“歸巢核酸內(nèi)切酶”或“（兆堿基）大范圍核酸酶”是一種切割得非常罕見的核酸內(nèi)切酶,。它們可以識別的DNA序列要比“經(jīng)典”限制酶可以識別的大得多，所以它們在人類基因組中產(chǎn)生解理的頻率低,。這使得它們有可能用于基因療法,，作為具有高度針對性的分子刀來瞄準(zhǔn)特定的基因。
在一項(xiàng)原理證明實(shí)驗(yàn)中,，Redondo等人設(shè)計(jì)出的“歸巢核酸內(nèi)切酶”有可能修復(fù)在“著色性干皮病”中發(fā)生突變的基因,。“著色性干皮病”影響核苷酸切除修復(fù)機(jī)制，削弱身體消除紫外線損傷的能力,。“著色性干皮病”患者發(fā)生突變的頻率高,，因此患皮膚癌的可能性大。新設(shè)計(jì)的酶名叫Amel3-4 和 Ini3-4,，是“歸巢核酸內(nèi)切酶” I-CreI的衍生物,，這種內(nèi)切酶在體外和體內(nèi)都能解理XPC人基因。結(jié)構(gòu)分析表明,，它們的解理催化機(jī)制與野生型homodimeric I-CreI的相似,，Amel3-4 和 Ini3-4在哺乳動(dòng)物細(xì)胞中都能誘導(dǎo)高水平的基因定向。這項(xiàng)工作展示了一種基因修復(fù)技術(shù),，該技術(shù)有可能修復(fù)“著色性干皮病”中產(chǎn)生其他單親遺傳疾病的基因,。（生物谷Bioon.com）
生物谷推薦原始出處：
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.
