Nucleic Acids Research Advance Access originally published online on December 22, 2008
Nucleic Acids Research 2009 37(3):877-890; doi:10.1093/nar/gkn1007
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Nucleic Acids Research, 2009, Vol. 37, No. 3 877-890
© 2008 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 |
Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation
1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, 2Northwest Genome Engineering Consortium, Seattle, WA 98101, 3Seattle Children's Hospital Research Institute and the Graduate Program in Molecular and Cellular Biology, M/S C9S, Seattle, WA 98101 and 4Center for RNA Molecular Biology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4960, USA
*To whom correspondence should be addressed. Tel: +1 206 667 4031; Fax: +1 206 667 3331; Email: bstoddar{at}fhcrc.org
Received November 4, 2008. Revised November 26, 2008. Accepted December 1, 2008.
The LAGLIDADG homing endonuclease (LHE) I-AniI has adopted an extremely efficient secondary RNA splicing activity that is beneficial to its host, balanced against inefficient DNA cleavage. A selection experiment identified point mutations in the enzyme that act synergistically to improve endonuclease activity. The amino-acid substitutions increase target affinity, alter the thermal cleavage profile and significantly increase targeted recombination in transfected cells. The RNA splicing activity is not affected by these mutations. The improvement in DNA cleavage activity is largely focused on one of the enzyme's two active sites, corresponding to a rearrangement of a lysine residue hypothesized to act as a general base. Most of the constructs isolated in the screen contain one or more mutations that revert an amino-acid identity to a residue found in one or more close homologues of I-AniI. This implies that mutations that have previously reduced the endonuclease activity of I-AniI are identified and reversed, sometimes in combination with additional ‘artificial’ mutations, to optimize its in vivo activity.
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