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Nucleic Acids Research Advance Access originally published online on August 24, 2007
Nucleic Acids Research 2007 35(17):5839-5850; doi:10.1093/nar/gkm624
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Nucleic Acids Research, 2007, Vol. 35, No. 17 5839-5850
© 2007 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

Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease

Jennifer L. Eklund1, Umut Y. Ulge3, Jennifer Eastberg3,4 and Raymond J. Monnat, Jr1,2,*

1Department of Genome Sciences, 2Department of Pathology, 3the Molecular and Cellular Biology Program, University of Washington, Seattle, WA and 4Fred Hutchinson Cancer Research Center, Seattle, WA, USA

*To whom correspondence should be addressed. Tel: 206 616 7392; Fax: 206 543 3967; Email: monnat{at}u.washington.edu

Received June 24, 2007. Revised July 27, 2007. Accepted July 30, 2007.

We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic homing endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or ‘homing’ site DNA in vivo. Native I-PpoI recognizes and cleaves a semi-palindromic 15-bp target site with high specificity in vivo and in vitro. This target site is present in the 28S or equivalent large subunit rDNA genes of all eukaryotes. I-PpoI variants able to bind mutant target site DNA had from 1 to 8 amino acid substitutions in the DNA–protein interface. Biochemical characterization of these proteins revealed a wide range of site–binding affinities and site discrimination. One-third of variants were able to cleave target site DNA, but there was no systematic relationship between site-binding affinity and site cleavage. Computational modeling of several variants provided mechanistic insight into how amino acid substitutions that contact, or are adjacent to, specific target site DNA base pairs determine I-PpoI site-binding affinity and site discrimination, and may affect cleavage efficiency.

Present address: Jennifer L. Eklund, University of Michigan School of Education, Ann Arbor, MI, USA.


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