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

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Roy TW,Bhagwat AS

doi

10.1093/nar/gkm1031

subject

Has Abstract

pub_date

2007-01-01 00:00:00

pages

e147

issue

21

eissn

0305-1048

issn

1362-4962

pii

gkm1031

journal_volume

35

pub_type

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