TY - JOUR

T1 - Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases

AU - Redondo, Pilar

AU - Prieto, Jesús

AU - Muñoz, Inés G

AU - Alibés, Andreu

AU - Stricher, Francois

AU - Serrano, Luis

AU - Cabaniols, Jean-Pierre

AU - Daboussi, Fayza

AU - Arnould, Sylvain

AU - Perez, Christophe

AU - Duchateau, Philippe

AU - Pâques, Frédéric

AU - Blanco, Francisco J

AU - Montoya, Guillermo

PY - 2008/11/6

Y1 - 2008/11/6

N2 - 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.

AB - 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.

KW - Animals

KW - CHO Cells

KW - Cell Line

KW - Cricetinae

KW - Cricetulus

KW - Crystallography, X-Ray

KW - DNA/chemistry

KW - DNA Repair

KW - DNA Restriction Enzymes/chemistry

KW - DNA-Binding Proteins/genetics

KW - Enzyme Stability

KW - Genetic Engineering

KW - Humans

KW - Models, Molecular

KW - Phosphorylation

KW - Protein Multimerization

KW - Substrate Specificity

KW - Xeroderma Pigmentosum/genetics

U2 - 10.1038/nature07343

DO - 10.1038/nature07343

M3 - Journal article

C2 - 18987743

VL - 456

SP - 107

EP - 111

JO - Nature

JF - Nature

SN - 0028-0836

IS - 7218

ER -