Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering.
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.
Marcaida
María José
MJ
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.
Prieto
Jesús
J
Redondo
Pilar
P
Nadra
Alejandro D
AD
Alibés
Andreu
A
Serrano
Luis
L
Grizot
Sylvestre
S
Duchateau
Philippe
P
Pâques
Frédéric
F
Blanco
Francisco J
FJ
Montoya
Guillermo
G
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
D004247:DNA; C081755:endodeoxyribonuclease I-Dmo I; D015253:Deoxyribonucleases, Type I Site-Specific