Article | Published:

Dynamic basis for dG•dT misincorporation via tautomerization and ionization

Nature volume 554, pages 195201 (08 February 2018) | Download Citation

Abstract

Tautomeric and anionic Watson–Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here, using NMR relaxation dispersion, we resolve a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson–Crick mismatches: two rapidly exchanging tautomeric species (Genol•T/UG•Tenol/Uenol; population less than 0.4%) and one anionic species (G•T/U; population around 0.001% at neutral pH). The sequence-dependent tautomerization or ionization step was inserted into a minimal kinetic mechanism for correct incorporation during replication after the initial binding of the nucleotide, leading to accurate predictions of the probability of dG•dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10−2 to 10−3-fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol, with contributions from anionic dG•dT dominant at pH 8.4 and above or for some mutagenic nucleotides.

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References

  1. 1.

    & The structure of DNA. Cold Spring Harb. Symp. Quant. Biol. 18, 123–131 (1953)

  2. 2.

    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)

  3. 3.

    & Structural basis of the translational elongation cycle. Annu. Rev. Biochem. 82, 203–236 (2013)

  4. 4.

    Active site tightness and substrate fit in DNA replication. Annu. Rev. Biochem. 71, 191–219 (2002)

  5. 5.

    , , , & A new understanding of the decoding principle on the ribosome. Nature 484, 256–259 (2012)

  6. 6.

    , & Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis. Proc. Natl Acad. Sci. USA 108, 17644–17648 (2011)

  7. 7.

    , & Replication infidelity via a mismatch with Watson–Crick geometry. Proc. Natl Acad. Sci. USA 108, 1862–1867 (2011)

  8. 8.

    , , , & Visualizing transient Watson–Crick-like mispairs in DNA and RNA duplexes. Nature 519, 315–320 (2015)

  9. 9.

    , , , & Structural insights into the translational infidelity mechanism. Nat. Commun. 6, 7251 (2015)

  10. 10.

    & Base pairing and fidelity in codon–anticodon interaction. Nature 263, 289–293 (1976)

  11. 11.

    & Complementary base pairing and the origin of substitution mutations. Nature 263, 285–289 (1976)

  12. 12.

    , , & Ensemble cryo-EM elucidates the mechanism of translation fidelity. Nature 546, 113–117 (2017)

  13. 13.

    , & Role of tautomerism in RNA biochemistry. RNA 21, 1–13 (2015)

  14. 14.

    , , & Ionization of bromouracil and fluorouracil stimulates base mispairing frequencies with guanine. J. Biol. Chem. 268, 15935–15943 (1993)

  15. 15.

    , & The spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β. Nucleic Acids Res. 42, 11233–11245 (2014)

  16. 16.

    , , , & New structural insights into translational miscoding. Trends Biochem. Sci. 41, 798–814 (2016)

  17. 17.

    , & Stem cell divisions, somatic mutations, cancer etiology, and cancer prevention. Science 355, 1330–1334 (2017)

  18. 18.

    & Effect of reaction pH on the fidelity and processivity of exonuclease-deficient Klenow polymerase. J. Biol. Chem. 268, 13462–13471 (1993)

  19. 19.

    , & The structural basis for the mutagenicity of O6-methyl-guanine lesions. Proc. Natl Acad. Sci. USA 103, 19701–19706 (2006)

  20. 20.

    , , & Kinetics of extension of O6-methylguanine paired with cytosine or thymine in defined oligonucleotide sequences. Biochemistry 30, 11595–11599 (1991)

  21. 21.

    , , & Nearest neighbor influences on DNA polymerase insertion fidelity. J. Biol. Chem. 264, 14415–14423 (1989)

  22. 22.

    & Fidelity of the human mitochondrial DNA polymerase. J. Biol. Chem. 281, 36236–36240 (2006)

  23. 23.

    & Mispairs with Watson–Crick base-pair geometry observed in ternary complexes of an RB69 DNA polymerase variant. Protein Sci. 23, 508–513 (2014)

  24. 24.

    , , & Selection of tRNA by the ribosome requires a transition from an open to a closed form. Cell 111, 721–732 (2002)

  25. 25.

    et al. DFT calculations on the effect of solvation on the tautomeric reactions for wobble gua–thy and canonical gua–cyt base-pairs. J. Mod. Phys. 4, 422–431 (2013)

  26. 26.

    & The nature of the transition mismatches with Watson–Crick architecture: the G*•T or G•T* DNA base mispair or both? A QM/QTAIM perspective for the biological problem. J. Biomol. Struct. Dyn. 33, 925–945 (2015)

  27. 27.

    , & . An average-magnetization analysis of R relaxation outside of the fast exchange limit. Mol. Phys. 101, 753–763 (2003)

  28. 28.

    , & Off-resonance R NMR studies of exchange dynamics in proteins with low spin-lock fields: an application to a Fyn SH3 domain. J. Am. Chem. Soc. 127, 713–721 (2005)

  29. 29.

    , , & Extending the range of microsecond-to-millisecond chemical exchange detected in labeled and unlabeled nucleic acids by selective carbon R NMR spectroscopy. J. Am. Chem. Soc. 131, 3818–3819 (2009)

  30. 30.

    , , , & Studying excited states of proteins by NMR spectroscopy. Nat. Struct. Biol. 8, 932–935 (2001)

  31. 31.

    , & Direct NMR evidence that transient tautomeric and anionic states in dG•dT form Watson–Crick-like base pairs. J. Am. Chem. Soc. 139, 4326–4329 (2017)

  32. 32.

    , & Tautomerism of 1-methyl derivatives of uracil, thymine, and 5-bromouracil. Is tautomerism the basis for the mutagenicity of 5-bromouridine? J. Phys. Chem. B 102, 5228–5233 (1998)

  33. 33.

    et al. Exceeding the limit of dynamics studies on biomolecules using high spin-lock field strengths with a cryogenically cooled probehead. J. Magn. Reson. 221, 1–4 (2012)

  34. 34.

    , , & Atomistic picture of conformational exchange in a T4 lysozyme cavity mutant: an experiment-guided molecular dynamics study. Chem. Sci. 7, 3602–3613 (2016)

  35. 35.

    & Effect of sequence context on O6-methylguanine repair and replication in vivo. Biochemistry 40, 14968–14975 (2001)

  36. 36.

    & . Theoretical study of R rotating-frame and R2 free-precession relaxation in the presence of n-site chemical exchange. J. Magn. Reson. 170, 104–112 (2004)

  37. 37.

    et al. Thermal fluctuations of immature SOD1 lead to separate folding and misfolding pathways. eLife 4, e07296 (2015)

  38. 38.

    & The base substitution fidelity of eukaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation. J. Biol. Chem. 261, 160–166 (1986)

  39. 39.

    , & Significant contribution of the 3′→5′ exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ε. Nucleic Acids Res. 42, 13853–13860 (2014)

  40. 40.

    & Fidelity of DNA synthesis. Annu. Rev. Biochem. 51, 429–457 (1982)

  41. 41.

    , , & Accuracy of initial codon selection by aminoacyl-tRNAs on the mRNA-programmed bacterial ribosome. Proc. Natl Acad. Sci. USA 112, 9602–9607 (2015)

  42. 42.

    & Structure and mechanism of DNA polymerases. Adv. Protein Chem. 71, 401–440 (2005)

  43. 43.

    & A new paradigm for DNA polymerase specificity. Biochemistry 45, 9675–9687 (2006)

  44. 44.

    , & Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry 30, 511–525 (1991)

  45. 45.

    , & Kinetic mechanism of DNA polymerization catalyzed by human DNA polymerase ε. Biochemistry 52, 7041–7049 (2013)

  46. 46.

    , & Kinetic mechanism of active site assembly and chemical catalysis of DNA polymerase β. Biochemistry 50, 9865–9875 (2011)

  47. 47.

    , & DNA polymerase λ active site favors a mutagenic mispair between the enol form of deoxyguanosine triphosphate substrate and the keto form of thymidine template: a free energy perturbation study. J. Phys. Chem. B 121, 7813–7822 (2017)

  48. 48.

    , , & Observing a DNA polymerase choose right from wrong. Cell 154, 157–168 (2013)

  49. 49.

    , , , & Capturing snapshots of APE1 processing DNA damage. Nat. Struct. Mol. Biol. 22, 924–931 (2015)

  50. 50.

    et al. Characterizing RNA excited states using NMR relaxation dispersion. Methods Enzymol. 558, 39–73 (2015)

  51. 51.

    & AIC model selection using Akaike weights. Psychon. Bull. Rev. 11, 192–196 (2004)

  52. 52.

    & Multimodel inference. Understanding AIC and BIC in model selection. Sociol. Methods Res. 33, 261–304 (2004)

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Acknowledgements

We thank members of the Al-Hashimi laboratory and T. Oas for discussions and input. We acknowledge technical support and resources from the Duke Magnetic Resonance Spectroscopy Center and the Duke Shared Cluster Resource. This work was supported by grants from the National Institutes of Health (NIH R01GM089846, P01GM0066275 and P50GM103297) and an Agilent Thought Leader Award to H.M.A., and a grant from the National Science Foundation (MCB-1716168) to Z.S. W.J.Z. was supported by a Pelotonia Graduate Fellowship from The Ohio State University Comprehensive Cancer Center.

Author information

Author notes

    • Isaac J. Kimsey
    • , Anisha Shakya
    • , Yi Xue
    •  & Bharathwaj Sathyamoorthy

    Present addresses: Nymirum, Durham, North Carolina 27713, USA (I.J.K.); Institute of Basic Science, Center for Soft and Living Matter, Ulsan, South Korea (A.S.); School of Life Sciences, Tsinghua University, Beijing, China (Y.X.); Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal 462 066, India (B.S.).

    • Isaac J. Kimsey
    •  & Eric S. Szymanski

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA

    • Isaac J. Kimsey
    • , Eric S. Szymanski
    • , Yi Xue
    • , Chia-Chieh Chu
    • , Bharathwaj Sathyamoorthy
    •  & Hashim M. Al-Hashimi
  2. Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA

    • Walter J. Zahurancik
    •  & Zucai Suo
  3. The Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA

    • Walter J. Zahurancik
    •  & Zucai Suo
  4. Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA

    • Anisha Shakya
  5. Department of Chemistry, Duke University, Durham, North Carolina 27710, USA

    • Hashim M. Al-Hashimi

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Contributions

I.J.K., E.S.S., and H.M.A. conceived the NMR research and kinetic model. W.J.Z. and Z.S. conceived the kinetic experiments. I.J.K., E.S.S., W.J.Z., Z.S. and H.M.A. wrote the manuscript. I.J.K. synthesized all DNA constructs as well as the RNA hpUG-CGC, hpUG-CGU, and xptG riboswitch constructs, and collected and analysed all NMR relaxation dispersion data. E.S.S. and H.M.A. constructed and tested the kinetic models of misincorporation with input from Z.S. W.J.Z. performed all kinetic experiments and provided input on the kinetic model and simulations with help from Z.S. A.S. synthesized A20G and A22G HIV-I TAR constructs. Y.X. synthesized and assigned p5abc and glnA riboswitch constructs. C.-C.C. synthesized and assigned the HIV-I RRE construct. B.S. synthesized and assigned the HIV-I SL1 dimer complex.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Zucai Suo or Hashim M. Al-Hashimi.

Reviewer Information Nature thanks J. Essigmann, M. Goodman and E. Westhof for their contribution to the peer review of this work.

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