title: Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases.
abstract: Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases-such as homing endonucleases, also known as meganucleases-constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules-Amel3-Amel4 and Ini3-Ini4-cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.
authors.affiliation: Macromolecular Crystallography Group, Spanish National Cancer Research Centre (CNIO), c/Melchor Fdez. Almagro 3, 28029 Madrid, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Redondo
authors.forename: Jesús
authors.initials: J
authors.lastname: Prieto
authors.forename: Inés G
authors.initials: IG
authors.lastname: Muñoz
authors.forename: Andreu
authors.initials: A
authors.lastname: Alibés
authors.forename: Francois
authors.initials: F
authors.lastname: Stricher
authors.forename: Luis
authors.initials: L
authors.lastname: Serrano
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Cabaniols
authors.forename: Fayza
authors.initials: F
authors.lastname: Daboussi
authors.forename: Sylvain
authors.initials: S
authors.lastname: Arnould
authors.forename: Christophe
authors.initials: C
authors.lastname: Perez
authors.forename: Philippe
authors.initials: P
authors.lastname: Duchateau
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pâques
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Blanco
authors.forename: Guillermo
authors.initials: G
authors.lastname: Montoya
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004260:DNA Repair; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D004268:DNA-Binding Proteins / Q000235:genetics*; D004795:Enzyme Stability; D005818:Genetic Engineering; D006801:Humans; D008958:Models, Molecular; D010766:Phosphorylation; D055503:Protein Multimerization; D013379:Substrate Specificity; D014983:Xeroderma Pigmentosum / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; C086984:XPC protein, human; D004247:DNA; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI