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Dynamic basis for dA•dGTP and dA•d8OGTP misincorporation via Hoogsteen base pairs

Nature Chemical Biology volume 19, pages 900–910 (2023)Cite this article

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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.

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Fig. 1: The conformational states of A•G and A•8OG found in the PDB with Aanti•Ganti as the GS.
Fig. 2: NMR R1ρ and CEST measurements reveal sparsely populated and short-lived Asyn•Ganti and Aanti+•Gsyn Hoogsteen bps in hpGAC.
Fig. 3: Chemical shift fingerprinting of Asyn•Ganti and Aanti+•Gsyn.
Fig. 4: A•8OG redistributes the ensemble, rendering Aanti•8OGsyn the GS.
Fig. 5: Kinetic modeling reveals Aanti+•Gsyn as a plausible pathway for dA•dGTP misincorporation by human polymerase β.
Fig. 6: The role of A•G and A•8OG dynamics in misincorporation and damage repair.

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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.

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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).

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Author notes

  1. Eric S. Szymanski

    Present address: Base4, Durham, NC, USA

  2. Atul K. Rangadurai

    Present address: Hospital for Sick Children, Toronto, Ontario, Canada

  3. Honglue Shi

    Present address: Innovative Genomics Institute, University of California, Berkeley, CA, USA

  4. Bei Liu

    Present address: Department of Chemistry, University of Chicago, Chicago, IL, USA

Authors and Affiliations

  1. Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA

    Stephanie Gu, Eric S. Szymanski, Atul K. Rangadurai, Bei Liu & Akanksha Manghrani

  2. Department of Chemistry, Duke University, Durham, NC, USA

    Honglue Shi

  3. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Hashim M. Al-Hashimi

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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.

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Correspondence to Hashim M. Al-Hashimi.

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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.

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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

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