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

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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 cancers1. 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.

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Figure 1: New heterodimer cleavage activity and specificity.
Figure 2: Crystal structures of the Ini3–Ini4 and Amel3–Amel4 heterodimers.
Figure 3: Structural comparison of the wild-type, Amel3–Amel4 and Ini3–Ini4 complexes.
Figure 4: Analysis of the efficiency of gene repair by customized meganucleases and genotoxicity controls.

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Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the PDB under accession numbers 2vbj, 2vbl, 2vbn and 2vbo.


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We would like to thank J. Casado and D. Megias for technical support and the beamline staff at the ESRF and SLS for advice during data collection. This project received financial support from the EU MEGATOOL project (LSHG-CT-2006-037226). A.A. is partly funded by a CRG-Novartis fellowship.

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Correspondence to Francisco J. Blanco or Guillermo Montoya.

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Redondo, P., Prieto, J., Muñoz, I. et al. Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases. Nature 456, 107–111 (2008) doi:10.1038/nature07343

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