Methylating agents generate cytotoxic and mutagenic DNA damage. Cells use 3-methyladenine-DNA glycosylases to excise some methylated bases from DNA, and suicidal O(6)-methylguanine-DNA methyltransferases to transfer alkyl groups from other lesions onto a cysteine residue. 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. Theoretical protein fold recognition had suggested that AlkB resembles the Fe(ii)- and alpha-ketoglutarate-dependent dioxygenases, which use iron-oxo intermediates to oxidize chemically inert compounds. 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, alpha-ketoglutarate and Fe(ii). The AlkB enzyme couples oxidative decarboxylation of alpha-ketoglutarate to the hydroxylation of these methylated bases in DNA, resulting in direct reversion to the unmodified base and the release of formaldehyde.

The Escherichia coli AlkB protein protects against the cytotoxicity of methylating agents by repair of the DNA lesions 1-methyladenine and 3-methylcytosine, which are generated in singlestranded stretches of DNA. AlkB is an ␣-ketoglutarate-and Fe(II)-dependent dioxygenase that oxidizes the relevant methyl groups and releases them as formaldehyde. Here, we identify two human AlkB homologs, ABH2 and ABH3, by sequence and fold similarity, functional assays, and complementation of the E. coli alkB mutant phenotype. The levels of their mRNAs do not appear to correlate with cell proliferation but tissue distributions are different. Both enzymes remove 1-methyladenine and 3-methylcytosine from methylated polynucleotides in an ␣-ketoglutarate-dependent reaction, and act by direct damage reversal with the regeneration of the unsubstituted bases. AlkB, ABH2, and ABH3 can also repair 1-ethyladenine residues in DNA with the release of acetaldehyde.A lthough single-stranded regions of DNA occur in vivo within replication forks and transcription bubbles, the susceptibility of single-stranded DNA to alkylating agents has been little investigated. The major lesions generated in single-stranded DNA are 1-alkyladenine and 3-alkylcytosine; these modification sites are protected by the complementary strand in duplex DNA (1). The 3-methylcytosine (3-meC) lesions block replication and are potentially cytotoxic (2). The Escherichia coli AlkB function counteracts toxicity by alkylating agents and its expression is induced by exposure to such agents (3, 4). Expression of E. coli AlkB in mammalian cells also confers increased resistance to alkylating agents (5). We have shown that AlkB specifically repairs alkylation damage in single-stranded DNA in vivo, and binds preferentially to single-stranded DNA in vitro (6). These results indicated that AlkB repairs 1-methyladenine (1-meA) and͞or 3-meC residues in DNA, but the reaction mechanism was unknown. In an important lead, protein fold analysis combined with weak sequence homology suggested that AlkB is a member of the family of ␣-ketoglutarate (␣KG)-and Fe(II)-dependent dioxygenases (7). These enzymes are involved in a variety of metabolic reactions; however, a fungal member of the family can perform catabolic oxidative demethylation of the free base 1-methylthymine (8). Biochemical assays with purified AlkB protein recently demonstrated that AlkB is indeed an ␣KG-and Fe(II)-dependent dioxygenase that oxidatively demethylates 1-meA and 3-meC residues in single-stranded as well as doublestranded DNA. The methyl group is released from the lesion as free formaldehyde, with accompanying regeneration of the unsubstituted base residue in DNA (9, 10).Because alkylating agents are environmental carcinogens, and also are used clinically as cytotoxic anticancer drugs, it was of interest to determine whether human cells have a counterpart to the E. coli AlkB protein. Here, we identify and characterize two human AlkB homologs encoded on different chromosomes. Materials and MethodsSingle-Stranded DNA ...

Methylation of histone tails is a key determinant in forming active and silent states of chromatin. Histone methylation was regarded as irreversible until the recent identification of a lysinespecific histone demethylase (LSD1), which acts specifically on mono-and dimethylated histone H3 lysine 4. Here, we propose that the fission yeast protein Epe1 is a putative histone demethylase that could act by oxidative demethylation. Epe1 modulates the stability of silent chromatin and contains a JmjC domain. The Epe1 protein can be modelled onto the structure of the 2-oxoglutarate-Fe(II)-dependent dioxygenase, factor inhibiting hypoxia inducible factor (FIH), which is a protein hydroxylase that also contains a JmjC domain. Thus, Epe1 and certain other chromatin-associated JmjC-domain proteins may be protein hydroxylases that catalyse a novel histone modification. Another intriguing possibility is that, by hydroxylating the methyl groups, Epe1 and certain other JmjC-domain proteins may be able to demethylate mono-, di-or trimethylated histones.

Heterochromatin normally has prescribed chromosomal positions and must not encroach on adjacent regions. We demonstrate that the fission yeast protein Epe1 stabilises silent chromatin, preventing the oscillation of heterochromatin domains. Epe1 loss leads to two contrasting phenotypes: alleviation of silencing within heterochromatin and expansion of silent chromatin into neighbouring euchromatin. Thus, we propose that Epe1 regulates heterochromatin assembly and disassembly, thereby affecting heterochromatin integrity, centromere function and chromosome segregation fidelity. Epe1 regulates the extent of heterochromatin domains at the level of chromatin, not via the RNAi pathway. Analysis of an ectopically silenced site suggests that heterochromatin oscillation occurs in the absence of heterochromatin boundaries. Epe1 requires predicted iron- and 2-oxyglutarate (2-OG)-binding residues for in vivo function, indicating that it is probably a 2-OG/Fe(II)-dependent dioxygenase. We suggest that, rather than being a histone demethylase, Epe1 may be a protein hydroxylase that affects the stability of a heterochromatin protein, or protein–protein interaction, to regulate the extent of heterochromatin domains. Thus, Epe1 ensures that heterochromatin is restricted to the domains to which it is targeted by RNAi.