Abstract
Replicative errors contribute to the genetic diversity needed for evolution but in high frequency can lead to genomic instability. Here, we show that DNA dynamics determine the frequency of misincorporating the A•G mismatch, and altered dynamics explain the high frequency of 8-oxoguanine (8OG) A•8OG misincorporation. NMR measurements revealed that Aanti•Ganti (population (pop.) of >91%) transiently forms sparsely populated and short-lived Aanti+•Gsyn (pop. of ~2% and kex = kforward + kreverse of ~137 s−1) and Asyn•Ganti (pop. of ~6% and kex of ~2,200 s−1) Hoogsteen conformations. 8OG redistributed the ensemble, rendering Aanti•8OGsyn the dominant state. A kinetic model in which Aanti+•Gsyn is misincorporated quantitatively predicted the dA•dGTP misincorporation kinetics by human polymerase β, the pH dependence of misincorporation and the impact of the 8OG lesion. Thus, 8OG increases replicative errors relative to G because oxidation of guanine redistributes the ensemble in favor of the mutagenic Aanti•8OGsyn Hoogsteen state, which exists transiently and in low abundance in the A•G mismatch.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The NMR data generated in this study are included in the published article and the Supplementary Information file.
Code availability
Code for the kinetic simulations is available on GitHub at https://github.com/alhashimilab/AG-Simulation.
References
Shendure, J. & Akey, J. M. The origins, determinants, and consequences of human mutations. Science 349, 1478–1483 (2015).
Sprouffske, K., Aguilar-Rodriguez, J., Sniegowski, P. & Wagner, A. High mutation rates limit evolutionary adaptation in Escherichia coli. PLoS Genet. 14, e1007324 (2018).
Cahill, D. P., Kinzler, K. W., Vogelstein, B. & Lengauer, C. Genetic instability and Darwinian selection in tumours. Trends Cell Biol. 9, M57–M60 (1999).
Tomasetti, C., Li, L. & Vogelstein, B. Stem cell divisions, somatic mutations, cancer etiology, and cancer prevention. Science 355, 1330–1334 (2017).
Rattray, A. J. & Strathern, J. N. Error-prone DNA polymerases: when making a mistake is the only way to get ahead. Annu. Rev. Genet. 37, 31–66 (2003).
Barbari, S. R. & Shcherbakova, P. V. Replicative DNA polymerase defects in human cancers: consequences, mechanisms, and implications for therapy. DNA Repair 56, 16–25 (2017).
Watson, J. D. & Crick, F. H. Genetical implications of the structure of deoxyribonucleic acid. Nature 171, 964–967 (1953).
Schendel, P. F. & Robins, P. E. Repair of O6-methylguanine in adapted Escherichia coli. Proc. Natl Acad. Sci. USA 75, 6017–6020 (1978).
Warren, J. J., Forsberg, L. J. & Beese, L. S. The structural basis for the mutagenicity of O6-methyl-guanine lesions. Proc. Natl Acad. Sci. USA 103, 19701–19706 (2006).
Kimsey, I. J., Petzold, K., Sathyamoorthy, B., Stein, Z. W. & Al-Hashimi, H. M. Visualizing transient Watson–Crick-like mispairs in DNA and RNA duplexes. Nature 519, 315–320 (2015).
Kimsey, I. J. et al. Dynamic basis for dG•dT misincorporation via tautomerization and ionization. Nature 554, 195–201 (2018).
Wang, W., Hellinga, H. W. & Beese, L. S. Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis. Proc. Natl Acad. Sci. USA 108, 17644–17648 (2011).
Bebenek, K., Pedersen, L. C. & Kunkel, T. A. Replication infidelity via a mismatch with Watson–Crick geometry. Proc. Natl Acad. Sci. USA 108, 1862–1867 (2011).
Koag, M. C., Nam, K. & Lee, S. The spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β. Nucleic Acids Res. 42, 11233–11245 (2014).
Topal, M. D. & Fresco, J. R. Complementary base pairing and the origin of substitution mutations. Nature 263, 285–289 (1976).
Koag, M. C., Jung, H. & Lee, S. Mutagenesis mechanism of the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine. Nucleic Acids Res. 48, 5119–5134 (2020).
Freudenthal, B. D., Beard, W. A. & Wilson, S. H. DNA polymerase minor groove interactions modulate mutagenic bypass of a templating 8-oxoguanine lesion. Nucleic Acids Res. 41, 1848–1858 (2013).
Kirby, T. W., DeRose, E. F., Beard, W. A., Wilson, S. H. & London, R. E. A thymine isostere in the templating position disrupts assembly of the closed DNA polymerase β ternary complex. Biochemistry 44, 15230–15237 (2005).
van Loon, B., Markkanen, E. & Hubscher, U. Oxygen as a friend and enemy: how to combat the mutational potential of 8-oxo-guanine. DNA Repair 9, 604–616 (2010).
Brieba, L. G. et al. Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase. EMBO J. 23, 3452–3461 (2004).
Alexandrov, L. B. et al. The repertoire of mutational signatures in human cancer. Nature 578, 94–101 (2020).
Kool, E. T. Active site tightness and substrate fit in DNA replication. Annu. Rev. Biochem. 71, 191–219 (2002).
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).
Rozov, A., Demeshkina, N., Westhof, E., Yusupov, M. & Yusupova, G. New structural insights into translational miscoding. Trends Biochem. Sci. 41, 798–814 (2016).
Shi, H. et al. NMR chemical exchange measurements reveal that N6-methyladenosine slows RNA annealing. J. Am. Chem. Soc. 141, 19988–19993 (2019).
Rangadurai, A., Szymaski, E. S., Kimsey, I. J., Shi, H. & Al-Hashimi, H. M. Characterizing micro-to-millisecond chemical exchange in nucleic acids using off-resonance R1ρ relaxation dispersion. Prog. Nucl. Magn. Reson. Spectrosc. 112–113, 55–102 (2019).
Nikolova, E. N. et al. Transient Hoogsteen base pairs in canonical duplex DNA. Nature 470, 498–502 (2011).
Nikolova, E. N., Goh, G. B., Brooks, C. L. III & Al-Hashimi, H. M. Characterizing the protonation state of cytosine in transient G•C Hoogsteen base pairs in duplex DNA. J. Am. Chem. Soc. 135, 6766–6769 (2013).
Baisden, J. T., Boyer, J. A., Zhao, B., Hammond, S. M. & Zhang, Q. Visualizing a protonated RNA state that modulates microRNA-21 maturation. Nat. Chem. Biol. 17, 80–88 (2021).
Chu, C. C., Plangger, R., Kreutz, C. & Al-Hashimi, H. M. Dynamic ensemble of HIV-1 RRE stem IIB reveals non-native conformations that disrupt the Rev-binding site. Nucleic Acids Res. 47, 7105–7117 (2019).
Dethoff, E. A., Petzold, K., Chugh, J., Casiano-Negroni, A. & Al-Hashimi, H. M. Visualizing transient low-populated structures of RNA. Nature 491, 724–728 (2012).
Sedgwick, B. Repairing DNA-methylation damage. Nat. Rev. Mol. Cell Biol. 5, 148–157 (2004).
Lane, A. N., Jenkins, T. C., Brown, D. J. & Brown, T. N.m.r. determination of the solution conformation and dynamics of the A•G mismatch in the d(CGCAAATTGGCG)2 dodecamer. Biochem. J. 279, 269–281 (1991).
Kouchakdjian, M. et al. NMR structural studies of the ionizing radiation adduct 7-hydro-8-oxodeoxyguanosine (8-oxo-7H-dG) opposite deoxyadenosine in a DNA duplex. 8-Oxo-7H-dGsyn•dAanti alignment at lesion site. Biochemistry 30, 1403–1412 (1991).
Rechkoblit, O. et al. Impact of conformational heterogeneity of OxoG lesions and their pairing partners on bypass fidelity by Y family polymerases. Structure 17, 725–736 (2009).
Balbo, P. B., Wang, E. C. & Tsai, M. D. Kinetic mechanism of active site assembly and chemical catalysis of DNA polymerase β. Biochemistry 50, 9865–9875 (2011).
Ahn, J., Kraynov, V. S., Zhong, X., Werneburg, B. G. & Tsai, M. D. DNA polymerase β: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes. Biochem. J. 331, 79–87 (1998).
Manlove, A. H., Nunez, N. N. & David, S. S. in Base Excision Repair Pathway: Molecular Mechanisms and Role in Disease Development and Therapeutic Design (ed. Wilson, D. M., III) 63–115 (World Scientific, 2017).
Ahn, J., Werneburg, B. G. & Tsai, M. D. DNA polymerase β: structure–fidelity relationship from pre-steady-state kinetic analyses of all possible correct and incorrect base pairs for wild type and R283A mutant. Biochemistry 36, 1100–1107 (1997).
Brown, J. A., Duym, W. W., Fowler, J. D. & Suo, Z. Single-turnover kinetic analysis of the mutagenic potential of 8-oxo-7,8-dihydro-2′-deoxyguanosine during gap-filling synthesis catalyzed by human DNA polymerases λ and β. J. Mol. Biol. 367, 1258–1269 (2007).
Burak, M. J., Guja, K. E., Hambardjieva, E., Derkunt, B. & Garcia-Diaz, M. A fidelity mechanism in DNA polymerase λ promotes error-free bypass of 8-oxo-dG. EMBO J. 35, 2045–2059 (2016).
Kirouac, K. N. & Ling, H. Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase ι. Proc. Natl Acad. Sci. USA 108, 3210–3215 (2011).
Batra, V. K., Shock, D. D., Beard, W. A., McKenna, C. E. & Wilson, S. H. Binary complex crystal structure of DNA polymerase β reveals multiple conformations of the templating 8-oxoguanine lesion. Proc. Natl Acad. Sci. USA 109, 113–118 (2012).
Batra, V. K., Beard, W. A., Shock, D. D., Pedersen, L. C. & Wilson, S. H. Structures of DNA polymerase β with active-site mismatches suggest a transient abasic site intermediate during misincorporation. Mol. Cell 30, 315–324 (2008).
Westhof, E., Yusupov, M. & Yusupova, G. Recognition of Watson–Crick base pairs: constraints and limits due to geometric selection and tautomerism. F1000Prime Rep. 6, 19 (2014).
Li, P., Rangadurai, A., Al-Hashimi, H. M. & Hammes-Schiffer, S. Environmental effects on guanine-thymine mispair tautomerization explored with quantum mechanical/molecular mechanical free energy simulations. J. Am. Chem. Soc. 142, 11183–11191 (2020).
Ganser, L. R., Kelly, M. L., Herschlag, D. & Al-Hashimi, H. M. The roles of structural dynamics in the cellular functions of RNAs. Nat. Rev. Mol. Cell Biol. 20, 474–489 (2019).
Fromme, J. C., Banerjee, A., Huang, S. J. & Verdine, G. L. Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase. Nature 427, 652–656 (2004).
Lee, S. & Verdine, G. L. Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase. Proc. Natl Acad. Sci. USA 106, 18497–18502 (2009).
Alvey, H. S., Gottardo, F. L., Nikolova, E. N. & Al-Hashimi, H. M. Widespread transient Hoogsteen base pairs in canonical duplex DNA with variable energetics. Nat. Commun. 5, 4786 (2014).
Lu, X. J., Bussemaker, H. J. & Olson, W. K. DSSR: an integrated software tool for dissecting the spatial structure of RNA. Nucleic Acids Res. 43, e142 (2015).
Leontis, N. B., Stombaugh, J. & Westhof, E. The non-Watson–Crick base pairs and their associated isostericity matrices. Nucleic Acids Res. 30, 3497–3531 (2002).
Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).
Hansen, A. L., Nikolova, E. N., Casiano-Negroni, A. & Al-Hashimi, H. M. Extending the range of microsecond-to-millisecond chemical exchange detected in labeled and unlabeled nucleic acids by selective carbon R1ρ NMR spectroscopy. J. Am. Chem. Soc. 131, 3818–3819 (2009).
McConnell, H. M. Reaction rates by nuclear magnetic resonance. J. Chem. Phys. 28, 430–431 (1958).
Koss, H., Rance, M. & Palmer, A. G. III. General expressions for R1ρ relaxation for N-site chemical exchange and the special case of linear chains. J. Magn. Reson. 274, 36–45 (2017).
Palmer, A. G. & Koss, H. in Methods in Enzymology, Vol. 615 (ed. Wand, A. J.) 177–236 (Academic Press, 2019).
Zhao, B., Hansen, A. L. & Zhang, Q. Characterizing slow chemical exchange in nucleic acids by carbon CEST and low spin-lock field R1ρ NMR spectroscopy. J. Am. Chem. Soc. 136, 20–23 (2014).
Liu, B., Rangadurai, A., Shi, H. & Al-Hashimi, H. M. Rapid assessment of Watson–Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino 1H CEST. Magn. Reson. 2, 715–731 (2021).
Schlagnitweit, J., Steiner, E., Karlsson, H. & Petzold, K. Efficient detection of structure and dynamics in unlabeled RNAs: the SELOPE approach. Chemistry 24, 6067–6070 (2018).
Acknowledgements
We thank members of the H.M.A.-H. laboratory for assistance and critical comments on the manuscript. This work was supported by the US National Institute for General Medical Sciences (R01GM089846).
Author information
Authors and Affiliations
Contributions
S.G., E.S.S. and H.M.A.-H. conceived the project and experimental design. E.S.S. and S.G. prepared the samples. S.G. and E.S.S. performed NMR experiments. S.G., A.K.R., H.S., B.L. and H.M.A.-H. analyzed the NMR data. S.G., A.M. and H.M.A.-H. performed and analyzed the computational modeling. S.G. and H.M.A.-H. wrote the paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Chemical Biology thanks Lewis Kay and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 NMR analysis reveals Aanti•Ganti as the ground-state in hpGAC.
(A) 2D [1H, 1H] NOESY imino walk of Aanti•Ganti at T = 1 °C. (B) 2D [1H, 1H] NOESY spectra of the H2ʹ/H2ʹʹ-H6/H8 region for Aanti•Ganti at T = 25 °C. (C) 2D [13C, 1H] HSQC spectra of the C1ʹ-H1ʹ, C2-H2, C6-H6, and C8-H8 regions for Aanti•Ganti at T = 25 °C.
Extended Data Fig. 2 Characterization of A•G dynamics in the hpGAT hairpin.
(A) Secondary structure of hpGAT and chemical shift assignments of the C1ʹ-H1ʹ, C2-H2, C6-H6, and C8-H8 regions of 2D [13C, 1H] HSQC spectra for hpGAT. (B) Three-state dynamic equilibrium of A•G in the hpGAT is like hpGAC. Asterix denotes population and exchange degeneracy in the slow exchange to Aanti+•Gsyn. (C) Off-resonance R1ρ profiles for A-C2, A-C1ʹ, and A-C8. Spin-lock powers used for R1ρ profiles are color-coded in panel (C). Solid lines in panel (C) denote the global 2-state fits to the data using B-M equations as described in Methods. Data for the R1ρ profiles in panel (C) were presented as values ± 1 s.d. from Monte Carlo simulations for one measurement as described in Methods. (D) Comparison of the RD-derived Δω between hpGAT (dark orange) and hpGAC (gold). The Δω data corresponding to the panel (D) is presented as mean values ± 1 s.d. from Monte Carlo simulations (number of iterations = 500) for one R1ρ measurement as described in Methods.
Extended Data Fig. 3 Exchange parameters for the A•G mismatch measured using R1ρ in hpGAC at 10 °C.
Shown are the off-resonance 13C R1ρ profiles measured for A-C1ʹ, A-C8, A-C2, and G-C8 collected at 10 °C and pH 7.4 in NMR buffer as described in Methods. Spin-lock powers used for R1ρ profiles are color-coded. Solid lines in the profiles denote the global 2-state fits to the data using B-M equations as described in Methods. Data for the R1ρ profiles were presented as values ± 1 s.d. from Monte Carlo simulations for one measurement as described in Methods. For the R1ρ profiles corresponding to A-C1ʹ and A-C8, the ± 1 s.d. is smaller than the data points. These exchange measurements at 10 °C only sense Asyn-Ganti exchange (top) since Aanti+-Gsyn exchange becomes too slow to have a substantial contribution (middle). Probes that sensed exchange at this condition are highlighted in green.
Extended Data Fig. 4 pH-dependence of conformational exchange involving the Aanti+•Gsyn ES.
(A) The pH-dependence of R2 + Rex profiles for A-C1ʹ, A-C8, A-C2, G-C1ʹ, and G-C8 for pH 6.9 and pH 7.4. Spin-lock powers used for R1ρ profiles are color-coded. (B) NMR-derived Δω of pH 6.9 (outlined) agrees with that of pH 7.4 (filled), indicating that the same Hoogsteen ESs are being detected at the lower pH. The Δω data in panel (B) are presented as mean values ± 1 s.d. from Monte Carlo simulations (number of iterations = 500) for one R1ρ or CEST measurement as described in Methods. (C) 13C R1ρ profile measured for A-C2 at pH 6.9. Spin-lock powers used for the R1ρ profile is color-coded. Solid lines denote the global fits to the data using B-M equations while fixing the population of the Aanti+•Gsyn ES to be the pKa-derived population. Data for the R1ρ profile in panel (C) were presented as values ± 1 s.d. (smaller than the data points) from Monte Carlo simulations for one measurement as described in Methods. (D) B-M simulations on 3-state exchange in A-C8 using exchange parameters determined at pH 7.4 and pH 6.9 indicate that the R2 + Rex for Aanti+•Gsyn are masked by exchange with Asyn•Ganti.
Extended Data Fig. 5 NMR spectra of mutant-mimics of the ESs.
Chemical shift overlays of the C1ʹ-H1ʹ, C2-H2, C6-H8, and C8-H8 regions of 2D [13C, 1H] HSQC spectra for the m1A (green), m1G (dark blue), and low pH (light blue) structural mimics relative to A•G (black).
Extended Data Fig. 6 Characterization of the A•8OG mismatch.
Left: 2D [1H, 1H] NOESY imino walk of A•8OG at 25 °C. Right: Chemical shift assignments of the C1ʹ-H1ʹ, C2-H2, C6-H6, and C8-H8 regions of 2D [13C, 1H] HSQC spectra for A•8OG.
Extended Data Fig. 7 Definition of rate constants in the kinetic mechanism used to model misincorporation.
Shown are kinetic mechanisms for (A) correct dA•dTTP Watson-Crick incorporation. (B) dA•dGTP misincorporation and (C) dA•d8OGTP misincorporation. In (B), k1, k-1(dNTP), k3, and k-3 are used in Models 1, 2, and 1 + 2. In Model 1, only Aanti+•Gsyn is accepted as the mutagenic intermediate and can proceed forward with rate constants k2 and k-2 while Asyn•Ganti can be unbound by the polymerase with rate constants k1 and k-1(ES1). Model 2 is the same as Model 1 but with Asyn•Ganti as the mutagenic form proceeding forward with rate constants k2 and k-2 and Aanti+•Gsyn unbound with rate constants k1 and k-1(ES2). In Model 1 + 2, both Aanti+•Gsyn and Asyn•Ganti can proceed forward with misincorporation with rate constants k2 and k-2. Neither ES species can unbind. (C) Kinetic mechanism of dA•d8OGTP misincorporation. All rate constants listed are used. E, Eʹʹ, Eʹ, and E* refer to DNA polymerase in the open, ajar, closed, and catalytically active conformations, respectively.
Extended Data Fig. 8 Misincorporation probability (Fpol) of dA•dGTP and dA•8OGTP from kinetic simulations.
Shown are the Fpol calculated from the simulated Kd and kpol for the reference Watson-Crick dA•dTTP bp and dA•dGTP or dA•8OGTP. Data presented for the reference experimental (Exp) values as solid bar graphs were obtained from their corresponding reference for A•G37 (left), its pH dependence39 (middle) and the impact of 8OG40 (right). Data presented for the computational kinetic modeling as the white bar graphs are presented as mean values ± 1 s.d. (not visible) from Monte Carlo simulations (number of iterations = 200) as described in Methods.
Supplementary information
Supplementary Information
Supplementary Tables 1–11.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Gu, S., Szymanski, E.S., Rangadurai, A.K. et al. Dynamic basis for dA•dGTP and dA•d8OGTP misincorporation via Hoogsteen base pairs. Nat Chem Biol 19, 900–910 (2023). https://doi.org/10.1038/s41589-023-01306-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41589-023-01306-5