Structure of the I-SceI nuclease complexed with its dsDNA target and three catalytic metal ions.
Homing endonucleases are highly specific DNA-cleaving enzymes that recognize and cleave long stretches of DNA. The engineering of these enzymes provides instruments for genome modification in a wide range of fields, including gene targeting. The homing endonuclease I-SceI from the yeast Saccharomyces cerevisiae has been purified after overexpression in Escherichia coli and its crystal structure has been determined in complex with its target DNA. In order to evaluate the number of ions that are involved in the cleavage process, thus determining the catalytic mechanism, crystallization experiments were performed in the presence of Mn(2+), yielding crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 80.11, b = 80.57, c = 130.87 Å, α = β = γ = 90°. The self-rotation function and the Matthews coefficient suggested the presence of two protein-DNA complexes in the asymmetric unit. The crystals diffracted to a resolution limit of 2.9 Å using synchrotron radiation. From the anomalous data, it was determined that three cations are involved in catalysis and it was confirmed that I-SceI follows a two-metal-ion DNA-strand cleavage mechanism.
Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), c/Melchor Fernandez Almagro 3, 28029 Madrid, Spain.
Jesús
J
Prieto
Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), c/Melchor Fernandez Almagro 3, 28029 Madrid, Spain.
Pilar
P
Redondo
CIC bioGUNE, Parque Tecnológico de Vizcaya, Edificio 800, 48160 Derio, Spain.
Nekane
N
Merino
CIC bioGUNE, Parque Tecnológico de Vizcaya, Edificio 800, 48160 Derio, Spain.
Maider
M
Villate
Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), c/Melchor Fernandez Almagro 3, 28029 Madrid, Spain.
Guillermo
G
Montoya
CIC bioGUNE, Parque Tecnológico de Vizcaya, Edificio 800, 48160 Derio, Spain.
Francisco J
FJ
Blanco
Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), c/Melchor Fernandez Almagro 3, 28029 Madrid, Spain.
Rafael
R
Molina
D002384:Catalysis; D004247:DNA / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
D008670:Metals; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific
DNA cleavage; I-SceI; Saccharomyces cerevisiae; gene targeting; genetics; homing endonuclease; protein–DNA interaction