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Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7

Nature Structural & Molecular Biology volume 11, pages 784790 (2004) | Download Citation

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Abstract

Ultraviolet-induced DNA damage poses a lethal block to replication. To understand the structural basis for this, we determined crystal structures of a replicative DNA polymerase from bacteriophage T7 in complex with nucleotide substrates and a DNA template containing a cis-syn cyclobutane pyrimidine dimer (CPD). When the 3′ thymine is the templating base, the CPD is rotated out of the polymerase active site and the fingers subdomain adopts an open orientation. When the 5′ thymine is the templating base, the CPD lies within the polymerase active site where it base-pairs with the incoming nucleotide and the 3′ base of the primer, while the fingers are in a closed conformation. These structures reveal the basis for the strong block of DNA replication that is caused by this photolesion.

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  • 11 July 2004

    Update figure 2 with the corrected image provided, added note in full and fig 2 legend, appended pdf

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References

  1. 1.

    , & DNA Repair and Mutagenesis (American Society for Microbiology, Washington, DC, 1995).

  2. 2.

    Unraveling the molecular pathway from sunlight to skin cancer. Acc. Chem. Res. 27, 76–82 (1994).

  3. 3.

    , & Direct and indirect effects of UV radiation on DNA and its components. J. Photochem. Photobiol. B 63, 88–102 (2001).

  4. 4.

    et al. The importance of repairing stalled replication forks. Nature 404, 37–41 (2000).

  5. 5.

    , & 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).

  6. 6.

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

  7. 7.

    & Structural insights into the origins of DNA polymerase fidelity. Structure 11, 489–496 (2003).

  8. 8.

    & The mechanism of action of T7 DNA polymerase. Curr. Opin. Struct. Biol. 8, 704–712 (1998).

  9. 9.

    Hydrogen bonding revisited: geometric selection as a principal determinant of DNA replication fidelity. Proc. Natl. Acad. Sci. USA 94, 10493–10495 (1997).

  10. 10.

    , , & Pyrene nucleotide as a mechanistic probe: evidence for a transient abasic site-like intermediate in the bypass of dipyrimidine photoproducts by T7 DNA polymerase. Biochemistry 39, 14603–14610 (2000).

  11. 11.

    , & The ability of a variety of polymerases to synthesize past site-specific cis-syn, trans-syn-II (6-4), and Dewar photoproducts of thymidylyl-(3′→5′)-thymidine. J. Biol. Chem. 273, 21933–21940 (1998).

  12. 12.

    New structural and mechanistic insight into the A-rule and the instructional and non-instructional behavior of DNA photoproducts and other lesions. Mutat. Res. 510, 55–70 (2002).

  13. 13.

    et al. Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis. Nature 404, 1014–1018 (2000).

  14. 14.

    , & Mechanism of nucleotide incorporation opposite a thymine-thymine dimer by yeast DNA polymerase η. Proc. Natl. Acad. Sci. USA 100, 12093–12098 (2003).

  15. 15.

    & DNA lesion bypass polymerases open up. Structure 9, 759–764 (2001).

  16. 16.

    , & Error-prone DNA polymerases: novel structures and the benefits of infidelity. Cell 107, 9–12 (2001).

  17. 17.

    Damage repair DNA polymerases Y. Curr. Opin. Struct. Biol. 13, 23–30 (2003).

  18. 18.

    , , , & Replication of a cis-syn thymine dimer at atomic resolution. Nature 424, 1083–1087 (2003).

  19. 19.

    & Compilation and alignment of DNA polymerase sequences. Nucleic Acids Res. 19, 4045–4057 (1991).

  20. 20.

    The 'A rule' of mutagen specificity: a consequence of DNA polymerase bypass of non-instructional lesions? Bioessays 13, 79–84 (1991).

  21. 21.

    & A specific partner for abasic damage in DNA. Nature 399, 704–708 (1999).

  22. 22.

    , , , & Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution. Nature 391, 251–258 (1998).

  23. 23.

    et al. Crystal structure of a DNA decamer containing a cis-syn thymine dimer. Proc. Natl. Acad. Sci. USA 99, 15965–15970 (2002).

  24. 24.

    , & Sequence-specific recognition of double helical nucleic acids by proteins. Proc. Natl. Acad. Sci. USA 73, 804–808 (1976).

  25. 25.

    Translesion DNA synthesis: polymerase response to altered nucleotides. Cancer Surv. 4, 493–516 (1985).

  26. 26.

    Conformational coupling in DNA polymerase fidelity. Annu. Rev. Biochem. 62, 685–713 (1993).

  27. 27.

    et al. Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment). J. Biol. Chem. 274, 3067–3075 (1999).

  28. 28.

    et al. The Y-family of DNA polymerases. Mol. Cell 8, 7–8 (2001).

  29. 29.

    Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu. Rev. Biochem. 71, 17–50 (2002).

  30. 30.

    , , & T-T cyclobutane dimers are misinstructive, rather than non-instructive, mutagenic lesions. Mol. Gen. Genet. 222, 166–168 (1990).

  31. 31.

    , & Mutagenic properties of the T-C cyclobutane dimer. J. Bacteriol. 179, 2835–2839 (1997).

  32. 32.

    & Preparation and characterization of a set of deoxyoligonucleotide 49-mers containing site-specific cis-syn, trans-syn-I (6-4), and Dewar photoproducts of thymidylyl(3′→5′)-thymidine. J. Biol. Chem. 268, 11143–11151 (1993).

  33. 33.

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

  34. 34.

    , & Rapid automated molecular replacement by evolutionary search. Acta. Crystallogr. D 55, 484–491 (1999).

  35. 35.

    , , & Improved methods for building protein models in electron density maps and the location of errors in these models. Acta. Crystallogr. A 47, 110–119 (1991).

  36. 36.

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

  37. 37.

    , & Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).

  38. 38.

    Ribbons. Methods Enzymol. 277, 493–505 (1997).

  39. 39.

    MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

  40. 40.

    & Raster3D: photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997).

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Acknowledgements

We thank our colleagues for help with X-ray data collection and stimulating discussions. Thanks to the staff at beamlines X-12C and X-25 of the National Synchrotron Light Source and beamline F1 of the Cornell High Energy Synchrotron Source for their assistance with X-ray data collection. This work was funded by research grants R01 GM55390 (to T.E.) and R01 CA40463 (to J.-S.T.), and by funding from the Giovanni Armenise Harvard Center for Structural Biology. Y.L. is a US National Institutes of Health Ruth L. Kirschstein postdoctoral fellow (F32 GM065746). T.E.E. is the Hsien Wu and Daisy Yen Wu professor at Harvard Medical School.

Author information

Author notes

    • Shuchismita Dutta

    Present address: Research Collaboratory for Structural Bioinformatics, Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, New Jersey 08854, USA.

    • Sylvie Doublié

    Present address: Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, The University of Vermont, 95 Carrigan Drive, Burlington, Vermont 05405, USA.

Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.

    • Ying Li
    • , Shuchismita Dutta
    • , Sylvie Doublié
    •  & Tom Ellenberger
  2. Department of Chemistry, Washington University in St. Louis, One Brookings Drive, St. Louis, Missouri 63130, USA.

    • Hussam Moh'd Bdour
    •  & John-Stephen Taylor

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The authors declare no competing financial interests.

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Correspondence to Tom Ellenberger.

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DOI

https://doi.org/10.1038/nsmb792

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