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Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

Nature volume 517, pages 635–639 (2015)Cite this article

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Abstract

Oxidative stress promotes genomic instability and human diseases1. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2′-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP)2,3. Nucleotide pools are especially vulnerable to oxidative damage4. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival5,6 and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner7. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics8. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis9,10. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine11. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

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Figure 1: 8-Oxo-dGTP specificity and insertion opposite adenine.
Figure 2: 8-Oxo-dGTP insertion opposite cytosine.
Figure 3: Product complex with 8-oxo-dGMP(anti):cytosine.
Figure 4: Charge modulation of the polymerase active site.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession numbers 4UAW, 4UAY, 4UAZ, 4UB1, 4UB2, 4UB3, 4UB4, 4UB5, 4UBB, and 4UBC.

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Acknowledgements

We thank the Collaborative Crystallography group at the National Institute of Environmental Health Sciences for help with data collection and analysis. We thank L. Pedersen for discussions. 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 National Institutes of Health, National Institute of Environmental Health Sciences (project numbers Z01-ES050158 (to S.W.), Z01-ES050161 (to S.W.), and ZIC-ES043010 (to L.P.)) and in association with National Institutes of Health grant 1U19CA105010. We are grateful for computational support for the molecular dynamics simulations from the HPC clusters at NYU as well as the Blue Gene at CCNI. Support from Philip Morris USA Inc. and Philip Morris International to T.S. is gratefully acknowledged.

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Authors and Affiliations

  1. Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, PO Box 12233, Research Triangle Park, North Carolina 27709-2233, USA,

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

  2. Department of Chemistry, New York University, and NYU-ECNU Center for Computational Chemistry at NYU Shanghai, 10th Floor Silver Center, 100 Washington Square East, New York, New York 10003, USA,

    Taejin Kim & Tamar Schlick

  3. Courant Institute of Mathematical Sciences, New York University, 251 Mercer Street, New York, New York 10012, USA,

    Taejin Kim & Tamar Schlick

Authors
  1. Bret D. Freudenthal
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Contributions

B.F., W.B., and S.W. designed the project. B.F. performed crystallography. D.S. did the kinetic analyses. T.K. and T.S. did the molecular dynamics simulations. L.P. did the quantum mechanical analysis. B.F., W.B., and S.W. prepared the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Samuel H. Wilson.

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Extended data figures and tables

Extended Data Figure 1 Mutagenic 8-oxo-dGTP insertion opposite adenine.

a, Overlay of the ternary complex for 8-oxo-dGTP(syn):Ad generated with Ca2+ or a dideoxy-terminated primer (Protein Data Bank accession number 3MBY) is shown in yellow and green, respectively (root mean squared deviation of 0.17 Å). b, The pol β active site is shown with a 2Fo − Fc map contoured at 1.5σ after a 20 s soak. Key active site residues are indicated and Mg2+ ions are shown as red spheres. The reactant 8-oxo-dGTP and product 8-oxo-dGMP are shown in green and yellow respectively. c, A focused view of b with the density removed. Coordinating waters (blue) and their distances (in ångströms) to active site metals are shown. d, The active site following a 40 s soak is shown with an omit map (3σ) for the Mgp and coordinating waters. e, The coordination distances (in ångströms) for the Nac, Mgp, and Mgn metals are indicated for the closed product complex after a 40 s soak. f, The 8-oxo-dGMP(anti):Ad contact between N3 and N6 of 8-oxo-dGMP and Ad respectively is shown for the open product complex after a 90 s soak.

Extended Data Figure 2 Pre-catalytic ground state with 8-oxo-dGTP and templating cytosine after a 5 s soak in MnCl2.

a, The pre-catalytic pol β active site is shown with an omit map (3σ). The ground state metal (Mng) has been removed for clarity. b, The view is a 90° rotation relative to a. An overlay of the 8-oxo-dGTP(anti) with Ca2+ and Mn2+ is shown in yellow and purple respectively. The anomalous density map contoured at 5σ for the Mn2+ ions is shown in purple. The Mng coordinating water molecules are shown in blue and the distances (in ångströms) are indicated.

Extended Data Figure 3 Reaction with 8-oxo-dGTP opposite templating cytosine.

a, Focused view of the active site following a 40 s soak is shown with key residues indicated; density has been removed for clarity (see Fig. 2e for density). b, An omit map (3σ) for Cag is shown. Coordinating waters are shown in blue (distances in ångströms). c, An omit map (3σ) for Mgp is shown.

Extended Data Figure 4 Quantum mechanical computational models for 8-oxo-dGTP(anti) and dGTP(anti).

The models used for the quantum mechanical computational studies with the calcium ions, oxygen, phosphates, carbon, nitrogen, and protons shown in green, red, orange, grey, blue, and white, respectively. The key atoms and Asp 190, Asp 192, and Asp 256 mimics are indicated. a, The 8-oxo-dGTP(anti) with three calcium ions and eight water molecules. b, The 8-oxo-dGTP(anti) with two calcium ions and three water molecules. c, The dGTP(anti) with two calcium ions and three water molecules.

Extended Data Figure 5 Molecular dynamics simulation analysis of 8-oxo-dGTP(anti) and dGTP(anti) opposite Cy.

a, The 8-oxo-dGTP(anti) opposite Cy at 80 ns superimposed upon the initial structure. A multicolour code based on atom type is used for the final molecular dynamics structure, whereas the reference initial structure is shown in light grey. The catalytic (Mgc), nucleotide (Mgn), and ground (Mgg) magnesium metal ions are shown in green, and average distances over the course of the simulation are indicated for Pα-O3′ and Mgc-O3′. b, Distance distributions between hydrogen atoms in the water shell and O8 in the 8-oxo-dGTP(anti):Cy simulation. A snapshot of the 8-oxo-dGTP, Mgg, and water shell (W(g1–g5)) is plotted at top. Black and red dotted lines indicate Mgg coordination and a hydrogen-bonding interaction between a water molecule and O8, respectively. Four of the five water molecules in the water shell (W(g1–g4)) contribute to hydrogen-bonding interactions with O8. Blue and orange lines indicate distances between hydrogen atoms in each water molecule and O8. The red line in the bottom plot indicates the minimum distance between hydrogen atoms in the water shell and O8. c, The dGTP(anti) opposite Cy at 80 ns superimposed upon the initial structure (grey). Distances and ion labelling are as for a. d, Root mean squared deviation of the evolving molecular dynamics structure for the entire polymerase/DNA complex (top) and for the active site only (bottom), with respect to the crystal structure.

Extended Data Table 1 Data collection and refinement statistics of pol β 8-oxo-dGTP insertion opposite adenine
Full size table
Extended Data Table 2 Key DNA and structural parameters near the pol β active site*
Full size table
Extended Data Table 3 Data collection and refinement statistics of pol β insertion opposite cytosine with 8-oxo-dGTP and dGTP
Full size table
Extended Data Table 4 Quantum mechanical point charges for each atom of 8-oxo-dGTP(anti) and dGTP(anti) with either two or three calcium ions
Full size table
Extended Data Table 5 Average distances in the active sites in 8-oxo-dGTP(anti) and dGTP(anti)
Full size table

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Freudenthal, B., Beard, W., Perera, L. et al. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide. Nature 517, 635–639 (2015). https://doi.org/10.1038/nature13886

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