1. Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK
2. Departments of Chemistry or Microbiology & Molecular Genetics, Michigan State University, East Lansing, Michigan 48824, USA
3. These authors contributed equally to this work

Correspondence to: Robert P. Hausinger³Barbara Sedgwick¹ Correspondence and requests for materials should be addressed to B.S. (e-mail: Email: b.sedgwick@cancer.org.uk) or R.P.H. (e-mail: Email: hausinge@msu.edu).

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 residue^{1, 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 DNA^{3, 4, 5}. Theoretical protein fold recognition had suggested that AlkB resembles the Fe(ii)- and -ketoglutarate-dependent dioxygenases⁶, which use iron-oxo intermediates to oxidize chemically inert compounds^{7, 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, -ketoglutarate and Fe(ii). The AlkB enzyme couples oxidative decarboxylation of -ketoglutarate to the hydroxylation of these methylated bases in DNA, resulting in direct reversion to the unmodified base and the release of formaldehyde.