Journal of the American Chemical Society Figure 1. Chemical structures of EA, m6A, m1A, and eA and proposed mechanisms of AlkB-mediated enzymatic reactions on alkyl-DNA adducts. (a) EA, (b) m6A, (c) m1A, and (d) eA. The AlkB protein is an α-ketoglutarate- and Fe(II)- dependent dioxygenase that can eﬃciently repair various alkyl lesions in both DNA and RNA, in single- and double-stranded contexts.11,12 It has at least nine human homologues: ABH1 through ABH8 and FTO, some of which have also been proposed or proven to act upon alkylated DNA and RNA.2,13−15 AlkB, together with Ada, AlkA, and AidB, is one of the four proteins in the E. coli adaptive response to alkylating agents.14 The reported substrate scope for the AlkB enzyme includes 1-methyladenine (m1A), 3-methylcytosine (m3C), 3- ethylcytosine (e3C), 1-methylguanine (m1G), 3-methylthy- mine (m3T), 3,N4-ethenocytosine (eC), eA, and EA.2,13−15 In the current work, we used high-resolution ESI-TOF mass spectrometry16 to investigate the ability of AlkB to repair EA. To our surprise we found that the oxidizing enzyme AlkB is capable of complete repair of the EA base (Figure 1a). The observation of AlkB’s complete conversion of EA to adenine along a deﬁned reaction path makes plausible the strategy of Article designing inhibitors to block the repair activity, thus potentially increasing the anticancer eﬃcacy of BCNU. The EA dealkylation process requires two oxidation reactions, one at N1 and a second at N6, to achieve complete restoration of the undamaged adenine base. The observation of N6 oxidation in the reaction of AlkB with EA suggested that the enzyme might act on other N6-modiﬁcations of adenine. One such modiﬁcation is N6-methyladenine (m6A) (Figure 1b). The m6A base is functionally similar to N4- and 5- methylcytosine (m4C and m5C), and is an important epigenetic signal for DNA replication and repair, protein− DNA interactions, host-pathogen interactions and other cellular processes;17−20 there is approximately one m6A for every 200 bases in the E. coli genome.21 As controls against which to compare the chemistry of AlkB on m6A (and EA), we used m1A and eA (Figure 1c and 1d), which are good substrates for AlkB both in vivo and in vitro.10,22 As we were preparing this manuscript, Jia et al. reported that the obesity-associated FTO protein (one of AlkB’s mammalian homologues) can remove the methyl group from m6A in both RNA and DNA; they indicated that the demethylation of FTO on m6A in mRNA correlates with epigenetic regulation in mammalian cells.23 In our present study, we observe that AlkB can also demethylate m6A. The implications of this discovery include the possibility that AlkB might be not only a DNA repair enzyme in E. coli but also a participant in cellular epigenetic control, which may help cells defend against alkylation damage to nucleic acids. ■ RESULTS This study utilized chemical synthesis to incorporate structurally deﬁned lesions into speciﬁc sites of oligonucleo- tides. The modiﬁed oligonucleotides were allowed to react with puriﬁed AlkB and snapshots were taken at various time points to identify the products and intermediates of reactions. This method enabled us to eﬃciently monitor the ability of AlkB to repair alkylation damage using a high-resolution mass spectrometry tool employed by us earlier with a number of DNA lesions.6,10,16 Four 16mer oligonucleotides were chemically synthesized containing the alkyl-DNA adducts shown in Figure 1. The sequence used was 5′-GAAGACCTXGGCGTCC-3′; the ﬂanking sequence of each lesion (X) was identical, ﬁxing the local sequence context for the repair reaction. The 16mers with individual lesions were incubated with the cofactors for the AlkB reaction: Fe(II) and α-ketoglutarate (see Experimental Section). In the presence of all cofactors, two sets of experiments were conducted for each lesion, one with the AlkB protein and one with the puriﬁcation buﬀer of AlkB but without the protein. Following the repair reaction, HPLC-ESI- TOF mass spectrometry was utilized to identify the products and intermediates. The 16mer oligonucleotides demonstrated a good signal in the −4 charge envelope of the mass spectra. To give one example to illustrate the method of analysis, the molecular weight (MW) of m1A in the 16mer is calculated as 4902.88 Da for the neutral species, and the MW of its monoisotopic peak (all 12C, 14N, etc.) when ionized with four negative charges (the −4 charge envelope) in the electric ﬁeld of the spectrometer is calculated as having an m/z of 1224.71 (all MWs are shown in Table 1). The multiple peaks in each −4 charge envelope reﬂect the number of 13C or other heavier isotopes (Figure 2a). Keeping with m1A as the example, its monoisotopic peak is observed as 1224.67, consistent with the theoretical calculation. The next peak in that envelope has an 8897 dx.doi.org/10.1021/ja3010094 | J. Am. Chem. Soc. 2012, 134, 8896−8901