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:

Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction

Nature Chemical Biology volume 13, pages 1074–1080 (2017)Cite this article

Subjects

Abstract

DNA polymerases catalyze efficient and high-fidelity DNA synthesis. While this reaction favors nucleotide incorporation, polymerases also catalyze a reverse reaction, pyrophosphorolysis, that removes the DNA primer terminus and generates deoxynucleoside triphosphates. Because pyrophosphorolysis can influence polymerase fidelity and sensitivity to chain-terminating nucleosides, we analyzed pyrophosphorolysis with human DNA polymerase β and found the reaction to be inefficient. The lack of a thio-elemental effect indicated that this reaction was limited by a nonchemical step. Use of a pyrophosphate analog, in which the bridging oxygen is replaced with an imido group (PNP), increased the rate of the reverse reaction and displayed a large thio-elemental effect, indicating that chemistry was now rate determining. Time-lapse crystallography with PNP captured structures consistent with a chemical equilibrium favoring the reverse reaction. These results highlight the importance of the bridging atom between the β- and γ-phosphates of the incoming nucleotide in reaction chemistry, enzyme conformational changes, and overall reaction equilibrium.

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: Single-turnover analysis of pyrophosphorolysis.
Figure 2: Qualitative assay of pol β reverse reaction with various PPi analogs.
Figure 3: Single-turnover analysis of PNP-dependent reverse reaction.
Figure 4: Removal of aberrant primer termini by pol β–dependent reverse reaction.
Figure 5: Observing the reverse reaction by time-lapse crystallography.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Bebenek, K. & Kunkel, T.A. Functions of DNA polymerases. Adv. Protein Chem. 69, 137–165 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Deutscher, M.P. & Kornberg, A. Enzymatic synthesis of deoxyribonucleic acid. 28. The pyrophosphate exchange and pyrophosphorolysis reactions of deoxyribonucleic acid polymerase. J. Biol. Chem. 244, 3019–3028 (1969).

    CAS  PubMed  Google Scholar 

  3. Parsons, J.L., Nicolay, N.H. & Sharma, R.A. Biological and therapeutic relevance of nonreplicative DNA polymerases to cancer. Antioxid. Redox Signal. 18, 851–873 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. McKenna, C.E., Kashemirov, B.A., Peterson, L.W. & Goodman, M.F. Modifications to the dNTP triphosphate moiety: from mechanistic probes for DNA polymerases to antiviral and anti-cancer drug design. Biochim. Biophys. Acta. 1804, 1223–1230 (2010).

    Article  CAS  PubMed  Google Scholar 

  5. Smith, A.J., Meyer, P.R., Asthana, D., Ashman, M.R. & Scott, W.A. Intracellular substrates for the primer-unblocking reaction by human immunodeficiency virus type 1 reverse transcriptase: detection and quantitation in extracts from quiescent- and activated-lymphocyte subpopulations. Antimicrob. Agents Chemother. 49, 1761–1769 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Urban, S., Urban, S., Fischer, K.P. & Tyrrell, D.L. Efficient pyrophosphorolysis by a hepatitis B virus polymerase may be a primer-unblocking mechanism. Proc. Natl. Acad. Sci. USA 98, 4984–4989 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hanes, J.W. & Johnson, K.A. A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase. Nucleic Acids Res. 35, 6973–6983 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Crespan, E., Maga, G. & Hübscher, U. A new proofreading mechanism for lesion bypass by DNA polymerase-λ. EMBO Rep. 13, 68–74 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Beard, W.A. & Wilson, S.H. Structure and mechanism of DNA polymerase β. Biochemistry 53, 2768–2780 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Perera, L., Beard, W.A., Pedersen, L.G. & Wilson, S.H. Chapter Four - Applications of quantum mechanical/molecular mechanical methods to the chemical insertion step of DNA and RNA polymerization. Adv. Protein Chem. Struct. Biol. 97, 83–113 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Freudenthal, B.D., Beard, W.A., Shock, D.D. & Wilson, S.H. Observing a DNA polymerase choose right from wrong. Cell 154, 157–168 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kirby, T.W. et al. Metal-induced DNA translocation leads to DNA polymerase conformational activation. Nucleic Acids Res. 40, 2974–2983 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Das, K. et al. Conformational states of HIV-1 reverse transcriptase for nucleotide incorporation vs pyrophosphorolysis—binding of foscarnet. ACS Chem. Biol. 11, 2158–2164 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. 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 

  15. Eckstein, F. Nucleoside phosphorothioates. Annu. Rev. Biochem. 54, 367–402 (1985).

    Article  CAS  PubMed  Google Scholar 

  16. Vande Berg, B.J., Beard, W.A. & Wilson, S.H. DNA structure and aspartate 276 influence nucleotide binding to human DNA polymerase β. Implication for the identity of the rate-limiting conformational change. J. Biol. Chem. 276, 3408–3416 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Liu, J. & Tsai, M.D. DNA polymerase β: pre-steady-state kinetic analyses of dATP α S stereoselectivity and alteration of the stereoselectivity by various metal ions and by site-directed mutagenesis. Biochemistry 40, 9014–9022 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Lipton, A. Emerging role of bisphosphonates in the clinic-antitumor activity and prevention of metastasis to bone. Cancer Treat. Rev. 34 (Suppl. 1), S25–S30 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Rozovskaya, T. et al. Pyrophosphate analogues in pyrophosphorolysis reaction catalyzed by DNA polymerases. FEBS Lett. 247, 289–292 (1989).

    Article  CAS  PubMed  Google Scholar 

  20. Penningroth, S.M., Olehnik, K. & Cheung, A. ATP formation from adenyl-5′-yl imidodiphosphate, a nonhydrolyzable ATP analog. J. Biol. Chem. 255, 9545–9548 (1980).

    CAS  PubMed  Google Scholar 

  21. Oertell, K. et al. Transition state in DNA polymerase β catalysis: rate-limiting chemistry altered by base-pair configuration. Biochemistry 53, 1842–1848 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. Oertell, K. et al. Effect of β,γ-CHF- and β,γ-CHCl-dGTP halogen atom stereochemistry on the transition state of DNA polymerase β. Biochemistry 51, 8491–8501 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Sucato, C.A. et al. Modifying the β,γ leaving-group bridging oxygen alters nucleotide incorporation efficiency, fidelity, and the catalytic mechanism of DNA polymerase β. Biochemistry 46, 461–471 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Sucato, C.A. et al. DNA polymerase β fidelity: halomethylene-modified leaving groups in pre-steady-state kinetic analysis reveal differences at the chemical transition state. Biochemistry 47, 870–879 (2008).

    Article  CAS  PubMed  Google Scholar 

  25. Batra, V.K., Beard, W.A., Pedersen, L.C. & Wilson, S.H. Structures of DNA polymerase mispaired DNA termini transitioning to pre-catalytic complexes support an induced-fit fidelity mechanism. Structure 24, 1863–1875 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Li, A., Gong, S. & Johnson, K.A. Rate-limiting pyrophosphate release by HIV reverse transcriptase improves fidelity. J. Biol. Chem. 291, 26554–26565 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Cruchaga, C., Ansó, E., Rouzaut, A. & Martínez-Irujo, J.J. Selective excision of chain-terminating nucleotides by HIV-1 reverse transcriptase with phosphonoformate as substrate. J. Biol. Chem. 281, 27744–27752 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Yanvarev, D.V. et al. Methylene bisphosphonates as the inhibitors of HIV RT phosphorolytic activity. Biochimie 127, 153–162 (2016).

    Article  CAS  PubMed  Google Scholar 

  30. Balbo, P.B., Wang, E.C.-W. & Tsai, M.-D. Kinetic mechanism of active site assembly and chemical catalysis of DNA polymerase β. Biochemistry 50, 9865–9875 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. Tsai, Y.-C. & Johnson, K.A. A new paradigm for DNA polymerase specificity. Biochemistry 45, 9675–9687 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Dahlberg, M.E. & Benkovic, S.J. Kinetic mechanism of DNA polymerase I (Klenow fragment): identification of a second conformational change and evaluation of the internal equilibrium constant. Biochemistry 30, 4835–4843 (1991).

    Article  CAS  PubMed  Google Scholar 

  33. Oertell, K. et al. Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity. Proc. Natl. Acad. Sci. USA 113, E2277–E2285 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Patel, S.S., Wong, I. & Johnson, K.A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry 30, 511–525 (1991).

    Article  CAS  PubMed  Google Scholar 

  35. Olson, A.C., Patro, J.N., Urban, M. & Kuchta, R.D. The energetic difference between synthesis of correct and incorrect base pairs accounts for highly accurate DNA replication. J. Am. Chem. Soc. 135, 1205–1208 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Perera, L. et al. Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse. Proc. Natl. Acad. Sci. USA 112, E5228–E5236 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yount, R.G. Adenylylimidodiphosphate and guanylylimidodiphosphate. Methods Enzymol. 38, 420–427 (1974).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  39. Kadina, A.P. et al. Two scaffolds from two flips: (α,β)/(β,γ) CH2/NH “Met-Im” Analogues of dTTP. Org. Lett. 17, 2586–2589 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Nakamura, T., Zhao, Y., Yamagata, Y., Hua, Y.J. & Yang, W. Watching DNA polymerase η make a phosphodiester bond. Nature 487, 196–201 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gao, Y. & Yang, W. Capture of a third Mg2+ is essential for catalyzing DNA synthesis. Science 352, 1334–1337 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Vyas, R., Reed, A.J., Tokarsky, E.J. & Suo, Z. Viewing human DNA polymerase β faithfully and unfaithfully bypass an oxidative lesion by time-dependent crystallography. J. Am. Chem. Soc. 137, 5225–5230 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Batra, V.K. et al. Magnesium-induced assembly of a complete DNA polymerase catalytic complex. Structure 14, 757–766 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Beard, W.A. & Wilson, S.H. Purification and domain-mapping of mammalian DNA polymerase β. Methods Enzymol. 262, 98–107 (1995).

    Article  CAS  PubMed  Google Scholar 

  45. Freudenthal, B.D. et al. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. Nature 517, 635–639 (2015).

    Article  CAS  PubMed  Google Scholar 

  46. Beard, W.A., Shock, D.D., Batra, V.K., Prasad, R. & Wilson, S.H. Substrate-induced DNA polymerase β activation. J. Biol. Chem. 289, 31411–31422 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  48. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the Collaborative Crystallography group at NIEHS for help with data collection and analysis. Use of the advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract W-31-109-Eng-38. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (project numbers Z01-ES050158 and Z01-ES050161) (S.H.W.) and was in association with the National Institutes of Health grant 1U19CA105010 (S.H.W.) and R00-ES024431 (B.D.F.).

Author information

Authors and Affiliations

  1. Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina, USA

    David D Shock, Bret D Freudenthal, William A Beard & Samuel H Wilson

  2. Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas, USA

    Bret D Freudenthal

Authors
  1. David D Shock
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bret D Freudenthal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William A Beard
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Samuel H Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.D.S., B.D.F., W.A.B., and S.H.W. designed the project. D.D.S. did the kinetic analyses. B.D.F. carried out crystallography. D.D.S., B.D.F., W.A.B., and S.H.W. prepared the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Samuel H Wilson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–2 and Supplementary Figures 1–8. (PDF 4379 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shock, D., Freudenthal, B., Beard, W. et al. Modulating the DNA polymerase β reaction equilibrium to dissect the reverse reaction. Nat Chem Biol 13, 1074–1080 (2017). https://doi.org/10.1038/nchembio.2450

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2450

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