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Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase


Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology1,2, including the oxidative demethylation of methylated nucleic acids and histones3,4. Escherichia coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids5,6, which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes7,8,9,10,11. Here we present structural characterization of oxidation intermediates for these demethylases. Using a chemical cross-linking strategy12,13, complexes of AlkB–double stranded DNA (dsDNA) containing 1,N6-etheno adenine (εA), N3-methyl thymine (3-meT) and N3-methyl cytosine (3-meC) are stabilized and crystallized, respectively. Exposing these crystals, grown under anaerobic conditions containing iron(II) and α-ketoglutarate (αKG), to dioxygen initiates oxidation in crystallo. Glycol (from εA) and hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-meC) is also proposed, based on crystallographic observations and computational analysis. The observation of these unprecedented intermediates provides direct support for the oxidative demethylation mechanism for these demethylases. This study also depicts a general mechanistic view of how a methyl group is oxidatively removed from different biological substrates.

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Figure 1: Oxidative repair of damaged nucleic acid bases by AlkB.
Figure 2: Intermediates trapped during in crystallo oxidation of εA and 3-meT.
Figure 3: A zwitterionic intermediate 3 is proposed for the demethylation of 3-meC.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates are deposited in the Protein Data Bank under accessionnumbers 3O1M,3O1O,3O1P, 3O1R,3O1S,3O1T,3O1Uand3O1V.


  1. Kovaleva, E. G. & Lipscomb, J. D. Versatility of biological non-heme Fe(II) centers in oxygen activation reactions. Nature Chem. Biol. 4, 186–193 (2008)

    CAS  Article  Google Scholar 

  2. Schofield, C. J. & Zhang, Z. Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes. Curr. Opin. Struct. Biol. 9, 722–731 (1999)

    CAS  Article  Google Scholar 

  3. Yi, C., Yang, C. G. & He, C. A non-heme iron-mediated chemical demethylation in DNA and RNA. Acc. Chem. Res. 42, 530–541 (2009)

    Article  Google Scholar 

  4. Klose, R. J. & Zhang, Y. Regulation of histone methylation by demethylimination and demethylation. Nature Rev. Mol. Cell Biol. 8, 307–318 (2007)

    CAS  Article  Google Scholar 

  5. Falnes, P. O., Johansen, R. F. & Seeberg, E. AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli . Nature 419, 178–182 (2002)

    CAS  ADS  Article  Google Scholar 

  6. Trewick, S. C., Henshaw, T. F., Hausinger, R. P., Lindahl, T. & Sedgwick, B. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage. Nature 419, 174–178 (2002)

    CAS  ADS  Article  Google Scholar 

  7. Westbye, M. P. et al. Human AlkB homolog 1 is a mitochondrial protein that demethylates 3-methylcytosine in DNA and RNA. J. Biol. Chem. 283, 25046–25056 (2008)

    CAS  Article  Google Scholar 

  8. Ringvoll, J. et al. Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA. EMBO J. 25, 2189–2198 (2006)

    CAS  Article  Google Scholar 

  9. Aas, P. A. et al. Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. Nature 421, 859–863 (2003)

    CAS  ADS  Article  Google Scholar 

  10. Sundheim, O. et al. Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage. EMBO J. 25, 3389–3397 (2006)

    CAS  Article  Google Scholar 

  11. Frayling, T. M. et al. A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science 316, 889–894 (2007)

    CAS  ADS  Article  Google Scholar 

  12. Yang, C. G. et al. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA. Nature 452, 961–965 (2008)

    CAS  ADS  Article  Google Scholar 

  13. Qi, Y. et al. Encounter and extrusion of an intrahelical lesion by a DNA repair enzyme. Nature 462, 762–766 (2009)

    CAS  ADS  Article  Google Scholar 

  14. Gerken, T. et al. The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase. Science 318, 1469–1472 (2007)

    CAS  ADS  Article  Google Scholar 

  15. Jia, G. et al. Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO. FEBS Lett. 582, 3313–3319 (2008)

    CAS  Article  Google Scholar 

  16. Koivisto, P., Robins, P., Lindahl, T. & Sedgwick, B. Demethylation of 3-methylthymine in DNA by bacterial and human DNA dioxygenases. J. Biol. Chem. 279, 40470–40474 (2004)

    CAS  Article  Google Scholar 

  17. Falnes, P. O. Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins. Nucleic Acids Res. 32, 6260–6267 (2004)

    CAS  Article  Google Scholar 

  18. Delaney, J. C. & Essigmann, J. M. Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli . Proc. Natl Acad. Sci. USA 101, 14051–14056 (2004)

    CAS  ADS  Article  Google Scholar 

  19. Mishina, Y., Yang, C. G. & He, C. Direct repair of the exocyclic DNA adduct 1,N6-ethenoadenine by the DNA repair AlkB proteins. J. Am. Chem. Soc. 127, 14594–14595 (2005)

    CAS  Article  Google Scholar 

  20. Delaney, J. C. et al. AlkB reverses etheno DNA lesions caused by lipid oxidation in vitro and in vivo . Nature Struct. Mol. Biol. 12, 855–860 (2005)

    CAS  Article  Google Scholar 

  21. Yu, B. et al. Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB. Nature 439, 879–884 (2006)

    CAS  ADS  Article  Google Scholar 

  22. Yu, B. & Hunt, J. F. Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB. Proc. Natl Acad. Sci. USA 106, 14315–14320 (2009)

    CAS  ADS  Article  Google Scholar 

  23. Holland, P. J. & Hollis, T. Structural and mutational analysis of Escherichia coli AlkB provides insight into substrate specificity and DNA damage searching. PLoS ONE 5, e8680 (2010)

    ADS  Article  Google Scholar 

  24. Han, Z. et al. Crystal structure of the FTO protein reveals basis for its substrate specificity. Nature 464, 1205–1209 (2010)

    CAS  ADS  Article  Google Scholar 

  25. Schlichting, I. et al. The catalytic pathway of cytochrome p450cam at atomic resolution. Science 287, 1615–1622 (2000)

    CAS  ADS  Article  Google Scholar 

  26. Kovaleva, E. G. & Lipscomb, J. D. Crystal structures of Fe2+ dioxygenase superoxo, alkylperoxo, and bound product intermediates. Science 316, 453–457 (2007)

    CAS  ADS  Article  Google Scholar 

  27. Burzlaff, N. I. et al. The reaction cycle of isopenicillin N synthase observed by X-ray diffraction. Nature 401, 721–724 (1999)

    CAS  ADS  Article  Google Scholar 

  28. David, S. S., O’Shea, V. L. & Kundu, S. Base-excision repair of oxidative DNA damage. Nature 447, 941–950 (2007)

    CAS  ADS  Article  Google Scholar 

  29. Hitomi, K., Iwai, S. & Tainer, J. A. The intricate structural chemistry of base excision repair machinery: implications for DNA damage recognition, removal, and repair. DNA Repair 6, 410–428 (2007)

    CAS  Article  Google Scholar 

  30. Fromme, J. C., Banerjee, A. & Verdine, G. L. DNA glycosylase recognition and catalysis. Curr. Opin. Struct. Biol. 14, 43–49 (2004)

    CAS  Article  Google Scholar 

  31. Mishina, Y. & He, C. Probing the structure and function of the Escherichia coli DNA alkylation repair AlkB protein through chemical cross-linking. J. Am. Chem. Soc. 125, 8730–8731 (2003)

    CAS  Article  Google Scholar 

  32. Mishina, Y., Chen, L. X. & He, C. Preparation and characterization of the native iron(II)-containing DNA repair AlkB protein directly from Escherichia coli . J. Am. Chem. Soc. 126, 16930–16936 (2004)

    CAS  Article  Google Scholar 

  33. Read, R. J. Pushing the boundaries of molecular replacement with maximum likelihood. Acta Crystallogr. D 57, 1373–1382 (2001)

    CAS  Article  Google Scholar 

  34. Collaborative Computational Project, N. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  35. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  36. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  37. DeLano, W. L. The PyMOL molecular graphics system. <> (2002)

  38. Searls, T. & McLaughlin, L. W. Synthesis of the analogue nucleoside 3-deaza-2′-deoxycytidine and its template activity with DNA polymerase. Tetrahedron 55, 11985–11996 (1999)

    CAS  Article  Google Scholar 

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This study was supported by the National Institutes of Health (GM071440 to C.H.; GM084028 to Q.C.), beamlines 23ID-B (General Medicine and Cancer Institutes Collaborative Access Team (GM/CA-CAT)), 19BM-D (Structual Biology Center (SBC-CAT)), 14BM-C (BioCARS) and 21ID-D (Life Sciences Collaborative Access Team (LS-CAT)) at the Advanced Photon Source at Argonne National Laboratory, the National Institutes of Health and the United States Department of Energy. Computational resources from the National Center for Supercomputing Applications at the University of Illinois and the Center of High Throughput Computing at UW–Madison are appreciated. We also thank X. Yang, Z. Ren and E. Duguid for crystallographic discussions.

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C.Y., G.J. and C.H. designed the experiments. Experiments were performed by C.Y., G.J., Q.D., W.Z., G.Z., X.J. and C.-G.Y.; computational analyses were performed by G.H. and Q.C. C.Y. and C.H. wrote the paper and G.H., Q.D. and Q.C. contributed to editing the manuscript.

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Correspondence to Chuan He.

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The authors declare no competing financial interests.

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Yi, C., Jia, G., Hou, G. et al. Iron-catalysed oxidation intermediates captured in a DNA repair dioxygenase. Nature 468, 330–333 (2010).

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