Nucleic Acids Research Advance Access published online on April 22, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm183
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Structural Biology |
The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage1
Spanish National Cancer Center (CNIO), Structural Biology and Biocomputing Programme, 1NMR Group and 2Macromolecular Crystallography Group, c/Melchor Fdez. Almagro 3, 28029-Madrid, Spain and 3CELLECTIS S.A., 102 route de Noisy 93235 Romainville, France
*To whom correspondence should be addressed. Tel: 00 34 912246900; Fax: 00 34 912246976; Email: gmontoya{at}cnio.es
Received December 12, 2006. Revised March 13, 2007. Accepted March 13, 2007.
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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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