Isolation and characterization of new homing endonuclease specificities at individual target site positions
Isolation and characterization of new homing endonuclease specificities at individual target site positions
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DOI：
10.1016/j.jmb.2004.07.031
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发表时间：
2004-09-03
影响因子：
5.6
通讯作者：
Seligman, LM
中科院分区：
文献类型：
--
作者：
Sussman, D;Chadsey, M;Seligman, LM
归巢核酸内切酶是高度特异性的DNA核酸内切酶，在移动的内含子或内含肽内编码，其诱导其同源靶位点的靶向重组、双链修复和基因转换。由于它们的生物学功能和高水平的靶特异性，这些酶作为基因靶向的工具正在进行深入的研究。这些研究需要重新设计天然存在的酶以识别新的靶位点。在这里，我们报告的研究中，同源二聚体LAGLIDADG归巢核酸内切酶I-CreI改变在个别侧链对应的接触点，以不同的碱基对在其靶位点。所得的酶构建体在体内驱动选定的DNA靶标的特异性消除，并在体外显示DNA结合和切割的转移特异性。这些构建体中的两个的晶体结构表明，可以发生单个侧链/DNA接触模式的取代，而几乎没有周围复合物的结构变形或重排，从而促进了归巢核酸内切酶活性和特异性的分离的模块化重新设计策略。(C)2004 Elsevier Ltd.保留所有权利。
Homing endonucleases are highly specific DNA endonucleases, encoded within mobile introns or inteins, that induce targeted recombination, double-strand repair and gene conversion of their cognate target sites. Due to their biological function and high level of target specificity, these enzymes are under intense investigation as tools for gene targeting. These studies require that naturally occurring enzymes be redesigned to recognize novel target sites. Here, we report studies in which the homodimeric LAGLIDADG homing endonuclease I-CreI is altered at individual side-chains corresponding to contact points to distinct base-pairs in its target site. The resulting enzyme constructs drive specific elimination of selected DNA targets in vivo and display shifted specificities of DNA binding and cleavage in vitro. Crystal structures of two of these constructs demonstrate that substitution of individual side-chain/DNA contact patterns can occur with almost no structural deformation or rearrangement of the surrounding complex, facilitating an isolated, modular redesign strategy for homing endonuclease activity and specificity. (C) 2004 Elsevier Ltd. All rights reserved.