期刊
NUCLEIC ACIDS RESEARCH
卷 40, 期 14, 页码 6936-6945出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gks320
关键词
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资金
- Ministerio de Ciencia e Innovacion [JCI-2011-09308, BFU2008-01344/BMC, BFU2011-23815/BMC, CSD2006-20642]
- Comunidad Autonoma de Madrid [CAM-P2006/Gen-0166]
- EU Marie Curie 'SMARTBREAKER' [2010-276953]
- Ministerio de Educacion [SB2010-0105]
- Spanish Ministry of Science
Homing endonucleases represent protein scaffolds that provide powerful tools for genome manipulation, as these enzymes possess a very low frequency of DNA cleavage in eukaryotic genomes due to their high specificity. The basis of protein-DNA recognition must be understood to generate tailored enzymes that target the DNA at sites of interest. Protein-DNA interaction engineering of homing endonucleases has demonstrated the potential of these approaches to create new specific instruments to target genes for inactivation or repair. Protein-DNA interface studies have been focused mostly on specific contacts between amino acid side chains and bases to redesign the binding interface. However, it has been shown that 4 bp in the central DNA sequence of the 22-bp substrate of a homing endonuclease (I-CreI), which do not show specific protein-DNA interactions, is not devoid of content information. Here, we analyze the mechanism of target discrimination in this substrate region by the I-CreI protein, determining how it can occur independently of the specific protein-DNA interactions. Our data suggest the important role of indirect readout in this substrate region, opening the possibility for a fully rational search of new target sequences, thus improving the development of redesigned enzymes for therapeutic and biotechnological applications.
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