Although DNA is the carrier of genetic information, it has limited chemical stability. Hydrolysis, oxidation and nonenzymatic methylation of DNA occur at significant rates in vivo, and are counteracted by specific DNA repair processes. The spontaneous decay of DNA is likely to be a major factor in mutagenesis, carcinogenesis and ageing, and also sets limits for the recovery of DNA fragments from fossils.

We report here that FTO (fat mass and obesity-associated protein) exhibits efficient oxidative demethylation activity of abundant N6-methyladenosine (m6A) residues in RNA in vitro. FTO knockdown with siRNA led to an increased level of m6A in mRNA, whereas overexpression of FTO resulted in a decreased level of m6A in human cells. We further show that FTO partially colocalizes with nuclear speckles, supporting m6A in nuclear RNA as a physiological substrate of FTO.

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)-and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)-and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the Copyright 2007 by the American Association for the Advancement of Science; all rights reserved. ||To whom correspondence should be addressed. E-mail: chris.ponting@dpag.ox.ac.uk (C.P.P.); frances.ashcroft@dpag.ox.ac.uk (F.M.A.); so104@medschl.cam.ac.uk (S.O.); christopher.schofield@chem.ox.ac.uk (C.J.S.). * These authors contributed equally to this work. † These authors contributed equally to this work. ‡ These authors contributed equally to this work. § These authors contributed equally to this work. Recent studies have revealed a strong association between common variants in the first intron of FTO and obesity in both children and adults, with ~16% of studied populations homozygous for the risk alleles (1-4). As adults, these individuals weigh ~3 kg more than those homozygous for the low risk alleles as a result of a specific increase in fat mass (2). FTO mRNA is expressed in a wide range of human tissues (2). The Fto gene was first cloned after identification of a fused-toe mutant mouse whose phenotype results from a 1.6-Mb deletion of six genes, including Fto (5).Sequence analysis predicts that FTO protein contains a double-stranded beta-helix (DSBH) fold homologous to those of Fe(II) and 2-oxoglutarate (2OG) oxygenases [for a review of these enzymes, see (6)] (Fig. 1). The predicted DSBH fold of FTO contains four conserved residues characteristic of Fe(II) and 2OG binding sites (7,8), and its sequence is highly conserved in organisms ranging from mammals to green algae ( Fig. 1 and fig. S1). 2OG oxygenases are involved in diverse processes, including DNA repair, fatty acid metabolism, and posttranslational modifications, for example, proline hydroxylation and histone lysine demethylation [reviewed in (6, 9)]. They require nonheme iron [Fe(II)] as a cofactor, use oxygen and, almost always, 2OG as cosubstrates, and produce succinate and carbon dioxide as by-products.To determine whether FTO is a 2OG oxygenase, we expressed the murine Fto gene in Escherichia coli and purified N-terminally hexa-His tagged Fto (10). Some 2OG oxygenases catalyze 2OG turnover without a "prime" substrate provided that a reducing agent, typically ascorbate, is present (uncoupled turnover We next considered the identity of the prime FTO substrate. Among 2OG oxygenases with known substrates, the FTO sequence is most similar to that of the E. coli enzyme AlkB (11) and its eukaryotic hom...

The abundant nuclear enzyme poly(ADP-ribose) polymerase catalyses the synthesis of poly(ADP-ribose) from nicotinamide adenine dinucleotide (NAD+). This protein has an N-terminal DNA-binding domain containing two zinc-fingers, which is linked to the C-terminal NAD(+)-binding domain by a short region containing several glutamic acid residues that are sites of auto-poly(ADP-ribosyl)ation. The intracellular production of poly(ADP-ribose) is induced by agents that generate strand interruptions in DNA. The branched homopolymer chains may attain a size of 200-300 residues but are rapidly degraded after synthesis. The function of poly(ADP-ribose) synthesis is not clear, although it seems to be required for DNA repair. Here we describe a human cell-free system that enables the role of poly(ADP-ribose) synthesis in DNA repair to be characterized. The results indicate that unmodified polymerase molecules bind tightly to DNA strand breaks; auto-poly(ADP-ribosyl)ation of the protein then effects its release and allows access to lesions for DNA repair enzymes.

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.

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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 ...