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Nucleic Acids Research Advance Access published online on November 14, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm1031
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© 2007 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.

Methods Online

Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethylation

Todd W. Roy and A. S. Bhagwat*

Department of Chemistry, Wayne State University, Detroit, MI 48202, USA

*To whom correspondence should be addressed. Tel: +1 313 577 2549; Fax: +1 313 577 8822; Email: axb{at}chem.wayne.edu

Received September 6, 2007. Revised October 16, 2007. Accepted October 29, 2007.

The Escherichia coli AlkB protein catalyzes the direct reversal of alkylation damage to DNA; primarily 1-methyladenine (1mA) and 3-methylcytosine (3mC) lesions created by endogenous or environmental alkylating agents. AlkB is a member of the non-heme iron (II) {alpha}-ketoglutarate-dependent dioxygenase superfamily, which removes the alkyl group through oxidation eliminating a methyl group as formaldehyde. We have developed a fluorescence-based assay for the dealkylation activity of this family of enzymes. It uses formaldehyde dehydrogenase to convert formaldehyde to formic acid and monitors the creation of an NADH analog using fluorescence. This assay is a great improvement over the existing assays for DNA demethylation in that it is continuous, rapid and does not require radioactively labeled material. It may also be used to study other demethylation reactions including demethylation of histones. We used it to determine the kinetic constants for AlkB and found them to be somewhat different than previously reported values. The results show that AlkB demethylates 1mA and 3mC with comparable efficiencies and has only a modest preference for a single-stranded DNA substrate over its double-stranded DNA counterpart.


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