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

Roy TW, Bhagwat AS - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.

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.

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Oxidative demethylation of DNA by AlkB coupled with FDH reaction. The demethylation#of 1mA or 3mC in DNA by AlkB and production of formaldehyde are shown schematically. Also shown are the hydroxylated base intermediates and the conversion of formaldehyde to formate by FDH through a coupled reaction. In the process, coenzyme NAD+ is converted to NADH.
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Figure 1: Oxidative demethylation of DNA by AlkB coupled with FDH reaction. The demethylation#of 1mA or 3mC in DNA by AlkB and production of formaldehyde are shown schematically. Also shown are the hydroxylated base intermediates and the conversion of formaldehyde to formate by FDH through a coupled reaction. In the process, coenzyme NAD+ is converted to NADH.

Mentions: The AlkB protein of Escherichia coli, and its orthologs in other organisms, catalyzes the removal of alkyl groups from DNA through oxidation in the form of aldehydes and its major substrates are 1-methyladenine (1mA) and 3-methylcytosine (3mC). Additionally, it also repairs 3-methylthymine, 1-methylguanine, 1,N6-ethenoadenine and 3,N4-etheno cytosine (1,4–6). These lesions occur preferentially in single-stranded (SS) DNA due to the availability of affected atoms (N1 of purines and N3 of pyrimidines) to participate in SN2 type additions. In duplex DNA, these atoms are involved in Watson–Crick base pairing and are inaccessible. AlkB is a member of the non-heme iron (II) α-ketoglutarate-dependent dioxygenase superfamily, which reverses alkylation damage to DNA through oxidation of the attached alkyl group creating a hydroxyalkyl intermediate that is unstable and decomposes to an oxidized alkylgroup (an aldehyde) and the regenerated base (5,7,8). The general scheme for this reaction is shown in Figure 1.Figure 1.


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

Roy TW, Bhagwat AS - Nucleic Acids Res. (2007)

Oxidative demethylation of DNA by AlkB coupled with FDH reaction. The demethylation#of 1mA or 3mC in DNA by AlkB and production of formaldehyde are shown schematically. Also shown are the hydroxylated base intermediates and the conversion of formaldehyde to formate by FDH through a coupled reaction. In the process, coenzyme NAD+ is converted to NADH.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175350&req=5

Figure 1: Oxidative demethylation of DNA by AlkB coupled with FDH reaction. The demethylation#of 1mA or 3mC in DNA by AlkB and production of formaldehyde are shown schematically. Also shown are the hydroxylated base intermediates and the conversion of formaldehyde to formate by FDH through a coupled reaction. In the process, coenzyme NAD+ is converted to NADH.
Mentions: The AlkB protein of Escherichia coli, and its orthologs in other organisms, catalyzes the removal of alkyl groups from DNA through oxidation in the form of aldehydes and its major substrates are 1-methyladenine (1mA) and 3-methylcytosine (3mC). Additionally, it also repairs 3-methylthymine, 1-methylguanine, 1,N6-ethenoadenine and 3,N4-etheno cytosine (1,4–6). These lesions occur preferentially in single-stranded (SS) DNA due to the availability of affected atoms (N1 of purines and N3 of pyrimidines) to participate in SN2 type additions. In duplex DNA, these atoms are involved in Watson–Crick base pairing and are inaccessible. AlkB is a member of the non-heme iron (II) α-ketoglutarate-dependent dioxygenase superfamily, which reverses alkylation damage to DNA through oxidation of the attached alkyl group creating a hydroxyalkyl intermediate that is unstable and decomposes to an oxidized alkylgroup (an aldehyde) and the regenerated base (5,7,8). The general scheme for this reaction is shown in Figure 1.Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48202, USA.

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.

Show MeSH
Related in: MedlinePlus