Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA

Nature volume 452, pages 961–965 (2008)Cite this article

Abstract

Escherichia coli AlkB and its human homologues ABH2 and ABH3 repair DNA/RNA base lesions by using a direct oxidative dealkylation mechanism. ABH2 has the primary role of guarding mammalian genomes against 1-meA damage by repairing this lesion in double-stranded DNA (dsDNA), whereas AlkB and ABH3 preferentially repair single-stranded DNA (ssDNA) lesions and can repair damaged bases in RNA. Here we show the first crystal structures of AlkB–dsDNA and ABH2–dsDNA complexes, stabilized by a chemical cross-linking strategy. This study reveals that AlkB uses an unprecedented base-flipping mechanism to access the damaged base: it squeezes together the two bases flanking the flipped-out one to maintain the base stack, explaining the preference of AlkB for repairing ssDNA lesions over dsDNA ones. In addition, the first crystal structure of ABH2, presented here, provides a structural basis for designing inhibitors of this human DNA repair protein.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Crystal structures of the active site cross-linked AlkB–DNA1 and ABH2–DNA2 complexes.
Figure 2: Crystal structures of AlkB–DNA4 and AlkB–DNA5 complexes with 1-meA recognized by an intact active site.
Figure 3: Close views of the base-flipping regions.
Figure 4: Crystal structure of the ABH2–DNA6 complex with 1-meA recognized by an intact active site.
Figure 5: Structural comparison of AlkB, ABH2 and ABH3 (stereo view).

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates are deposited in Protein Data Bank under accession numbers 3BKZ ((Mn/2KG) AlkB–DNA1), 3BI3 ((Mn/2KG) AlkB–DNA5), 3BIE ((Mn/2KG)AlkB–DNA4), 3BTX (ABH2–DNA2), 3BTZ (ABH2–DNA3), 3BU0 ((Mn/2KG)ABH2–DNA2), 3BTY (ABH2–DNA6) and 3BUC ((Mn/2KG)ABH2–DNA6).

References

  1. Friedberg, E. C., Walker, G. C. & Siede, W. DNA Repair and Mutagenesis Ch. 1 (ASM Press, Washington DC, 1995)

    Google Scholar 

  2. Wood, R. D., Mitchell, M., Sgouros, J. & Lindahl, T. Human DNA repair genes. Science 291, 1284–1289 (2001)

    Article  ADS  CAS  Google Scholar 

  3. Sedgwick, B. Repairing DNA-methylation damage. Nature Rev. Mol. Cell Biol. 5, 148–157 (2004)

    Article  CAS  Google Scholar 

  4. Mishina, Y. & He, C. Oxidative dealkylation DNA repair mediated by the monomuclear non-heme iron AlkB proteins. J. Inorg. Biochem. 100, 670–678 (2006)

    Article  CAS  Google Scholar 

  5. Lindahl, T., Sedgewick, B., Sekiguchi, M. & Nakabeppu, Y. Regulation and expression of the adaptive response to alkylating agents. Annu. Rev. Biochem. 57, 133–157 (1988)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  7. 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)

    Article  ADS  CAS  Google Scholar 

  8. Aravind, L. & Koonin, E. V. The DNA-repair protein AlkB, EGL-9, and leprecan define new families of 2-oxoglutarate- and iron-dependent dioxygenases. Genome Biol. 2, research0007 (2001)

    Article  CAS  Google Scholar 

  9. 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)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. 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)

    Article  ADS  CAS  Google Scholar 

  12. 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)

    Article  CAS  Google Scholar 

  13. 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)

    Article  CAS  Google Scholar 

  14. Duncan, T. et al. Reversal of DNA alkylation damage by two human dioxygenases. Proc. Natl Acad. Sci. USA 99, 16660–16665 (2002)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  16. Lee, D.-H. et al. Repair of methylation damage in DNA and RNA by mammalian AlkB homologues. J. Biol. Chem. 280, 39448–39459 (2005)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  18. 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)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. 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)

    Article  CAS  Google Scholar 

  22. Dinglay, S., Trewick, S. C., Lindahl, T. & Sedgwick, B. Defective processing of methylated single-stranded DNA by E. coli AlkB mutants. Genes Dev. 14, 2097–2105 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase at 2.7 Å resolution: implications of comformational changes for polymerization and inhibition mechanism. Science 282, 1669–1675 (1998)

    Article  ADS  CAS  Google Scholar 

  24. Verdine, G. L. & Norman, D. P. G. Covalent trapping of protein–DNA complexes. Annu. Rev. Biochem. 72, 337–366 (2003)

    Article  CAS  Google Scholar 

  25. 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)

    Article  CAS  Google Scholar 

  26. Duguid, E. M., Mishina, Y. & He, C. How do DNA repair proteins locate potential base lesions? A chemical crosslinking method to investigate the damage-searching mechanism of O6-alkylguanine-DNA alkyltransferases. Chem. Biol. 10, 827–835 (2003)

    Article  CAS  Google Scholar 

  27. Mishina, Y., Lee, C. H. & He, C. Interaction of human and bacterial AlkB proteins with DNA as probed through chemical cross-linking studies. Nucleic Acids Res. 32, 1548–1554 (2004)

    Article  CAS  Google Scholar 

  28. 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)

    Article  CAS  Google Scholar 

  29. Lange, S. J. & Que, L. Oxygen activating nonheme iron enzymes. Curr. Opin. Chem. Biol. 2, 159–172 (1998)

    Article  CAS  Google Scholar 

  30. Elkins, J. M. et al. X-ray crystal structure of Escherichia coli taurine/α-ketoglutarate dioxygenase complexed to ferrous iron and substrates. Biochemistry 41, 5185–5192 (2002)

    Article  CAS  Google Scholar 

  31. Lau, A. Y., Scharer, O. D., Samson, L., Verdine, G. L. & Ellenberger, T. Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanismf for nucleotide flipping and base excision. Cell 95, 249–258 (1998)

    Article  CAS  Google Scholar 

  32. Fromme, J. C., Banerjee, A., Huang, S. J. & Verdine, G. L. Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase. Nature 427, 652–656 (2004)

    Article  ADS  CAS  Google Scholar 

  33. Banerjee, A., Santos, W. L. & Verdine, G. L. Structure of a DNA glycocylase searching for lesions. Science 311, 1153–1157 (2006)

    Article  ADS  CAS  Google Scholar 

  34. Slupphaug, G. et al. A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA. Nature 384, 87–92 (1996)

    Article  ADS  CAS  Google Scholar 

  35. Bruner, S. D., Norman, D. P. G. & Verdine, G. L. Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA. Nature 403, 859–866 (2000)

    Article  ADS  CAS  Google Scholar 

  36. Parker, J. B. et al. Enzymatic capture of an extrahelical thymine in the search for uracil in DNA. Nature 449, 433–437 (2007)

    Article  ADS  CAS  Google Scholar 

  37. Kissinger, C. R., Gehlhaar, D. K. & Fogel, D. B. Rapid automated molecular replacement by evolutionary search. Acta Crystallogr. D 55, 484–491 (1999)

    Article  CAS  Google Scholar 

  38. Read, R. J. Pushing the boundaries of molecular replacement with maximum likelihood. Erratum. Acta Crystallogr. D 59, 404 (2003)

    Article  Google Scholar 

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

    Article  Google Scholar 

  40. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D. 50, 760–763 (1994)

  41. Otwinowski, Z. W. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  42. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)

    Article  CAS  Google Scholar 

  43. Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  44. DeLano, W. L. The PyMOL molecular graphics system (DeLano Scientific, Palo Alto, California, 2002)

Download references

Acknowledgements

We thank: R. Zhang and other beamline staff for assistance with data collection; Y. Luo and the Proteomics and Informatics Services Facility at Research Resources Center (University of Illinois at Chicago) for liquid chromatography–mass spectrometry analysis; and X. Yang, H. Chen and P. R. Chen for discussions. We also thank T. Lindahl and B. Sedgwick for the gift of the abh2 gene. This work was supported by National Institutes of Health (GM071440 to C.H. and a PCBio fellowship for C.T.S.), the W. M. Keck Foundation Distinguished Young Scholar in Medical Research Program (C.H.), and the Arnold and Mabel Beckman Foundation Young Investigator Program (C.H.). Data collection was performed at beamlines 19BM (Structure Biology Center) and 14BM (BioCARS) at the Advanced Photon Source at Argonne National Laboratory; financial support for these beamlines comes from the National Institutes of Health and the United States Department of Energy.

Author Contributions C.-G.Y. and C.Y. solved all AlkB–dsDNA and ABH2–dsDNA structures with help from E.M.D. (crystallography), C.T.S. (initial construct of ABH2 and crystallography) and X.J. (biochemistry). P.A.R. contributed to protein crystallography. C.H. designed the overall project and wrote the manuscript with C.-G.Y. and C.Y. All authors discussed results and commented on the manuscript.

Author information

Author notes

  1. Cai-Guang Yang and Chengqi Yi: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA,

    Cai-Guang Yang, Chengqi Yi, Erica M. Duguid, Christopher T. Sullivan, Xing Jian & Chuan He

  2. Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA,

    Phoebe A. Rice

Authors
  1. Cai-Guang Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chengqi Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Erica M. Duguid
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher T. Sullivan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xing Jian
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Phoebe A. Rice
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chuan He
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chuan He.

Supplementary information

Supplementary information

This file contains Supplementary Tables 1-2, Supplementary Figures 1-16 with Legends, Supplementary Method and Result, and additional references (PDF 7316 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yang, CG., Yi, C., Duguid, E. et al. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA. Nature 452, 961–965 (2008). https://doi.org/10.1038/nature06889

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature06889

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing