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Nucleic Acids Research Advance Access originally published online on April 1, 2008
Nucleic Acids Research 2008 36(9):2917-2925; doi:10.1093/nar/gkn131
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Nucleic Acids Research, 2008, Vol. 36, No. 9 2917-2925
© 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

Differential stabilization of reaction intermediates: specificity checkpoints for M.EcoRI revealed by transient fluorescence and fluorescence lifetime studies

Ben Youngblood1, Eleanor Bonnist2,3, David T.F. Dryden2,3, Anita C. Jones2,3 and Norbert O. Reich1,4,*

1Program in Biomolecular Science and Engineering, University of California, Santa Barbara, CA 93106-9510, USA, 2School of Chemistry, 3Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre (COSMIC), The University of Edinburgh, West Mains Road, Edinburgh EH9 3JZ, UK and 4The Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106-9510, USA

*To whom correspondence should be addressed. Tel: +1 805 893 8368; Fax: + 1 805 893 4120; Email: reich{at}chem.ucsb.edu

Received February 6, 2008. Revised March 5, 2008. Accepted March 10, 2008.

M.EcoRI, a bacterial sequence-specific S-adenosyl-L-methionine-dependent DNA methyltransferase, relies on a complex conformational mechanism to achieve its remarkable specificity, including DNA bending, base flipping and intercalation into the DNA. Using transient fluorescence and fluorescence lifetime studies with cognate and noncognate DNA, we have characterized several reaction intermediates involving the WT enzyme. Similar studies with a bending-impaired, enhanced-specificity M.EcoRI mutant show minimal differences with the cognate DNA, but significant differences with noncognate DNA. These results provide a plausible explanation of the way in which destabilization of reaction intermediates can lead to changes in substrate specificity.


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