Homing endonuclease I-CreI derivatives with novel DNA target specificities
| Autor: | Brendan Springstubb, Barry L. Stoddard, Selma Masri, Django Sussman, Holly A. Morrison, Laura E. Rosen, Michael J. Brown, Lenny M. Seligman |
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| Rok vydání: | 2006 |
| Předmět: |
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Molecular I-CreI Protein Engineering Homing endonuclease Substrate Specificity 03 medical and health sciences Endonuclease chemistry.chemical_compound Genetics Flap endonuclease 030304 developmental biology 0303 health sciences biology Nucleic Acid Enzymes 030302 biochemistry & molecular biology Water DNA DNA Restriction Enzymes DNA/RNA non-specific endonuclease chemistry Amino Acid Substitution biology.protein Primer (molecular biology) In vitro recombination |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 |
| Popis: | Homing endonucleases are highly specific enzymes, capable of recognizing and cleaving unique DNA sequences in complex genomes. Since such DNA cleavage events can result in targeted allele-inactivation and/or allele-replacement in vivo, the ability to engineer homing endonucleases matched to specific DNA sequences of interest would enable powerful and precise genome manipulations. We have taken a step-wise genetic approach in analyzing individual homing endonuclease I-CreI protein/DNA contacts, and describe here novel interactions at four distinct target site positions. Crystal structures of two mutant endonucleases reveal the molecular interactions responsible for their altered DNA target specificities. We also combine novel contacts to create an endonuclease with the predicted target specificity. These studies provide important insights into engineering homing endonucleases with novel target specificities, as well as into the evolution of DNA recognition by this fascinating family of proteins. |
| Databáze: | OpenAIRE |
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