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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • DNA / chemistry*
  • DNA / metabolism
  • DNA Cleavage*
  • DNA Restriction Enzymes / metabolism*
  • Metals / chemistry
  • Molecular Dynamics Simulation
  • Protein Binding


  • Metals
  • DNA
  • DNA Restriction Enzymes
  • endodeoxyribonuclease CreI

Associated data

  • PDB/4AAB
  • PDB/4AAD
  • PDB/4AAE
  • PDB/4AAF
  • PDB/4AAG