AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli

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Abstract

The bacterial AlkB protein is known to be involved in cellular recovery from alkylation damage; however, the function of this protein remains unknown. AlkB homologues have been identified in several organisms, including humans, and a recent sequence alignment study has suggested that these proteins may belong to a superfamily of 2-oxoglutarate-dependent and iron-dependent oxygenases (2OG-Fe(ii)-oxygenases)1. Here we show that AlkB from Escherichia coli is indeed a 2-oxoglutarate-dependent and iron-dependent DNA repair enzyme that releases replication blocks in alkylated DNA by a mechanism involving oxidative demethylation of 1-methyladenine residues. This mechanism represents a new pathway for DNA repair and the third type of DNA damage reversal mechanism so far discovered.

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Figure 1: Repair of MMS-treated M13mp18 ssDNA by treatment with AlkB.
Figure 2: AlkB-mediated release of radioactivity from [3H]methyl DNA.
Figure 3: Reverse phase HPLC elution profiles of radioactivity released from [3H]methyl ssDNA by AlkB and/or acid treatment.
Figure 4: Oxidative demethylation of 1-methyladenine lesions in DNA by the AlkB protein.

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Acknowledgements

We thank M. Bjørås, L. Eide, K. Baynton, K. I. Kristiansen, J. Myllyharju, K. Skarstad and J. Klaveness for help and discussions, and L. Eide, K. Baynton and A. Klungland for critical reading of the manuscript. We are grateful to M. Bjørås for preparation of [3H]MNU-labelled DNA substrates, to L. Eide for the construction of the plasmid for expression of GST–AlkB, and to D. Daoudi for technical assistance. This work was supported by the Research Council of Norway and the Norwegian Cancer Society. E.S. also acknowledges support from the European Commission.

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Correspondence to Pål Ø. Falnes.

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Falnes, P., Johansen, R. & Seeberg, E. AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli. Nature 419, 178–182 (2002) doi:10.1038/nature01048

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