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:

DNA binding and nucleotide flipping by the human DNA repair protein AGT

Nature Structural & Molecular Biology volume 11, pages 714–720 (2004)Cite this article

Abstract

O6-alkylguanine-DNA alkyltransferase (AGT), or O6-methylguanine-DNA methyltransferase (MGMT), prevents mutations and apoptosis resulting from alkylation damage to guanines. AGT irreversibly transfers the alkyl lesion to an active site cysteine in a stoichiometric, direct damage reversal pathway. AGT expression therefore elicits tumor resistance to alkylating chemotherapies, and AGT inhibitors are in clinical trials. We report here structures of human AGT in complex with double-stranded DNA containing the biological substrate O6-methylguanine or crosslinked to the mechanistic inhibitor N1,O6-ethanoxanthosine. The prototypical DNA major groove–binding helix-turn-helix (HTH) motif mediates unprecedented minor groove DNA binding. This binding architecture has advantages for DNA repair and nucleotide flipping, and provides a paradigm for HTH interactions in sequence-independent DNA-binding proteins like RecQ and BRCA2. Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Recognition and repair of O6-alkylguanines by AGT.
Figure 2: AGT-DNA binding interface.
Figure 3: Minor groove binding, nucleotide flipping and O6-akylguanine recognition by AGT.
Figure 4: Reaction mechanism.
Figure 5: Directionality of ssDNA repair by AGT.
Figure 6: The minor groove DNA-binding mechanism of AGT expands the HTH repertoire.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Lindahl, T. & Wood, R.D. Quality control by DNA repair. Science 286, 1897–1905 (1999).

    Article  CAS  Google Scholar 

  2. 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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Meikrantz, W., Bergom, M.A., Memisoglu, A. & Samson, L. O6-alkylguanine DNA lesions trigger apoptosis. Carcinogenesis 19, 369–372 (1998).

    Article  CAS  Google Scholar 

  6. Hickman, M.J. & Samson, L.D. Apoptotic signaling in response to a single type of DNA lesion, O6-methylguanine. Mol. Cell 14, 105–116 (2004).

    Article  CAS  Google Scholar 

  7. Mitra, S. & Kaina, B. Regulation of repair of alkylation damage in mammalian genomes. Prog. Nucleic Acid Res. Mol. Biol. 44, 109–142 (1993).

    Article  CAS  Google Scholar 

  8. Gerson, S.L. Clinical relevance of MGMT in the treatment of cancer. J. Clin. Oncol. 20, 2388–2399 (2002).

    Article  CAS  Google Scholar 

  9. Noll, D.M. & Clarke, N.D. Covalent capture of a human O6-alkylguanine alkyltransferase–DNA complex using N1,O6-ethanoxanthosine, a mechanism-based crosslinker. Nucleic Acids Res. 29, 4025–4034 (2001).

    Article  CAS  Google Scholar 

  10. Wibley, J.E., Pegg, A.E. & Moody, P.C. Crystal structure of the human O6-alkylguanine-DNA alkyltransferase. Nucleic Acids Res. 28, 393–401 (2000).

    Article  CAS  Google Scholar 

  11. Daniels, D.S. & Tainer, J.A. Conserved structural motifs governing the stoichiometric repair of alkylated DNA by O6-alkylguanine-DNA alkyltransferase. Mutat. Res. 460, 151–163 (2000).

    Article  CAS  Google Scholar 

  12. Daniels, D.S. et al. Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding. EMBO J. 19, 1719–1730 (2000).

    Article  CAS  Google Scholar 

  13. Vora, R.A., Pegg, A.E. & Ealick, S.E. A new model for how O6-methylguanine-DNA methyltransferase binds DNA. Proteins 32, 3–6 (1998).

    Article  CAS  Google Scholar 

  14. Goodtzova, K., Kanugula, S., Edara, S. & Pegg, A.E. Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltransferase. Biochemistry 37, 12489–12495 (1998).

    Article  CAS  Google Scholar 

  15. Pegg, A.E., Morimoto, K. & Dolan, M.E. Investigation of the specificity of O6-alkylguanine-DNA-alkyltransferase. Chem. Biol. Interact. 65, 275–281 (1988).

    Article  CAS  Google Scholar 

  16. Rasimas, J.J., Pegg, A.E. & Fried, M.G. DNA-binding mechanism of O6-alkylguanine-DNA alkyltransferase. Effects of protein and DNA alkylation on complex stability. J. Biol. Chem. 278, 7973–7980 (2003).

    Article  CAS  Google Scholar 

  17. Glassner, B.J., Rasmussen, L.J., Najarian, M.T., Posnick, L.M. & Samson, L.D. Generation of a strong mutator phenotype in yeast by imbalanced base excision repair. Proc. Natl. Acad. Sci. U. S. A. 95, 9997–10002 (1998).

    Article  CAS  Google Scholar 

  18. Frosina, G. Overexpression of enzymes that repair endogenous damage to DNA. Eur. J. Biochem. 267, 2135–2149 (2000).

    Article  CAS  Google Scholar 

  19. Dodson, G. & Wlodawer, A. Catalytic triads and their relatives. Trends Biochem. Sci. 23, 347–352 (1998).

    Article  CAS  Google Scholar 

  20. Guengerich, F.P., Fang, Q., Liu, L., Hachey, D.L. & Pegg, A.E. O6-alkylguanine-DNA alkyltransferase: low pKa and high reactivity of cysteine 145. Biochemistry 42, 10965–10970 (2003).

    Article  CAS  Google Scholar 

  21. Luu, K.X., Kanugula, S., Pegg, A.E., Pauly, G.T. & Moschel, R.C. Repair of oligodeoxyribonucleotides by O6-alkylguanine-DNA alkyltransferase. Biochemistry 41, 8689–8697 (2002).

    Article  CAS  Google Scholar 

  22. Moody, P.C. & Demple, B. Crystallization of O6-methylguanine-DNA methyltransferase from Escherichia coli. J. Mol. Biol. 200, 751–752 (1988).

    Article  CAS  Google Scholar 

  23. von Hippel, P.H. & Berg, O.G. Facilitated target location in biological systems. J. Biol. Chem. 264, 675–678 (1989).

    CAS  PubMed  Google Scholar 

  24. Stanford, N.P., Szczelkun, M.D., Marko, J.F. & Halford, S.E. One- and three-dimensional pathways for proteins to reach specific DNA sites. EMBO J. 19, 6546–6557 (2000).

    Article  CAS  Google Scholar 

  25. Ali, R.B. et al. Implication of localization of human DNA repair enzyme O6-methylguanine-DNA methyltransferase at active transcription sites in transcription-repair coupling of the mutagenic O6-methylguanine lesion. Mol. Cell. Biol. 18, 1660–1669 (1998).

    Article  CAS  Google Scholar 

  26. Banavali, N.K. & MacKerell, A.D. Jr. Free energy and structural pathways of base flipping in a DNA GCGC containing sequence. J. Mol. Biol. 319, 141–160 (2002).

    Article  CAS  Google Scholar 

  27. Huang, N., Banavali, N.K. & MacKerell, A.D. Jr. Protein-facilitated base flipping in DNA by cytosine-5-methyltransferase. Proc. Natl. Acad. Sci. USA 100, 68–73 (2003).

    Article  CAS  Google Scholar 

  28. Hollis, T., Ichikawa, Y. & Ellenberger, T. DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA. EMBO J. 19, 758–766 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Barrett, T.E. et al. Crystal structure of a G:T/U mismatch-specific DNA glycosylase: mismatch recognition by complementary-strand interactions. Cell 92, 117–129 (1998).

    Article  CAS  Google Scholar 

  31. Parikh, S.S. et al. Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA. EMBO J. 17, 5214–5226 (1998).

    Article  CAS  Google Scholar 

  32. Mol, C.D., Izumi, T., Mitra, S. & Tainer, J.A. DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination [corrected]. Nature 403, 451–456 (2000).

    Article  CAS  Google Scholar 

  33. Hosfield, D.J., Guan, Y., Haas, B.J., Cunningham, R.P. & Tainer, J.A. Structure of the DNA repair enzyme endonuclease IV and its DNA complex: double-nucleotide flipping at abasic sites and three-metal-ion catalysis. Cell 98, 397–408 (1999).

    Article  CAS  Google Scholar 

  34. Klimasauskas, S., Kumar, S., Roberts, R.J. & Cheng, X. HhaI methyltransferase flips its target base out of the DNA helix. Cell 76, 357–369 (1994).

    Article  CAS  Google Scholar 

  35. Moschel, R.C., McDougall, M.G., Dolan, M.E., Stine, L. & Pegg, A.E. Structural features of substituted purine derivatives compatible with depletion of human O6-alkylguanine-DNA alkyltransferase. J. Med. Chem. 35, 4486–4491 (1992).

    Article  CAS  Google Scholar 

  36. Gajiwala, K.S. & Burley, S.K. Winged helix proteins. Curr. Opin. Struct. Biol. 10, 110–116 (2000).

    Article  CAS  Google Scholar 

  37. Gajiwala, K.S. et al. Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding. Nature 403, 916–921 (2000).

    Article  CAS  Google Scholar 

  38. Luscombe, N.M., Austin, S.E., Berman, H.M. & Thornton, J.M. An overview of the structures of protein–DNA complexes. Genome Biol. 1, reviews001.1–001.37 (2000).

    Article  Google Scholar 

  39. Yang, H. et al. BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure. Science 297, 1837–1848 (2002).

    Article  CAS  Google Scholar 

  40. Sibanda, B.L. et al. Crystal structure of an Xrcc4–DNA ligase IV complex. Nat. Struct. Biol. 8, 1015–1019 (2001).

    Article  CAS  Google Scholar 

  41. Bernstein, D.A., Zittel, M.C. & Keck, J.L. High-resolution structure of the E. coli RecQ helicase catalytic core. EMBO J. 22, 4910–4921 (2003).

    Article  CAS  Google Scholar 

  42. Leslie, A.G.W. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography Vol. 26 (Daresbury Laboratory, Warrington, UK, 1992).

    Google Scholar 

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

  44. Holton, J. & Alber, T. Automated protein crystal structure determination using ELVES. Proc. Natl. Acad. Sci. USA 101, 1537–1542 (2004).

    Article  CAS  Google Scholar 

  45. Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A. 50, 157–163 (1994).

    Article  Google Scholar 

  46. McRee, D.E. XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  48. Lavery, R. & Sklenar, H. The definition of generalized helicoidal parameters and of axis curvature for irregular nucleic acids. J. Biomol. Struct. Dyn. 6, 63–91 (1988).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C.L. Brooks III, M.E. Stroupe and A.J. Das for critical discussion and the staff and facilities of the Stanford Synchrotron Radiation Laboratory. This work was supported by grants from the US National Institutes of Health (J.A.T.), US National Cancer Institute (A.E.P.), Patterson Trust (D.M.N.), Alexander and Margaret Stewart Trust (D.M.N.) and Skaggs Institute for Chemical Biology (D.S.D.).

Author information

Author notes

  1. Tammy T Woo

    Present address: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120, Heidelberg, Germany

Authors and Affiliations

  1. Skaggs Institute for Chemical Biology, The Scripps Research Institute, MB-4, 10550 North Torrey Pines Road, La Jolla, 92037, California, USA

    Douglas S Daniels, Tammy T Woo & John A Tainer

  2. Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, 17033, Pennsylvania, USA

    Kieu X Luu & Anthony E Pegg

  3. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, School of Medicine, 725 North Wolfe Street, Baltimore, 21205, Maryland, USA

    David M Noll & Neil D Clarke

  4. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    John A Tainer

Authors
  1. Douglas S Daniels
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tammy T Woo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kieu X Luu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David M Noll
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Neil D Clarke
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anthony E Pegg
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John A Tainer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John A Tainer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Daniels, D., Woo, T., Luu, K. et al. DNA binding and nucleotide flipping by the human DNA repair protein AGT. Nat Struct Mol Biol 11, 714–720 (2004). https://doi.org/10.1038/nsmb791

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb791

This article is cited by

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