid, Spain
3 European Molecular Biology Laboratory (EMBL)-CRG Systems Biology Unit, Centre de Regulació Genòmica (CRG), Universitat Pompeu Fabra (UPF), Dr Aiguader 88, 08003 Barcelona, Spain
4 Institució Catalana de Recerca i Estudis Avan?ats (ICREA),
5 CELLECTIS S.A., 102 Avenue Gaston Roussel, 93235 Romainville, France
6 These authors contributed equally to this work.
Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers1. The use of rare cutting DNA endonucleases—such as homing endonucleases, also known as meganucleases—constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules—Amel3–Amel4 and Ini3–Ini4—cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.
