Journal of the American Chemical Society BCNU. Knowledge of intermediates along the repair pathway would enable those studies. Hypothesis on the Biological Function of m6A Demethylation by AlkB. Unlike m1A, eA, or EA, the m6A moiety is normally not considered a lesion. Rather, it is an important postreplicative DNA modiﬁcation17−20 and is considered the “sixth element of DNA” (m5C being the ﬁfth element in addition to the regular four DNA bases).19 The results presented here imply that AlkB might not only act as a repair enzyme to defend against exogenous or endogenous attacks by alkylating agents but may also help the cell to control its replication cycle. Besides its direct reversal repair of alkyl lesions, such as m1A and eA, AlkB, which is strongly induced by DNA damage, might also demethylate m6A to stop the cell replication cycle temporarily and provide extra time for the cell to repair alkyl adducts. After the majority of alkyl lesions are removed, AlkB and other repair enzymes can be degraded or down-regulated, followed by progression of the normal cell cycle. The epigenetic control of oxidative demethylation of m6A and its inﬂuence on cellular function regulation is also supported by the recent discovery of FTO’s demethylation of m6A.23 ■ CONCLUSIONS In this study, we demonstrated that AlkB can demethylate m6A and completely convert EA to undamaged adenine and other intermediates. These results indicate that AlkB can act not only as a DNA repair enzyme but also as a component that may modulate the cellular replication cycle, thus aiding E. coli in surviving alkylation attacks. Further investigation of the cellular functions of AlkB will provide insights into AlkB’s defense mechanisms for the cellular response to environmental attacks as well as the design and development of inhibitors for AlkB’s human homologues to improve treatments for cancer therapy. ■ EXPERIMENTAL SECTION Oligonucleotide Synthesis. Oligonucleotides containing the adducts in Figure 1 were prepared using the solid-phase phosphor- amidite method, and were deprotected, puriﬁed, and identiﬁed as described previously.10,22 For all four adducts, the oligonucleotide sequence of the 16mers was 5′-GAAGACCTXGGCGTCC-3′, (X = adduct). The calculated MWs of the oligonucleotides and intermediates are shown in Table 1. DNA concentration was measured by UV absorbance using the extinction coeﬃcients (ε) at 260 nm. For any alkyl-modiﬁed base, we substituted its extinction coeﬃcient with the extinction coeﬃcient of its unmodiﬁed counterpart due to the tiny diﬀerence between the values in the context of a 16mer oligonucleotide. In vitro Incubation Reactions of DNA Adducts with AlkB. All assays were carried out with AlkBΔN11 protein, a truncated version of AlkB in which the ﬁrst eleven residues were removed. AlkBΔN11 protein was puriﬁed as described and shown previously to have similar activity to the wild type protein.6 All AlkB incubation reactions utilized similar conditions as previously described.10,16 Reactions were performed at 37 °C in 45 mM HEPES (pH 8.0), 0.9 mM α- ketoglutarate, 67 μM Fe(NH4)2(SO4)2·6H2O, and 1.8 mM ascorbate, followed by dry ice storage until HPLC-ESI-TOF MS analysis. A typical reaction was performed with 5 μM DNA, all cofactors, and with or without 2.5 μM AlkB in a 10-μL volume for 1 h. In the case of a reaction with no AlkB protein added, the elution buﬀer for AlkB puriﬁcation was added instead. For the time and volumetric dependency reactions (Figures 4 in text and S1−S4 in SI), the reaction time and volumetric ratio varied correspondingly. HPLC-ESI-TOF MS analysis. Oligonucleotide analyses were carried out on an Agilent ESI-TOF mass spectrometer (Palo Alto, ■ ■ ■ Article CA). ESI was performed using a needle voltage of 3.5 kV. Nitrogen gas was set with drying at 10 L/min, the heated capillary at 325 °C and the nebulizer set at 15 psig. HPLC separations were performed by using a Zorbax SB-Aq column (2.1 mm × 150 mm; 3.5 μm; Agilent Technologies, Palo Alto, CA) with a ﬂow rate of 0.2 mL/min. Solvent A used 10 mM ammonium acetate in water; solvent B used 100% acetonitrile. A linear gradient was carried out in the following steps: 2 to 30% B over 30 min, 30 to 2% B over 5 min, and 2 to 2% B over 10 min. LC−MS analyses were normally carried out at room temperature except for the data presented in Figure 4, which were collected with the temperature of the autosampler set at 5 °C. Data analyses used the Agilent MassHunter Workstation software7. ■ ASSOCIATED CONTENT *S Supporting Information Table S1 and Figures S1−S4. This material is available free of charge via the Internet at http://pubs.acs.org. 8900 dx.doi.org/10.1021/ja3010094 | J. Am. Chem. Soc. 2012, 134, 8896−8901 AUTHOR INFORMATION Corresponding Author jessig@mit.edu Present Addresses #Visterra Inc., Cambridge, MA 02139. ¶Chemistry Department, Yale University, New Haven, CT, 06520. ○School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, Peoples Republic of China 300072. □Harvard Medical School, 25 Shattuck Street, Boston, MA 02115. △Johnson & Johnson Pharmaceutical Research & Develop- ment, 930 Route 202 South, Raritan, NJ 08869. Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants CA080024, CA26731, and ES02109. C.L.D. is a Howard Hughes Medical Institute Investigator. We thank Dr. John. S. Wishnok for helpful discussions. We also thank the Wishnok/Tannenbaum Mass Lab and the MIT Center for Environmental Health Sciences for providing the ESI-TOF mass spectrometry facility and Agilent Technologies for providing the UHPLC system. REFERENCES (1) Loeb, L. A.; Harris, C. C. Cancer Res. 2008, 68, 6863. (2) Shrivastav, N.; Li, D.; Essigmann, J. M. Carcinogenesis 2010, 31, 59. (3) Ludlum, D. B. IARC Sci. Publ. 1986, 70, 137. (4) Ludlum, D. B. IARC Sci. Publ. 1986, 78, 71. (5) Ludlum, D. B. Mutat. Res. 1990, 233, 117. (6) Frick, L. E.; Delaney, J. C.; Wong, C.; Drennan, C. L.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 755. (7) Guliaev, A. B.; Hang, B.; Singer, B. Nucleic Acids Res. 2002, 30, 3778. (8) Hang, B.; Chenna, A.; Guliaev, A. B.; Singer, B. Mutat. Res. 2003, 531, 191. (9) Guliaev, A. B.; Singer, B.; Hang, B. DNA Repair 2004, 3, 1311. (10) Delaney, J. C.; Smeester, L.; Wong, C.; Frick, L. E.; Taghizadeh, K.; Wishnok, J. S.; Drennan, C. L.; Samson, L. D.; Essigmann, J. M. Nat. Struct. Mol. Biol. 2005, 12, 855. (11) Trewick, S. C.; Henshaw, T. F.; Hausinger, R. P.; Lindahl, T.; Sedgwick, B. Nature 2002, 419, 174. (12) Falnes, P. O.; Bjoras, M.; Aas, P. A.; Sundheim, O.; Seeberg, E. Nucleic Acids Res. 2004, 32, 3456.