Skip Navigation


Nucleic Acids Research Advance Access originally published online on November 14, 2007
Nucleic Acids Research 2007 35(21):e147; doi:10.1093/nar/gkm1031
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (260K) Freely available
Right arrow Screen PDF (260K) Freely available
Right arrow Supplementary Data
Right arrowOA All Versions of this Article:
35/21/e147    most recent
gkm1031v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Roy, T. W.
Right arrow Articles by Bhagwat, A. S.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Roy, T. W.
Right arrow Articles by Bhagwat, A. S.
Related Collections
Right arrow Chromatin
Right arrow DNA characterisation
Right arrow Genomics
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2007, Vol. 35, No. 21 e147
© 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.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer:
Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.