Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Macromolecular Crystallography Group, Madrid, Spain.
Nucleic Acids Res. 2012 Aug;40(14):6936-45. doi: 10.1093/nar/gks320. Epub 2012 Apr 11.
Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4 bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.
归巢内切核酸酶代表了蛋白质支架，为基因组操作提供了强大的工具，因为这些酶由于其高度特异性，在真核基因组中 DNA 切割的频率非常低。为了产生靶向感兴趣的 DNA 位点的定制酶，必须理解蛋白质-DNA 识别的基础。归巢内切核酸酶的蛋白质-DNA 相互作用工程已经证明了这些方法具有创造新的特定工具来靶向基因失活或修复的潜力。蛋白质-DNA 界面研究主要集中在氨基酸侧链和碱基之间的特定接触上，以重新设计结合界面。然而，已经表明，在归巢内切核酸酶（I-CreI）的 22 碱基底物的中央 DNA 序列中的 4 个碱基（不显示特定的蛋白质-DNA 相互作用），并不是没有内容信息。在这里，我们通过 I-CreI 蛋白分析了该底物区域的靶标区分机制，确定了它如何能够独立于特定的蛋白质-DNA 相互作用而发生。我们的数据表明，在这个底物区域中，间接读取起着重要的作用，为新的靶序列的完全合理搜索开辟了可能性，从而提高了用于治疗和生物技术应用的重新设计酶的开发。
