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.

Structural basis for the suppression of skin cancers by DNA polymerase η

Abstract

DNA polymerase η (Polη) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of Polη (also known as POLH) in humans causes the variant form of xeroderma pigmentosum (XPV). We present the crystal structures of Saccharomyces cerevisiae Polη (also known as RAD30) in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures reveal that the ability of Polη to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the two Ts of the T-T dimer are accommodated in an active site cleft that is much more open than in other polymerases. We also show by structural, biochemical and genetic analysis that the two Ts are maintained in a stable configuration in the active site via interactions with Gln 55, Arg 73 and Met 74. Together, these features define the basis for Polη’s action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Polη–DNA–dATP ternary complexes.
Figure 2: Close-up views of the active site regions.
Figure 3: Conformational changes in Polη ternary complex.
Figure 4: Comparison between Polη and Polκ.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors files have been deposited in the Protein Data Bank under accession codes 3MFH and 3MFI.

References

  1. Johnson, R. E., Kondratick, C. M., Prakash, S. & Prakash, L. hRAD30 mutations in the variant form of xeroderma pigmentosum. Science 285, 263–265 (1999)

    CAS  Article  Google Scholar 

  2. Masutani, C. et al. The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase η. Nature 399, 700–704 (1999)

    ADS  CAS  Article  Google Scholar 

  3. Broughton, B. C. et al. Molecular analysis of mutations in DNA polymerase η in xeroderma pigmentosum-variant patients. Proc. Natl Acad. Sci. USA 99, 815–820 (2002)

    ADS  CAS  Article  Google Scholar 

  4. Gratchev, A., Strein, P., Utikal, J. & Sergij, G. Molecular genetics of Xeroderma pigmentosum variant. Exp. Dermatol. 12, 529–536 (2003)

    CAS  Article  Google Scholar 

  5. Inui, H. et al. Xeroderma pigmentosum-variant patients from America, Europe, and Asia. J. Invest. Dermatol. 128, 2055–2068 (2008)

    CAS  Article  Google Scholar 

  6. Tanioka, M. et al. Molecular analysis of DNA polymerase eta gene in Japanese patients diagnosed as xeroderma pigmentosum variant type. J. Invest. Dermatol. 127, 1745–1751 (2007)

    CAS  Article  Google Scholar 

  7. Johnson, R. E., Prakash, S. & Prakash, L. Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Polη. Science 283, 1001–1004 (1999)

    ADS  CAS  Article  Google Scholar 

  8. Prakash, S., Johnson, R. E. & Prakash, L. Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function. Annu. Rev. Biochem. 74, 317–353 (2005)

    CAS  Article  Google Scholar 

  9. Trincao, J. et al. Structure of the catalytic core of S. cerevisiae DNA polymerase η: implications for translesion DNA synthesis. Mol. Cell 8, 417–426 (2001)

    CAS  Article  Google Scholar 

  10. Kondratick, C. M., Washington, M. T., Prakash, S. & Prakash, L. Acidic residues critical for the activity and biological function of yeast DNA polymerase η. Mol. Cell. Biol. 21, 2018–2025 (2001)

    CAS  Article  Google Scholar 

  11. Doublié, S., Tabor, S., Long, A. M., Richardson, C. C. & Ellenberger, T. Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution. Nature 391, 251–258 (1998)

    ADS  Article  Google Scholar 

  12. Li, Y., Korolev, S. & Waksman, G. Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation. EMBO J. 17, 7514–7525 (1998)

    CAS  Article  Google Scholar 

  13. Steitz, T. A. DNA polymerases: structural diversity and common mechanisms. J. Biol. Chem. 274, 17395–17398 (1999)

    CAS  Article  Google Scholar 

  14. Ling, H., Boudsocq, F., Woodgate, R. & Yang, W. Crystal structure of a Y-family DNA polymerase in action: a mechanism for error-prone and lesion-bypass replication. Cell 107, 91–102 (2001)

    CAS  Article  Google Scholar 

  15. Nair, D. T., Johnson, R. E., Prakash, S., Prakash, L. & Aggarwal, A. K. Replication by human DNA polymerase-ι occurs by Hoogsteen base-pairing. Nature 430, 377–380 (2004)

    ADS  CAS  Article  Google Scholar 

  16. Nair, D. T., Johnson, R. E., Prakash, L., Prakash, S. & Aggarwal, A. K. Human DNA polymerase ι incorporates dCTP opposite template G via a G.C+ Hoogsteen base pair. Structure 13, 1569–1577 (2005)

    CAS  Article  Google Scholar 

  17. Nair, D. T., Johnson, R. E., Prakash, L., Prakash, S. & Aggarwal, A. K. Rev1 employs a novel mechanism of DNA synthesis using a protein template. Science 309, 2219–2222 (2005)

    ADS  CAS  Article  Google Scholar 

  18. Lone, S. et al. Human DNA polymerase κ encircles DNA: implications for mismatch extension and lesion bypass. Mol. Cell 25, 601–614 (2007)

    CAS  Article  Google Scholar 

  19. Alt, A. et al. Bypass of DNA lesions generated during anticancer treatment with cisplatin by DNA polymerase η. Science 318, 967–970 (2007)

    ADS  CAS  Article  Google Scholar 

  20. Rothwell, P. J. & Waksman, G. Structure and mechanism of DNA polymerases. Adv. Protein Chem. 71, 401–440 (2005)

    CAS  Article  Google Scholar 

  21. Washington, M. T., Prakash, L. & Prakash, S. Yeast DNA polymerase η utilizes an induced-fit mechanism of nucleotide incorporation. Cell 107, 917–927 (2001)

    CAS  Article  Google Scholar 

  22. Zhou, B. L., Pata, J. D. & Steitz, T. A. Crystal structure of a DinB lesion bypass DNA polymerase catalytic fragment reveals a classic polymerase catalytic domain. Mol. Cell 8, 427–437 (2001)

    CAS  Article  Google Scholar 

  23. Vaisman, A., Ling, H., Woodgate, R. & Yang, W. Fidelity of Dpo4: effect of metal ions, nucleotide selection and pyrophosphorolysis. EMBO J. 24, 2957–2967 (2005)

    CAS  Article  Google Scholar 

  24. Gietz, R. D. & Sugino, A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527–534 (1988)

    CAS  Article  Google Scholar 

  25. Johnson, R. E., Prakash, L. & Prakash, S. Yeast and human translesion DNA synthesis polymerases: expression, purification, and biochemical characterization. Methods Enzymol. 408, 390–407 (2006)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  27. 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 

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

    Article  Google Scholar 

  29. Winn, M. D., Murshudov, G. N. & Papiz, M. Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 (2003)

    CAS  Article  Google Scholar 

  30. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007)

    ADS  Article  Google Scholar 

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

    Article  Google Scholar 

Download references

Acknowledgements

We thank the staff at Brookhaven National Laboratory (beamlines X6A and X29) and the Advanced Photon Source (24ID) for facilitating X-ray data collection. We thank D. T. Nair, S. Lone, R. Vasquez-Del Carpio and M. Swan for discussions. This work was supported by NIH grants to A.K.A., S.P. and L.P.

Author information

Authors and Affiliations

Authors

Contributions

A.K.A. and T.D.S. designed the crystallographic studies; S.P., L.P. and R.E.J. designed the biochemical and genetic studies; T.D.S., R.E.J., R.J. and L.P. performed the experiments; all of the authors contributed to the writing of the paper.

Corresponding authors

Correspondence to Satya Prakash or Aneel K. Aggarwal.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-3 and Supplementary Figures 1-11 with legends. (PDF 22461 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Silverstein, T., Johnson, R., Jain, R. et al. Structural basis for the suppression of skin cancers by DNA polymerase η. Nature 465, 1039–1043 (2010). https://doi.org/10.1038/nature09104

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09104

Further reading

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

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer