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J Biol Chem. 2015 Nov 27;290(48):28727-36. doi: 10.1074/jbc.M115.678342. Epub 2015 Sep 11.

Crystal Structure of the Homing Endonuclease I-CvuI Provides a New Template for Genome Modification.

Author information

1
From the Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Macromolecular Crystallography Group, C/Melchor Fernández Almagro 3, 28029 Madrid, Spain.
2
the Protein Structure & Function Programme, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
3
the Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Bizkaia 800, 48160 Derio, Spain.
4
the Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Bizkaia 800, 48160 Derio, Spain, IKERBASQUE, Basque Foundation for Science, María Díaz de Haro 3, 48013 Bilbao, Spain, and.
5
CELLECTIS S. A., 8 rue de la croix Jarry, 75013 Paris, France.
6
From the Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Macromolecular Crystallography Group, C/Melchor Fernández Almagro 3, 28029 Madrid, Spain, jprieto@cnio.es.
7
From the Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Macromolecular Crystallography Group, C/Melchor Fernández Almagro 3, 28029 Madrid, Spain, the Protein Structure & Function Programme, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark, guillermo.montoya@cpr.ku.dk.

Abstract

Homing endonucleases recognize and generate a DNA double-strand break, which has been used to promote gene targeting. These enzymes recognize long DNA stretches; they are highly sequence-specific enzymes and display a very low frequency of cleavage even in complete genomes. Although a large number of homing endonucleases have been identified, the landscape of possible target sequences is still very limited to cover the complexity of the whole eukaryotic genome. Therefore, the finding and molecular analysis of homing endonucleases identified but not yet characterized may widen the landscape of possible target sequences. The previous characterization of protein-DNA interaction before the engineering of new homing endonucleases is essential for further enzyme modification. Here we report the crystal structure of I-CvuI in complex with its target DNA and with the target DNA of I-CreI, a homologue enzyme widely used in genome engineering. To characterize the enzyme cleavage mechanism, we have solved the I-CvuI DNA structures in the presence of non-catalytic (Ca(2+)) and catalytic ions (Mg(2+)). We have also analyzed the metal dependence of DNA cleavage using Mg(2+) ions at different concentrations ranging from non-cleavable to cleavable concentrations obtained from in vitro cleavage experiments. The structure of I-CvuI homing endonuclease expands the current repertoire for engineering custom specificities, both by itself as a new scaffold alone and in hybrid constructs with other related homing endonucleases or other DNA-binding protein templates.

KEYWORDS:

DNA endonuclease; X-ray crystallography; enzyme; gene targeting; genetics; homing endonucleases; protein-DNA interaction

PMID:
26363068
PMCID:
PMC4661390
DOI:
10.1074/jbc.M115.678342
[Indexed for MEDLINE]
Free PMC Article

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