Oxidative demethylation of DNA damage by Escherichia coli AlkB and its human homologs ABH2 and ABH3

The E. coli AlkB protein was implicated in the repair or tolerance of DNA methylation damage. However, despite the early isolation of an E. coli alkB mutant, the function of the AlkB protein had not been resolved (Kataoka et al., 1983). The E. coli alkB mutant is defective in processing methylated single stranded DNA, therefore, it was suggested that the AlkB protein either repairs or tolerates lesions generated in single stranded DNA, such as 1-methyladenine (1-meA) or 3-methylcytosine (3-meC), or that AlkB only acts on single stranded DNA (Dinglay et al, 2000). However, despite extensive testing, no enzymatic activity could be assigned to the AlkB protein. Recently, theoretical protein fold recognition suggested that the AlkB protein resembles members of the α-ketoglutarate-Fe(II) dependent dioxygenase superfamily (Aravind and Koonin, 2001). Here, the biochemical function of the enigmatic E. coli AlkB protein and its two human homologs ABH2 and ABH3 are elucidated. An in vitro assay was developed for the AlkB, ABH2 and ABH3 proteins and it was demonstrated that the activities of these proteins are dependent on α-ketoglutarate and Fe(II) and are stimulated by ascorbic acid. The requirement of AlkB, ABH2 and ABH3 for these distinctive co-factors strongly supports the proposal that these proteins are members of the α-ketoglutarate-Fe(II) dependent dioxygenase superfamily, which employ iron-oxo intermediates to oxidise chemically inert compounds. The AlkB, ABH2 and ABH3 proteins are shown to act specifically on 1-meA and 3-meC in both double and single stranded DNA. It was demonstrated that these proteins convert 1-meA and 3-meC to their unsubstituted parent residues in DNA and therefore act by a direct reversal mechanism. It is proposed that the E. coli AlkB protein and its human homologs directly revert DNA damage by oxidative demethylation, an unprecedented mechanism of DNA repair. This discovery may have implications for the treatment of cancer because antagonists of the human ABH2 and ABH3 proteins could be useful adjuncts to cancer chemotherapy that uses simple alkylating agents.