Sussman Django, Chadsey Meg, Fauce Steve, Engel Alex, Bruett Anna, Monnat Ray, Stoddard Barry L, Seligman Lenny M
Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N. A3-025 Seattle, WA 98109, USA.
J Mol Biol. 2004 Sep 3;342(1):31-41. doi: 10.1016/j.jmb.2004.07.031.
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.
归巢内切酶是高度特异性的DNA内切酶，由移动内含子或蛋白质内含子编码，可诱导其同源靶位点的靶向重组、双链修复和基因转换。由于其生物学功能和高水平的靶标特异性，这些酶作为基因靶向工具正受到深入研究。这些研究要求对天然存在的酶进行重新设计，以识别新的靶位点。在此，我们报告了相关研究，其中同二聚体LAGLIDADG归巢内切酶I-CreI在与靶位点中不同碱基对的接触点相对应的单个侧链上发生改变。所得的酶构建体在体内驱动对选定DNA靶标的特异性消除，并在体外显示出DNA结合和切割特异性的改变。其中两个构建体的晶体结构表明，单个侧链/DNA接触模式的替换几乎不会导致周围复合物的结构变形或重排，这有利于采用孤立的模块化重新设计策略来实现归巢内切酶的活性和特异性。
