Article thumbnail

Non-specific protein-DNA interactions control I-CreI target binding and cleavage

By Rafael Molina, Pilar Redondo, Stefano Stella, Marco Marenchino, Francesco Luigi Gervasio, Jean Charles Epinat, Julien Valton, Silvestre Grizot, Phillipe Duchateau and Guillermo Montoya


Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipula-tion, as these enzymes possess a very low fre-quency 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 po-tential 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 4bp in the central DNA sequence of the 22-bp sub-strate 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 develop-ment of redesigned enzymes for therapeutic and biotechnological applications

Year: 2012
DOI identifier: 10.1093/nar/gks320
OAI identifier: oai:CiteSeerX.psu:
Provided by: CiteSeerX
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • (external link)
  • (external link)
  • (external link)
  • Suggested articles

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.