Published online 23 April 2004
Nucleic Acids Research, 2004, Vol. 32, No. 7 2223-2230
© 2004 Oxford University Press
Unusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase
School of Chemistry, University of Edinburgh, The King’s Buildings, Edinburgh EH9 3JJ and 1 School of Cell and Molecular Biosciences, The University of Newcastle, Newcastle-upon-Tyne NE2 4HH, UK
*To whom correspondence should be addressed. Tel: +44 131 650 4735; Fax: +44 131 650 6453; Email: david.dryden{at}ed.ac.uk
Received January 9, 2004; Revised March 2, 2004; Accepted March 18, 2004
The methyltransferase, M.EcoKI, recognizes the DNA sequence 5'-AACNNNNNNGTGC-3' and methylates adenine at the underlined positions. DNA methylation has been shown by crystallography to occur via a base flipping mechanism and is believed to be a general mechanism for all methyltransferases. If no structure is available, the fluorescence of 2-aminopurine is often used as a signal for base flipping as it shows enhanced fluorescence when its environment is perturbed. We find that 2-aminopurine gives enhanced fluorescence emission not only when it is placed at the M.EcoKI methylation sites but also at a location adjacent to the target adenine. Thus it appears that 2-aminopurine fluorescence intensity is not a clear indicator of base flipping but is a more general measure of DNA distortion. Upon addition of the cofactor S-adenosyl-methionine to the M.EcoKI:DNA complex, the 2-aminopurine fluorescence changes to that of a new species showing excitation at 345 nm and emission at 450 nm. This change requires a fully active enzyme, the correct cofactor and the 2-aminopurine located at the methylation site. However, the new fluorescent species is not a covalently modified form of 2-aminopurine and we suggest that it represents a hitherto undetected physicochemical form of 2-aminopurine.
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