Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

• DOI: 10.1073/pnas.0804795105
• Publication Date: 2008-10-31T00:54:15Z
• Authors: María José Marcaida; Jesús Prieto; Pilar Redondo; Alejandro D. Nadra; Andreu Alibés; Luis Serrano; Sylvestre Grizot; Philippe Duchateau; Frédéric Pâques; Francisco J. Blanco; Guillermo Montoya
• Keywords: Models, Molecular; 0301 basic medicine; Binding Sites; Base Sequence; Protein Conformation; Molecular Sequence Data; Deoxyribonucleases, Type I Site-Specific; DNA; Crystallography, X-Ray; Protein Engineering; Substrate Specificity; 03 medical and health sciences; Nucleic Acid Conformation; DNA Cleavage; Dimerization

Homing endonucleases, also known as meganucleases, are sequence-specific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 Å resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward the generation of custom endonucleases for targeted genome engineering using the monomeric I-DmoI scaffold.