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Nucleic Acids Research 2005 33(22):6953-6960; doi:10.1093/nar/gki995
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Published online 9 December 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Article

Time-resolved fluorescence of 2-aminopurine as a probe of base flipping in M.HhaI–DNA complexes

Robert K. Neely1,2, Dalia Daujotyte3, Saulius Grazulis4, Steven W. Magennis2, David T. F. Dryden1,2, Saulius Klimasauskas3,5 and Anita C. Jones1,2,*

1School of Chemistry, The University of Edinburgh West Mains Road, Edinburgh EH9 3JJ, UK 2Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre (COSMIC), The University of Edinburgh West Mains Road, Edinburgh EH9 3JZ, UK 3Laboratory of Biological DNA Modification, Institute of Biotechnology LT-02241 Vilnius, Lithuania 4Laboratory of DNA–Protein Interactions, Institute of Biotechnology LT-02241 Vilnius, Lithuania 5Department of Biochemistry and Biophysics, Faculty of Natural Sciences, Vilnius University LT-2009 Vilnius, Lithuania

*To whom correspondence should be addressed. Tel: +44 131 6506449; Fax: +44 131 6504743; Email: a.c.jones{at}ed.ac.uk

Received September 22, 2005. Revised October 25, 2005. Accepted November 15, 2005.

DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (~100 ps) decay component and the large increase in the amplitude of the long (~10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures.

Correspondence may also be addressed to Saulius Klimasauskas. Tel: +370 5 2602114; Fax: +370 5 2602116; Email: klimasau{at}ibt.lt

PDB accession nos+

+2c7o, 2c7p, 2c7q and 2c7r


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