Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering
- 4 November 2008
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 105 (44), 16888-16893
- https://doi.org/10.1073/pnas.0804795105
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 Å 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.Keywords
This publication has 41 references indexed in Scilit:
- Crystal structures of I-SceI complexed to nicked DNA substrates: snapshots of intermediates along the DNA cleavage reaction pathwayNucleic Acids Research, 2008
- Computer design of obligate heterodimer meganucleases allows efficient cutting of custom DNA sequencesNucleic Acids Research, 2008
- Crystallization and preliminary X-ray diffraction analysis on the homing endonuclease I-Dmo-I in complex with its target DNAActa Crystallographica Section F Structural Biology and Crystallization Communications, 2007
- An improved zinc-finger nuclease architecture for highly specific genome editingNature Biotechnology, 2007
- A combinatorial approach to create artificial homing endonucleases cleaving chosen sequencesNucleic Acids Research, 2006
- From Monomeric to Homodimeric Endonucleases and Back: Engineering Novel Specificity of LAGLIDADG EnzymesJournal of Molecular Biology, 2006
- Engineering of Large Numbers of Highly Specific Homing Endonucleases that Induce Recombination on Novel DNA TargetsJournal of Molecular Biology, 2006
- Mechanistic Insights from the Structures of HincII Bound to Cognate DNA Cleaved from Addition of Mg2+ and Mn2+Journal of Molecular Biology, 2004
- Flexible DNA Target Site Recognition by Divergent Homing Endonuclease Isoschizomers I-CreI and I-MsoIJournal of Molecular Biology, 2003
- Crystal structure of the thermostable archaeal intron-encoded endonuclease I-DmoIJournal of Molecular Biology, 1999