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:

Enzymatic capture of an extrahelical thymine in the search for uracil in DNA

Nature volume 449, pages 433–437 (2007)Cite this article

Abstract

The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U•A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U•A or U•G base pairs in a background of approximately 109 T•A or C•G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T•A and U•A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.

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: Extrahelical uracil recognition by UNG and reaction coordinate tuning.
Figure 2: Stabilization of extrahelical thymine in the EI′ state in complex with UNG.
Figure 3: Conformational changes in the sugar and base along the flipping reaction coordinate.
Figure 4: Interaction map for the EI′ complex and imino proton exchange profiles for wild-type UNG and several mutant forms.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Stivers, J. T. & Jiang, Y. L. A mechanistic perspective on the chemistry of DNA repair glycosylases. Chem. Rev. 103, 2729–2759 (2003)

    Article  CAS  Google Scholar 

  3. Seiple, L., Jaruga, P., Dizdaroglu, M. & Stivers, J. T. Linking uracil base excision repair and 5-fluorouracil toxicity in yeast. Nucleic Acids Res. 34, 140–151 (2006)

    Article  CAS  Google Scholar 

  4. Kavli, B., Otterlei, M., Slupphaug, G. & Krokan, H. E. Uracil in DNA—General mutagen, but normal intermediate in acquired immunity. DNA Repair 6, 505–516 (2006)

    Article  Google Scholar 

  5. 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 

  6. Parikh, S. S. et al. Uracil-DNA glycosylase–DNA substrate and product structures: conformational strain promotes catalytic efficiency by coupled stereoelectronic effects. Proc. Natl Acad. Sci. USA 97, 5083–5088 (2000)

    Article  ADS  CAS  Google Scholar 

  7. Kavli, B. et al. Excision of cytosine and thymine from DNA by mutants of human uracil-DNA glycosylase. EMBO J. 15, 3442–3447 (1996)

    Article  MathSciNet  CAS  Google Scholar 

  8. Cao, C., Jiang, Y. L., Stivers, J. T. & Song, F. Dynamic opening of DNA during the enzymatic search for a damaged base. Nature Struct. Mol. Biol. 11, 1230–1236 (2004)

    Article  CAS  Google Scholar 

  9. Cao, C., Jiang, Y. L., Krosky, D. J. & Stivers, J. T. The catalytic power of uracil DNA glycosylase in the opening of thymine base pairs. J. Am. Chem. Soc. 128, 13034–13035 (2006)

    Article  CAS  Google Scholar 

  10. Krosky, D. J., Song, F. & Stivers, J. T. The origins of high-affinity enzyme binding to an extrahelical DNA base. Biochemistry 44, 5949–5959 (2005)

    Article  CAS  Google Scholar 

  11. Krosky, D. J., Schwarz, F. P. & Stivers, J. T. Linear free energy correlations for enzymatic base flipping: How do damaged base pairs facilitate specific recognition? Biochemistry 43, 4188–4195 (2004)

    Article  CAS  Google Scholar 

  12. Moran, S., Ren, R. X. F., Sheils, C. J., Rumney, S. & Kool, E. T. Non-hydrogen bonding 'terminator' nucleosides increase the 3′-end homogeneity of enzymatic RNA and DNA synthesis. Nucleic Acids Res. 24, 2044–2052 (1996)

    Article  CAS  Google Scholar 

  13. Jiang, Y. L. & Stivers, J. T. Mutational analysis of the base flipping mechanism of uracil DNA glycosylase. Biochemistry 41, 11236–11247 (2002)

    Article  CAS  Google Scholar 

  14. Banerjee, A., Yang, W., Karplus, M. & Verdine, G. L. Structure of a repair enzyme interrogating undamaged DNA elucidates recognition of damaged DNA. Nature 434, 612–618 (2005)

    Article  ADS  CAS  Google Scholar 

  15. Chen, L., Haushalter, K. A., Lieber, C. M. & Verdine, G. L. Direct visualization of a DNA glycosylase searching for damage. Chem. Biol. 9, 345–350 (2002)

    Article  CAS  Google Scholar 

  16. Gueron, M. & Leroy, J. Studies of base pair kinetics by NMR measurement of proton exchange. Methods Enzymol. 261, 383–413 (1995)

    Article  CAS  Google Scholar 

  17. Snoussi, K. & Leroy, J. L. Alteration of A•T base-pair opening kinetics by the ammonium cation in DNA A-tracts. Biochemistry 41, 12467–12474 (2002)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  19. Gueron, M. et al. Applications of imino proton exchange to nucleic acid kinetics and structures. In Structure and Methods (ed. Sarma, R. H.) 113–137 (Adenine press, New York, 1990)

    Google Scholar 

  20. Moe, J. G. & Russu, I. M. Kinetics and energetics of base-pair opening in 5′-d(CGCGAATTCGCG)-3′ and a substituted dodecamer containing ĠT mismatches. Biochemistry 31, 8421–8428 (1992)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Ren, R. X.-F., Chaudhuri, N. C., Paris, P. L., Rumney, S. & Kool, E. T. Naphthalene, phenanthrene, and pyrene as DNA base analogues: Synthesis, structure, and fluorescence in DNA. J. Am. Chem. Soc. 118, 7671–7678 (1996)

    Article  CAS  Google Scholar 

  23. Drohat, A. C., Jagadeesh, J., Ferguson, E. & Stivers, J. T. The role of electrophilic and base catalysis in the mechanism of Escherichia coli uracil DNA glycosylase. Biochemistry 38, 11866–11875 (1999)

    Article  CAS  Google Scholar 

  24. Drohat, A. C. et al. Hetronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase: Electrophilic catalysis of uracil expulsion by a neutral histidine 187. Biochemistry 38, 11876–11886 (1999)

    Article  CAS  Google Scholar 

  25. Xiao, G. et al. Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited. Proteins 35, 13–24 (1999)

    Article  CAS  Google Scholar 

  26. Slupphaug, G. et al. Properties of a recombinant human uracil-DNA glycosylase from the UNG gene and evidence that UNG encodes the major uracil-DNA glycosylase. Biochemistry 34, 128–138 (1995)

    Article  CAS  Google Scholar 

  27. Otwinoski, Z. & Minor, W. Processing of X-ray diffraction data in oscillation mode. Methods Enzymol. 276, 307–325 (1997)

    Article  Google Scholar 

  28. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C. & Read, R. J. Likelihood-enhanced fast translation functions. Acta Crystallogr. D 61, 458–464 (2005)

    Article  Google Scholar 

  29. Collaborative Computational Project, Number 4. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D 50, 760–776 (1994)

  30. Esnouf, R. M. Further additions to Molscript version 1.4, including reading and contouring of electron density maps. Acta Crystallogr. D 55, 938–940 (1999)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Majumdar for assistance with NMR experiments. This work was supported by NIH grants (J.T.S. and L.M.A.) and a major research instrumentation grant from the NSF.

Author Contributions J.B.P. prepared all mutant enzymes and performed the NMR experiments; M.A.B. collected, processed and refined X-ray data and performed structural analyses; D.J.K. conceived the structural approach and purified and crystallized the complexes; J.I.F. performed NMR studies on the L272G mutant; L.M.A. performed structural analyses, discussed and commented on the manuscript; J.T.S. analysed data and wrote the paper.

Coordinates and structure factor files for the EI complex and the abasic DNA complex have been deposited in the Protein Data Bank with accession numbers 2OXM and 2OYT, respectively.

Author information

Authors and Affiliations

  1. Departments of Pharmacology and Molecular Sciences and,,

    Jared B. Parker, Daniel J. Krosky, Joshua I. Friedman & James T. Stivers

  2. Biophysics and Biophysical Chemistry of the Johns Hopkins Medical School, 725 North Wolfe Street, Baltimore, Maryland 21205, USA,

    Mario A. Bianchet & L. Mario Amzel

Authors
  1. Jared B. Parker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mario A. Bianchet
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel J. Krosky
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joshua I. Friedman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. L. Mario Amzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. James T. Stivers
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to James T. Stivers.

Ethics declarations

Competing interests

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-4 with Legends and Supplementary Tables 1-3. The Supplementary Figures contain: (1) a scheme describing the general findings, (2) electron density maps of the flipped thymine and the abasic sugar excision product, (3) imino proton spectra of the free T/A 10 mer DNA duplex and its complexes with wild-type and mutant UNG enzymes, and (4) magnetization transfer time courses for the imino protons of residues G3, G4 and T2 of free and UNG-bound DNA. The Supplementary Tables detail (1) crystallographic data collection and refinement statistics, (2) imino proton exchange rates of free and bound DNA, and (3) chemical shifts of imino protons in each enzyme complex. (PDF 1951 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Parker, J., Bianchet, M., Krosky, D. et al. Enzymatic capture of an extrahelical thymine in the search for uracil in DNA. Nature 449, 433–437 (2007). https://doi.org/10.1038/nature06131

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature06131

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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