Nucleic Acids Research Advance Access originally published online on September 13, 2006
Nucleic Acids Research 2006 34(17):4791-4800; doi:10.1093/nar/gkl645
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Nucleic Acids Research, 2006, Vol. 34, No. 17 4791-4800
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Homing endonuclease I-CreI derivatives with novel DNA target specificities
1 Department of Biology and Program in Molecular Biology, Pomona College 175 West 6th Street, Claremont, CA 91711, USA 2 Division of Basic Sciences, Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North A3-025 Seattle, WA 98109, USA
*To whom correspondence should be addressed. Tel: +1 909 621 8608; Fax: +1 909 621 8878; Email: lms14747{at}pomona.edu
Received June 9, 2006. Accepted August 21, 2006.
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.
Present addresses: Holly A. Morrison, Department of Molecular and Cell Biology, UC Berkeley, Berkeley, CA 94720, USA
Selma Masri, Department of Surgical Research, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, CA 91010, USA
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