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Unusual 2-aminopurine fluorescence from a complex of DNA and the EcoKI methyltransferase

Lookup NU author(s): Professor Bernard Connolly

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Abstract

The methyltransferase, M.EcoKI, recognizes the DNA sequence 5′-AACNNNNNNGTGC-3′ and methylates adenine at the underlined positions. DNA 00methylation 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-adenosylmethionine 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. © Oxford University Press 2004; all rights reserved.

Publication metadata

Author(s): Su T-J, Connolly BA, Darlington C, Mallin R, Dryden DTF

Publication type: Article

Publication status: Published

Journal: Nucleic Acids Research

Year: 2004

Volume: 32

Issue: 7

Pages: 2223-2230

Print publication date: 01/01/2004

Date deposited: 08/02/2012

ISSN (print): 0305-1048

ISSN (electronic): 1362-4962

Publisher: Oxford University Press

URL: http://dx.doi.org/10.1093/nar/gkh531

DOI: 10.1093/nar/gkh531

PubMed id: 15107490

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