title: The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage.
abstract: Meganucleases are sequence-specific endonucleases with large cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These enzymes open novel perspectives for genome engineering in a wide range of fields, including gene therapy. A new crystal structure of the I-CreI dimer without DNA has allowed the comparison with the DNA-bound protein. The C-terminal loop displays a different conformation, which suggests its implication in DNA binding. A site-directed mutagenesis study in this region demonstrates that whereas the C-terminal helix is negligible for DNA binding, the final C-terminal loop is essential in DNA binding and cleavage. We have identified two regions that comprise the Ser138-Lys139 and Lys142-Thr143 pairs whose double mutation affect DNA binding in vitro and abolish cleavage in vivo. However, the mutation of only one residue in these sites allows DNA binding in vitro and cleavage in vivo. These findings demonstrate that the C-terminal loop of I-CreI endonuclease plays a fundamental role in its catalytic mechanism and suggest this novel site as a region to take into account for engineering new endonucleases with tailored specificity.
authors: Spanish National Cancer Center (CNIO), Structural Biology and Biocomputing Programme, NMR Group and Macromolecular Crystallography Group, c/Melchor Fdez. Almagro 3, 28029-Madrid, Spain.
authors: Jesús
authors: J
authors: Prieto
authors: Pilar
authors: P
authors: Redondo
authors: Daniel
authors: D
authors: Padró
authors: Sylvain
authors: S
authors: Arnould
authors: Jean-Charles
authors: JC
authors: Epinat
authors: Frédéric
authors: F
authors: Pâques
authors: Francisco J
authors: FJ
authors: Blanco
authors: Guillermo
authors: G
authors: Montoya
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry
chemical_list: D013912:Threonine; D012694:Serine; D004247:DNA; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI; D008239:Lysine