Design, activity, and structure of a highly specific artificial endonuclease

TitleDesign, activity, and structure of a highly specific artificial endonuclease
Publication TypeJournal Article
Year of Publication2002
AuthorsChevalier, B. S., Kortemme T., Chadsey M. S., Baker D., Monnat R. J., & Stoddard B. L.
JournalMolecular cell
Volume10
Issue4
Pagination895-905
Date Published2002 Oct
ISSN1097-2765
KeywordsAlgorithms, Base Sequence, Binding Sites, Catalysis, Collaborative Publication, Crystallography, X-Ray, Deoxyribonucleases, Type I Site-Specific, DNA, DNA Restriction Enzymes, Endonucleases, Models, Molecular, Protein Engineering, Protein Folding, Protein Structure, Tertiary, Recombinant Fusion Proteins, Structure-Activity Relationship, Substrate Specificity, Thermodynamics
Abstract

We have generated an artificial highly specific endonuclease by fusing domains of homing endonucleases I-DmoI and I-CreI and creating a new 1400 A(2) protein interface between these domains. Protein engineering was accomplished by combining computational redesign and an in vivo protein-folding screen. The resulting enzyme, E-DreI (Engineered I-DmoI/I-CreI), binds a long chimeric DNA target site with nanomolar affinity, cleaving it precisely at a rate equivalent to its natural parents. The structure of an E-DreI/DNA complex demonstrates the accuracy of the protein interface redesign algorithm and reveals how catalytic function is maintained during the creation of the new endonuclease. These results indicate that it may be possible to generate novel highly specific DNA binding proteins from homing endonucleases.

Alternate JournalMol. Cell
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