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- Title
Non-specific protein-DNA interactions control I-CreI target binding and cleavage.
- Authors
Molina, Rafael; Redondo, Pilar; Stella, Stefano; Marenchino, Marco; D'Abramo, Marco; Gervasio, Francesco Luigi; Epinat, Jean Charles; Valton, Julien; Grizot, Silvestre; Duchateau, Phillipe; Prieto, Jesús; Montoya, Guillermo
- 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.
- Publication
Nucleic acids research, 2012, Vol 40, Issue 14, p6936
- ISSN
1362-4962
- Publication type
Journal Article
- DOI
10.1093/nar/gks320