title: Engineering a Nickase on the Homing Endonuclease I-DmoI Scaffold.
abstract: Homing endonucleases are useful tools for genome modification because of their capability to recognize and cleave specifically large DNA targets. These endonucleases generate a DNA double strand break that can be repaired by the DNA damage response machinery. The break can be repaired by homologous recombination, an error-free mechanism, or by non-homologous end joining, a process susceptible to introducing errors in the repaired sequence. The type of DNA cleavage might alter the balance between these two alternatives. The use of "nickases" producing a specific single strand break instead of a double strand break could be an approach to reduce the toxicity associated with non-homologous end joining by promoting the use of homologous recombination to repair the cleavage of a single DNA break. Taking advantage of the sequential DNA cleavage mechanism of I-DmoI LAGLIDADG homing endonuclease, we have developed a new variant that is able to cut preferentially the coding DNA strand, generating a nicked DNA target. Our structural and biochemical analysis shows that by decoupling the action of the catalytic residues acting on each strand we can inhibit one of them while keeping the other functional.
authors.affiliation: From the Macromolecular Crystallography Group and.
authors.forename: Rafael
authors.initials: R
authors.lastname: Molina
authors.affiliation: From the Macromolecular Crystallography Group and.
authors.forename: María José
authors.initials: MJ
authors.lastname: Marcaida
authors.affiliation: From the Macromolecular Crystallography Group and.
authors.forename: Pilar
authors.initials: P
authors.lastname: Redondo
authors.affiliation: NMR Unit, Structural Biology and Biocomputing Program, Spanish National Cancer Research Center (CNIO), c/Melchor Fernández Almagro 3, 28029 Madrid, Spain.
authors.forename: Marco
authors.initials: M
authors.lastname: Marenchino
authors.affiliation: CELLECTIS S.A., 8 Rue de la Croix Jarry, 75013 Paris, France.
authors.forename: Phillippe
authors.initials: P
authors.lastname: Duchateau
authors.affiliation: Department of Chemistry, University of Rome "La Sapienza," Piazzale Aldo Moro 5, 00185, Rome, Italy, and.
authors.forename: Marco
authors.initials: M
authors.lastname: D'Abramo
authors.affiliation: From the Macromolecular Crystallography Group and Novo Nordisk Foundation Center for Protein Research, Protein Structure and Function Program, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark guillermo.montoya@cpr.ku.dk.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Montoya
authors.affiliation: From the Macromolecular Crystallography Group and jprieto@cnio.es.
authors.forename: Jesús
authors.initials: J
authors.lastname: Prieto
mesh_terms: D020816:Amino Acid Motifs; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting; D059765:Homologous Recombination / Q000235:genetics; D056004:Molecular Dynamics Simulation; D015202:Protein Engineering
chemical_list: D004268:DNA-Binding Proteins; C081755:endodeoxyribonuclease I-Dmo I; D003850:Deoxyribonuclease I; D015253:Deoxyribonucleases, Type I Site-Specific
keywords: gene targeting; genetics; nucleases; protein engineering; protein-DNA interaction; protein-nucleic acid interaction; x-ray crystallography