title: Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering.
abstract: 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 A 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.
authors.affiliation: Macromolecular Crystallography and Nuclear Magnetic Resonance Groups, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre, c/Melchor Fdez. Almagro 3, 28029 Madrid, Spain.
authors.forename: María José
authors.initials: MJ
authors.lastname: Marcaida
authors.forename: Jesús
authors.initials: J
authors.lastname: Prieto
authors.forename: Pilar
authors.initials: P
authors.lastname: Redondo
authors.forename: Alejandro D
authors.initials: AD
authors.lastname: Nadra
authors.forename: Andreu
authors.initials: A
authors.lastname: Alibés
authors.forename: Luis
authors.initials: L
authors.lastname: Serrano
authors.forename: Sylvestre
authors.initials: S
authors.lastname: Grizot
authors.forename: Philippe
authors.initials: P
authors.lastname: Duchateau
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pâques
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Blanco
authors.forename: Guillermo
authors.initials: G
authors.lastname: Montoya
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053837:DNA Cleavage; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D013379:Substrate Specificity
chemical_list: D004247:DNA; C081755:endodeoxyribonuclease I-Dmo I; D015253:Deoxyribonucleases, Type I Site-Specific