Journal of Biological Chemistry
Volume 286, Issue 37, 16 September 2011, Pages 32617-32627
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Enzymology
Mining Endonuclease Cleavage Determinants in Genomic Sequence Data*

https://doi.org/10.1074/jbc.M111.259572Get rights and content
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Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold. Many of these grafts exhibited novel and unexpected specificities. These findings show that the information present in genomic data can be exploited for endonuclease specificity redesign.

Computer Modeling
Enzyme Mutation
Gene Therapy
Nucleic Acid Enzymology
Protein-DNA Interaction
Computational Enzyme Design
Evolutionary Sequence Information
Genome Engineering
Homing Endonuclease

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*

This work was supported, in whole or in part, by National Institutes of Health Grants GM084433 and RL1CA133832, the Foundation for the National Institutes of Health through the Gates Foundation Grand Challenges in Global Health Initiative, and the Howard Hughes Medical Institute.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S3 and Figs. S1–S8.