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:

Template strand scrunching during DNA gap repair synthesis by human polymerase λ

Nature Structural & Molecular Biology volume 16, pages 967–972 (2009)Cite this article

Abstract

Family X polymerases such as DNA polymerase λ (Pol λ) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5′ and 3′ ends of short gaps. DNA binding and gap filling are well characterized for 1-nucleotide (nt) gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that, when bound to a 2-nt gap, Pol λ scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket that comprises three conserved amino acids. Replacing these amino acids with alanine results in less processive gap filling and less efficient NHEJ when 2-nt gaps are involved. Thus, akin to scrunching by RNA polymerase during transcription initiation, scrunching occurs during gap filling DNA synthesis associated with DNA repair.

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: Structure of Pol λ bound to a 2-nt gap.
Figure 2: Binding pocket for the scrunched nucleotide.
Figure 3: In vitro gap filling.
Figure 4: The triple alanine mutant is deficient in NHEJ of longer gaps.
Figure 5: Final conformation of the template strand after molecular dynamics simulations with a 4-nt gap substrate.
Figure 6: Scrunching during gap filling.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Moon, A.F. et al. The X family portrait: structural insights into biological functions of X family polymerases. DNA Repair (Amst.) 6, 1709–1725 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  2. Beard, W.A. & Wilson, S.H. Structure and mechanism of DNA polymerase β. Chem. Rev. 106, 361–382 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. García-Díaz, M. et al. DNA polymerase λ, a novel DNA repair enzyme in human cells. J. Biol. Chem. 277, 13184–13191 (2002).

    Article  PubMed  Google Scholar 

  4. García-Díaz, M., Bebenek, K., Kunkel, T.A. & Blanco, L. Identification of an intrinsic 5′-deoxyribose-5-phosphate lyase activity in human DNA polymerase λ: a possible role in base excision repair. J. Biol. Chem. 276, 34659–34663 (2001).

    Article  PubMed  Google Scholar 

  5. Braithwaite, E.K. et al. DNA polymerase λ protects mouse fibroblasts against oxidative DNA damage and is recruited to sites of DNA damage/repair. J. Biol. Chem. 280, 31641–31647 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Tano, K. et al. Interplay between DNA polymerases β and λ in repair of oxidation DNA damage in chicken DT40 cells. DNA Repair (Amst.) 6, 869–875 (2007).

    Article  CAS  Google Scholar 

  7. Nick McElhinny, S.A. et al. A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. Mol. Cell 19, 357–366 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Lee, J.W. et al. Implication of DNA polymerase λ in alignment-based gap filling for nonhomologous DNA end joining in human nuclear extracts. J. Biol. Chem. 279, 805–811 (2004).

    Article  CAS  PubMed  Google Scholar 

  9. Lieber, M.R., Lu, H., Gu, J. & Schwarz, K. Flexibility in the order of action and in the enzymology of the nuclease, polymerases, and ligase of vertebrate non-homologous DNA end joining: relevance to cancer, aging, and the immune system. Cell Res. 18, 125–133 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Weterings, E. & Chen, D.J. The endless tale of non-homologous end-joining. Cell Res. 18, 114–124 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Garcia-Diaz, M., Bebenek, K., Krahn, J.M., Kunkel, T.A. & Pedersen, L.C. A closed conformation for the Pol λ catalytic cycle. Nat. Struct. Mol. Biol. 12, 97–98 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Singhal, R.K. & Wilson, S.H. Short gap-filling synthesis by DNA polymerase β is processive. J. Biol. Chem. 268, 15906–15911 (1993).

    CAS  PubMed  Google Scholar 

  13. Sawaya, M.R., Prasad, R., Wilson, S.H., Kraut, J. & Pelletier, H. Crystal structures of human DNA polymerase β complexed with gapped and nicked DNA: evidence for an induced fit mechanism. Biochemistry 36, 11205–11215 (1997).

    Article  CAS  PubMed  Google Scholar 

  14. Moon, A.F. et al. Structural insight into the substrate specificity of DNA polymerase μ. Nat. Struct. Mol. Biol. 14, 45–53 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. van Gent, D.C. & van der Burg, M. Non-homologous end-joining, a sticky affair. Oncogene 26, 7731–7740 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Klungland, A. & Lindahl, T. Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1). EMBO J. 16, 3341–3348 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Davis, B.J., Havener, J.M. & Ramsden, D.A. End-bridging is required for Pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining. Nucleic Acids Res. 36, 3085–3094 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Brissett, N.C. et al. Structure of a NHEJ polymerase-mediated DNA synaptic complex. Science 318, 456–459 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Garcia-Diaz, M. et al. A structural solution for the DNA polymerase λ-dependent repair of DNA gaps with minimal homology. Mol. Cell 13, 561–572 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Cheetham, G.M. & Steitz, T.A. Structure of a transcribing T7 RNA polymerase initiation complex. Science 286, 2305–2309 (1999).

    Article  CAS  PubMed  Google Scholar 

  21. Kapanidis, A.N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Ikeda, R.A. & Richardson, C.C. Interactions of the RNA polymerase of bacteriophage T7 with its promoter during binding and initiation of transcription. Proc. Natl. Acad. Sci. USA 83, 3614–3618 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bebenek, K., Garcia-Diaz, M., Blanco, L. & Kunkel, T.A. The frameshift infidelity of human DNA polymerase λ. Implications for function. J. Biol. Chem. 278, 34685–34690 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Garcia-Diaz, M., Bebenek, K., Krahn, J.M., Pedersen, L.C. & Kunkel, T.A. Structural analysis of strand misalignment during DNA synthesis by a human DNA polymerase. Cell 124, 331–342 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Bebenek, K. et al. Substrate-induced DNA strand misalignment during catalytic cycling by DNA polymerase λ. EMBO Rep. 9, 459–464 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Garcia-Diaz, M. & Kunkel, T.A. Mechanism of a genetic glissando: structural biology of indel mutations. Trends Biochem. Sci. 31, 206–214 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Tang, G.Q., Roy, R., Ha, T. & Patel, S.S. Transcription initiation in a single-subunit RNA polymerase proceeds through DNA scrunching and rotation of the N-terminal subdomains. Mol. Cell 30, 567–577 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  29. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    Article  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  31. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. 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).

    Article  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  34. Bebenek, K., Garcia-Diaz, M., Patishall, S.R. & Kunkel, T.A. Biochemical properties of Saccharomyces cerevisiae DNA polymerase IV. J. Biol. Chem. 280, 20051–20058 (2005).

    Article  CAS  PubMed  Google Scholar 

  35. Kokoska, R.J., McCulloch, S.D. & Kunkel, T.A. The efficiency and specificity of apurinic/apyrimidinic site bypass by human DNA polymerase η and Sulfolobus solfataricus Dpo4. J. Biol. Chem. 278, 50537–50545 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. Osheroff, W.P., Jung, H.K., Beard, W.A., Wilson, S.H. & Kunkel, T.A. The fidelity of DNA polymerase β during distributive and processive DNA synthesis. J. Biol. Chem. 274, 3642–3650 (1999).

    Article  CAS  PubMed  Google Scholar 

  37. Pérez, A. et al. Refinement of the AMBER force field for nucleic acids: improving the description of α/γ conformers. Biophys. J. 92, 3817–3829 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Duan, Y. et al. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J. Comput. Chem. 24, 1999–2012 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Essmann, U. et al. A smooth particle mesh Ewald method. J. Chem. Phys. 103, 8577–8593 (1995).

    Article  CAS  Google Scholar 

  40. Case, D.A. et al. AMBER 10 (University of California, San Francisco, CA, 2008).

Download references

Acknowledgements

We thank L. Pedersen, W. Beard and B. Van Houten for critical reading of the manuscript. This work was partly supported by Project Z01 ES065070 to T.A.K. from the Division of Intramural Research of the US National Institutes of Health (NIH), National Institute of Environmental Health Sciences and by NIH grant CA 097096 to D.A.R. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38.

Author information

Author notes

  1. Miguel Garcia-Diaz

    Present address: Present Address: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York, USA.,

Authors and Affiliations

  1. Department of Health and Human Services, Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, North Carolina, USA

    Miguel Garcia-Diaz, Katarzyna Bebenek, Andres A Larrea, Lalith Perera, Joseph M Krahn, Lars C Pedersen & Thomas A Kunkel

  2. Department of Health and Human Services, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, North Carolina, USA

    Miguel Garcia-Diaz, Katarzyna Bebenek, Andres A Larrea & Thomas A Kunkel

  3. Department of Biochemistry and Biophysics, and Curriculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Jody M Havener & Dale A Ramsden

Authors
  1. Miguel Garcia-Diaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katarzyna Bebenek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andres A Larrea
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jody M Havener
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lalith Perera
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Joseph M Krahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lars C Pedersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dale A Ramsden
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Thomas A Kunkel
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.A.K. and D.A.R. supervised the study, designed experiments and analyzed data; M.G.-D., K.B., A.A.L. and J.M.H. performed experiments and analyzed data; L.C.P. contributed to structure refinement; J.M.K. and L.P. performed molecular dynamics simulations; J.M.K. prepared the Supplementary Video 1; all authors contributed to writing the manuscript.

Corresponding author

Correspondence to Thomas A Kunkel.

Supplementary information

Supplementary Video 1

Model of gap filling by Pol λ. The first incoming dNTP and matching template base are in green. The second incoming and template bases are in magenta. Residues at the scrunched binding site are in orange (Leu277, His511, Arg514). Side chains are shown for Arg517 near the template strand, and the following residues, from left to right: Asp490, Asp427, Asp429, Arg386, Tyr505 and Arg420. The dRP domain is light purple, the fingers domain is light cyan, the palm domain is light green and the thumb domain is beige. The animation shows transitions between nine models, each using experimental coordinates except for DNA base substitutions modeled to maintain the same sequence. The catalytic transitions use coordinates from superimposed models to produce the complete set of coordinates. Transitions were modeled with CNS, using stepped distance restraints as a tractor-beam, along with standard geometry restraints. This is meant as a tool to aid in visualizing the current series of structures. It is not a rigorous dynamics simulation, and may be missing intermediate details not present in the known structures. (AVI 5578 kb)

1) Scrunching and incoming dNTP (starts from 1RZT, ends at current structure)

2) Activation #1 of scrunched structure (active site with Mg from 2PFO)

3) Catalysis #1 (active site from 1XSP1)

4) Translocation from scrunched product to 1nt-gap (1XSL), PPi+Mg release

5) dNTP binding to 1-nt gap with DNA template-strand docking (1XSN)

6) Activation #2 of 1nt-gap (active site with Mg from 2PFO)

7) Catalysis #2 (active site from 1XSP1)

8) Relaxation after Catalysis #2 (ending at 1XSP2), and PPi+Mg release

Rights and permissions

Reprints and permissions

About this article

Cite this article

Garcia-Diaz, M., Bebenek, K., Larrea, A. et al. Template strand scrunching during DNA gap repair synthesis by human polymerase λ. Nat Struct Mol Biol 16, 967–972 (2009). https://doi.org/10.1038/nsmb.1654

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1654

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