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 ...

Highlights d High-binding-efficiency ligands of METTL3-14-WTAP are identified in silico d The binding mode of compounds overlaps with the METTL3-14-WTAP active site d The compounds are experimentally characterized as METTL3-14-WTAP activators d The compounds affect m 6 A level in mRNA and rRNA in cells

The Escherichia coli AlkB protein, and two human homologs ABH2 and ABH3, directly demethylate 1-methyladenine and 3-methylcytosine in DNA. They couple Fe(II)-dependent oxidative demethylation of these damaged bases to decarboxylation of ␣-ketoglutarate. Here, we have determined the kinetic parameters for AlkB oxidation of 1-methyladenine in poly(dA), short oligodeoxyribonucleotides, nucleotides, and nucleoside triphosphates. Methylated poly(dA) was the preferred AlkB substrate of those tested. The oligonucleotide trimer d(Tp1meApT) and even 5-phosphorylated 1-medAMP were relatively efficiently demethylated, and competed with methylated poly(dA) for AlkB activity. A polynucleotide structure was clearly not essential for AlkB to repair 1-methyladenine effectively, but a nucleotide 5 phosphate group was required. Consequently, 1-me-dAMP(5) was identified as the minimal effective AlkB substrate. The nucleoside triphosphate, 1-medATP, was inefficiently but actively demethylated by AlkB; a reaction with 1-me-ATP was even slower. E. coli DNA polymerase I Klenow fragment could employ 1-medATP as a precursor for DNA synthesis in vitro, suggesting that demethylation of alkylated deoxynucleoside triphosphates by AlkB could have biological significance. Although the human enzymes, ABH2 and ABH3, demethylated 1-methyladenine residues in poly(dA), they were inefficient with shorter substrates. Thus, ABH3 had very low activity on the trimer, d(Tp1meApT), whereas no activity was detected with ABH2. AlkB is known to repair methyl and ethyl adducts in DNA; to extend this substrate range, AlkB was shown to reduce the toxic effects of DNA damaging agents that generate hydroxyethyl, propyl, and hydroxypropyl adducts.Alkylating agents occur in the environment and arise endogenously during cellular metabolism. They damage DNA at multiple sites, and the lesions generated may result in mutagenesis and cell death. The importance of preventing these adverse effects is highlighted by the variety of DNA repair mechanisms that have evolved to remove alkylated bases from DNA. These repair functions are conserved from bacteria to humans. In Escherichia coli, the activities are induced up to 1000-fold (1) in the Ada response (the adaptive response to alkylating agents).3-Methyladenine-DNA glycosylases excise cytotoxic 3-methyladenine and related lesions from DNA in the first step of base excision repair (2). In contrast, suicidal O 6 -methylguanine-DNA methyltransferases directly demethylate the highly mutagenic and toxic lesion O 6 -methylguanine by transferring the methyl group onto a cysteine residue in the protein (3). A third type of DNA repair mechanism specific for alkylated bases was recently resolved. The E. coli AlkB protein (4) and its human homologs, ABH2 and ABH3, oxidize the methyl groups of 1-methyladenine (1-meA) 1 and 3-methylcytosine (3-meC) in DNA to directly regenerate unmodified adenine and cytosine residues. The methyl adduct is released as formaldehyde (5-8). AlkB and its human counterparts are members of the ␣-ketoglutar...

Rare DNA lesions that are chemically stable and refractory to repair may add disproportionately to the accumulation of mutations in long lived cells. 3-Methylthymine is a minor lesion that is induced by DNA-methylating agents and for which no repair process has been described previously. Here we demonstrate that this lesion can be directly demethylated in vitro by bacterial and human DNA dioxygenases. The Escherichia coli AlkB and human ABH3 proteins repaired 3-methylthymine in both single-stranded and double-stranded polydeoxynucleotides, whereas the human ABH2 protein preferred a duplex substrate. Thus, the known substrates of these enzymes now include 3-methylthymine in DNA, as well as 1-methyladenine and 3-methylcytosine, which all have structurally similar sites of alkylation. Repair of 3-methylthymine by AlkB and ABH3 was optimal at pH 6, but inefficient. At physiological pH, 3-methylthymine, which is a minor methylated lesion, was more slowly repaired than the major lesion generated in single-stranded DNA, 3-methylcytosine. Our data suggest that 3-methylthymine residues in DNA will be repaired inefficiently in vivo and therefore may occur at a low steady-state level, but the residues should not gradually accumulate to high levels in long lived cells.

The 2-oxoglutarate (2OG)-and Fe 2 þ -dependent dioxygenase AlkB couples the demethylation of modified DNA to the decarboxylation of 2OG. Extensive crystallographic analyses have shown no evidence of significant structural differences between complexes binding either 2OG or succinate. By using nuclear magnetic resonance spectroscopy, we have shown that the AlkB-succinate and AlkB-2OG complexes have significantly different dynamic properties in solution. 2OG makes the necessary contacts between the metal site and the large b-sheet to maintain a fully folded conformation. Oxidative decarboxylation of 2OG to succinate leads to weakening of a main contact with the large b-sheet, resulting in an enhanced dynamic state. These conformational fluctuations allow for the replacement of succinate in the central core of the protein and probably contribute to the effective release of unmethylated DNA. We also propose that the inherent dynamics of the co-product complex and the subsequent increased molecular ordering of the co-substrate complex have a role in DNA damage recognition.

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Butadiene (BD) is a high production volume chemical and is known to be tumorigenic in rodents. BD is metabolized to butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). These epoxides are genotoxic and alkylate DNA both in vitro and in vivo, mainly at the N7 position of guanine. In this study, a 32P-post-labeling/thin-layer chromatography (TLC)/high-pressure liquid chromatography (HPLC) assay for BDE and DEB adducts at the N7 of guanine was developed and was used in determining the enantiomeric composition of the adducts and the organ dose of BD exposure in lung. Exposure of 2'-deoxyguanosine (dGuo), 2'-deoxyguanosine-5'-phosphate (5'-dGMP) and 2'-deoxyguanosine-3'-phosphate (3'-dGMP) to racemic BDE followed by neutral thermal hydrolysis gave two products (products 1 and 2) that were identified by MS and UV and NMR spectroscopy as a diastereomeric pair of N7-(2,3,4-trihydroxybutan-1-yl)-guanines. Exposure of dGuo nucleotides to RR/SS DEB (also referred to as dl DEB) followed by thermal depurination resulted in a single product coeluting with the BDE product 1. If the reaction mixture of BDE and 5'-dGMP was analyzed by HPLC before hydrolysis of the glycosidic bond, four major nucleotide alkylation products (A, B, C and D) with identical UV sepectra were detected. The products were isolated and hydrolyzed, after which A and C coeluted with product 1 and B and D coeluted with the product 2. The major adduct of DEB-exposed 5'-dGMP was N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP (product E). A 32P-post-labeling assay was used to detect BDE- and DEB-derived N7-dGMP adducts in DNA. Levels of adducts increased with a dose of BDE and DEB and exhibited a half life of 30 +/- 3 (r = 0.98) and 31 +/- 4 h (r = 0.95), respectively. Incubation of DEB-modified DNA at 37 degrees C at neutral pH for up to 142 h did not lead to an increase of N7-(2,3,4-trihydroxybutan-1-yl)-dGMP in the DNA. These observations led to the conclusion that the N7-(2,3, 4-trihydroxybutan-1-yl)-dGMP adducts in DNA can be used as a marker of BDE exposure and that N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP adducts are related to DEB exposure. Dose-related levels of BDE- and DEB-derived adducts were detected in lungs of mice inhaling butadiene. Most of the N7-dGMP adducts (73%; product D) were derived from the 2R-diol-3S-epoxide of 1,3-butadiene. The data presented in this paper indicate that in vivo, 98% of N7-dGMP alkylation after BD exposure is derived from BDE, and approximately 2% of the adducts were derived from DEB and BMO.

We present here the first mass spectroscopic (MS) identification of the main tamoxifen-induced DNA adducts in rat liver. The two main adducts were isolated by high-performance liquid chromatography (HPLC) and identified by MS, MS-MS and ultraviolet spectroscopy. Adduct 1 was the N-desmethyltamoxifen-deoxyguanosine adduct in which the alpha-position of the metabolite N-desmethyltamoxifen is linked covalently to the amino group of deoxyguanosine. Adduct 2 was confirmed to be the trans isomer of alpha-(N2-deoxyguanosinyl)tamoxifen, as previously suggested by co-chromatography.