Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage

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Abstract

Methylating agents generate cytotoxic and mutagenic DNA damage. Cells use 3-methyladenine-DNA glycosylases to excise some methylated bases from DNA, and suicidal O6-methylguanine-DNA methyltransferases to transfer alkyl groups from other lesions onto a cysteine residue1,2. Here we report that the highly conserved AlkB protein repairs DNA alkylation damage by means of an unprecedented mechanism. AlkB has no detectable nuclease, DNA glycosylase or methyltransferase activity; however, Escherichia coli alkB mutants are defective in processing methylation damage generated in single-stranded DNA3,4,5. Theoretical protein fold recognition had suggested that AlkB resembles the Fe(ii)- and α-ketoglutarate-dependent dioxygenases6, which use iron-oxo intermediates to oxidize chemically inert compounds7,8. We show here that purified AlkB repairs the cytotoxic lesions 1-methyladenine and 3-methylcytosine in single- and double-stranded DNA in a reaction that is dependent on oxygen, α-ketoglutarate and Fe(ii). The AlkB enzyme couples oxidative decarboxylation of α-ketoglutarate to the hydroxylation of these methylated bases in DNA, resulting in direct reversion to the unmodified base and the release of formaldehyde.

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Figure 1: Release of ethanol-soluble material from methylated poly(dA) by AlkB in the presence of Fe(ii) and α-ketoglutarate.
Figure 2: Repair of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) by AlkB.
Figure 3: Production of formaldehyde and consumption of O2 by AlkB.
Figure 4: Schematic representation of the repair of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues in DNA by oxidative demethylation catalysed by AlkB.

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Acknowledgements

We thank J. McCracken for advice, and T. Duncan and P. Robins for technical help. This work was supported by Cancer Research UK, and by NIH grants to R.P.H. and J. McCracken.

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Correspondence to Robert P. Hausinger or Barbara Sedgwick.

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The authors declare that they have no competing financial interests.

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Trewick, S., Henshaw, T., Hausinger, R. et al. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage. Nature 419, 174–178 (2002) doi:10.1038/nature00908

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