title: Non-specific protein-DNA interactions control I-CreI target binding and cleavage.
abstract: 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.
authors.affiliation: Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Macromolecular Crystallography Group, Madrid, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Molina
authors.forename: Pilar
authors.initials: P
authors.lastname: Redondo
authors.forename: Stefano
authors.initials: S
authors.lastname: Stella
authors.forename: Marco
authors.initials: M
authors.lastname: Marenchino
authors.forename: Marco
authors.initials: M
authors.lastname: D'Abramo
authors.forename: Francesco Luigi
authors.initials: FL
authors.lastname: Gervasio
authors.forename: Jean Charles
authors.initials: JC
authors.lastname: Epinat
authors.forename: Julien
authors.initials: J
authors.lastname: Valton
authors.forename: Silvestre
authors.initials: S
authors.lastname: Grizot
authors.forename: Phillipe
authors.initials: P
authors.lastname: Duchateau
authors.forename: Jesús
authors.initials: J
authors.lastname: Prieto
authors.forename: Guillermo
authors.initials: G
authors.lastname: Montoya
mesh_terms: D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053837:DNA Cleavage; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D008670:Metals / Q000737:chemistry; D056004:Molecular Dynamics Simulation; D011485:Protein Binding
chemical_list: D008670:Metals; D004247:DNA; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI