Unexpected non-Hoogsteen–based mutagenicity mechanism of FaPy-DNA lesions

Journal name:
Nature Chemical Biology
Volume:
9,
Pages:
455–461
Year published:
DOI:
doi:10.1038/nchembio.1254
Received
Accepted
Published online

Abstract

8-Oxopurines (8-oxodG and 8-oxodA) and formamidopyrimidines (FaPydG and FaPydA) are major oxidative DNA lesions involved in cancer development and aging. Their mutagenicity is believed to result from a conformational shift of the N9-C1′ glycosidic bonds from anti to syn, which allows the lesions to form noncanonical Hoogsteen-type base pairs with incoming triphosphates during DNA replication. Here we present biochemical data and what are to our knowledge the first crystal structures of carbocyclic FaPydA and FaPydG containing DNA in complex with a high-fidelity polymerase. Crystallographic snapshots show that the cFaPy lesions keep the anti geometry of the glycosidic bond during error-free and error-prone replication. The observed dG·dC→dT·dA transversion mutations are the result of base shifting and tautomerization.

At a glance

Figures

  1. Representation of the two main oxidation products of 2′-deoxyguanosine (8-oxodG, FaPydG) and FaPydA.
    Figure 1: Representation of the two main oxidation products of 2′-deoxyguanosine (8-oxodG, FaPydG) and FaPydA.

    (a) For 8-oxodG, the crystallographically observed 8-oxodG·dC and 8-oxodG·dA base pairs are shown. (b,c) Additionally, the putative base pairs formed by FaPydG and FaPydA are depicted. In the natural FaPy lesions, X = O, whereas in the stabilized carbocyclic analogs of FaPydG (b) and FaPydA (c), X = CH2. (d) Depiction of the through-space interactions between C8H and the sugar protons in the anti conformer (red) and the syn conformer (blue). The reference NOESY signal between interactions was quantified, giving the same syn/anti distribution for both dG and cdG.

  2. Nucleotide insertion and bypass of oxidative lesions.
    Figure 2: Nucleotide insertion and bypass of oxidative lesions.

    (a) Single-nucleotide insertion reactions opposite cFaPydA and cFaPydG in comparison to those opposite of 8-oxodA and 8-oxodG. The primer was hybridized to a lesion-free control DNA or lesion (X)-containing template strand. F, fluorescein; M, marker. (b) Schematic presentation of the analysis of the misinsertion frequency and mutagenic signature. The biotinylated primer strands from the primer extension reaction with Bst Pol I and all four dNTPs were isolated and subjected to pyrosequencing analysis. (c) Relative frequency of correct and incorrect lesion bypass from the pyrosequencing experiment. The given percentage values are averages of three independent experiments.

  3. Error-free reading of Bst Pol I through cFaPydA-containing template DNA.
    Figure 3: Error-free reading of Bst Pol I through cFaPydA-containing template DNA.

    (a,b) Schematic view (a) and superposition of cFaPydA-DNA (red) in the pre-IS of the polymerase (gold; PDB code 4B9L) with the polymerase in complex with undamaged DNA (gray; PDB code 1L3S34) (b). (cf) Structure after one (c,d; PDB code 4B9M) and three (e,f; PDB code 4B9N) rounds of correct template extension with the cFaPydA·dT base pair (red) at the post-IS (gold) n−1 and n−3, respectively. Overlaid in gray is the polymerase in complex with undamaged DNA (PDB codes 1L3T and 1L5U34).

  4. Error-free reading of Bst Pol I through cFaPydG-containing template DNA.
    Figure 4: Error-free reading of Bst Pol I through cFaPydG-containing template DNA.

    (a,b) Schematic view (a) and superposition of cFaPydG-DNA outside of the pre-IS (orange, yellow; PDB code 4B9S) with the polymerase in complex with 8-oxodG in the pre-IS (gray; PDB code 1U45 (ref. 6)) (b). (c,d) Schematic view (c) and structure after correct dCTP insertion with the cFaPydG·dC base pair (orange, yellow; PDB code 4B9T) in comparison with a dG·dC base pair (gray; PDB code 1L5U34) at the n−1 post-IS position (d).

  5. Erroneous bypass and extension of cFaPydG.
    Figure 5: Erroneous bypass and extension of cFaPydG.

    (a,b) Schematic view (a) and structure of Bst Pol I (yellow) in complex with cFaPydG (orange) after incorporation of a dATP in the crystal (PDB code 4B9U), superimposed with the polymerase in complex with the cognate dG·dC base pair (gray, PDB code 1L5U34) (b). (c,d) Schematic view (c) and structure after elongating the cFaPydG·dA base pair (orange) at n−2 (PDB code 4B9V) in comparison with structure involving undamaged DNA (gray; PDB code 1L5U) (d). (e,f) Side view of the base pairs. The cFaPydG·dA (orange, yellow) base pair is markedly buckled, the template strand is displaced, and the DNA is widened in comparison with the cognate base pair (gray) (e). After further strand elongation, the displacement is slightly relieved, but the cFaPydG·dA base pair remains out of planarity (f). Distances in e and f are given in Å.

Compounds

22 compounds View all compounds
  1. (1'S,2'R,4'R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((5-formamido-6-(tritylamino)pyrimidin-4-yl)amino)cyclopentyl-(2-cyanoethyl)diisopropylphosphoramidite
    Compound 1 (1'S,2'R,4'R)-2-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((5-formamido-6-(tritylamino)pyrimidin-4-yl)amino)cyclopentyl-(2-cyanoethyl)diisopropylphosphoramidite
  2. (1'S,2'R,4'R)-4-((2-Acetamido-5-formamido-6-oxo-1,6-dihydropyrimidin-4-yl)-amino)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)cyclopentyl(2-cyanoethyl)diisopropylphosphoramidite
    Compound 2 (1'S,2'R,4'R)-4-((2-Acetamido-5-formamido-6-oxo-1,6-dihydropyrimidin-4-yl)-amino)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)cyclopentyl(2-cyanoethyl)diisopropylphosphoramidite
  3. (1S,2R,4R)-4-Ammonium-2-(hydroxymethyl)cyclopentanol-trifluoroacetate
    Compound 3 (1S,2R,4R)-4-Ammonium-2-(hydroxymethyl)cyclopentanol-trifluoroacetate
  4. tert-Butyl ((1R,3S,4R)-3-hydroxy-4-(hydroxymethyl)cyclopentyl)carbamate
    Compound 4 tert-Butyl ((1R,3S,4R)-3-hydroxy-4-(hydroxymethyl)cyclopentyl)carbamate
  5. 4-Amino-6-chloro-5-nitropyrimidine
    Compound 5 4-Amino-6-chloro-5-nitropyrimidine
  6. Pyrimidine-4,6-diol
    Compound 6 Pyrimidine-4,6-diol
  7. 4,6-Dihydroxy-5-nitropyrimidine
    Compound 7 4,6-Dihydroxy-5-nitropyrimidine
  8. 4,6-Dichloro-5-nitropyrimidine
    Compound 8 4,6-Dichloro-5-nitropyrimidine
  9. N-4-{[(1'R,3'S,4'R)-3'-Hydroxy-4'-(hydroxymethyl)cyclopentyl]amino}-5-nitro-6-aminopyrimidine
    Compound 9 N-4-{[(1'R,3'S,4'R)-3'-Hydroxy-4'-(hydroxymethyl)cyclopentyl]amino}-5-nitro-6-aminopyrimidine
  10. N-4-{[(1'R,3'S,4'R)-3'-[(tert-Butyldimethylsilyl)oxy]-4'-{[(tert-butyldimethylsilyl)oxy]methyl}cyclopentyl]amino}-5-nitro-6-aminopyrimidine
    Compound 9a N-4-{[(1'R,3'S,4'R)-3'-[(tert-Butyldimethylsilyl)oxy]-4'-{[(tert-butyldimethylsilyl)oxy]methyl}cyclopentyl]amino}-5-nitro-6-aminopyrimidine
  11. N-4-{[(1'R,3'S,4'R)-3'-[(tert-Butyldimethylsilyl)oxy]-4'-{[(tert-butyldimethylsilyl)oxy]methyl}cyclopentyl]amino}-5-formylamino-6-aminopyrimidine
    Compound 10 N-4-{[(1'R,3'S,4'R)-3'-[(tert-Butyldimethylsilyl)oxy]-4'-{[(tert-butyldimethylsilyl)oxy]methyl}cyclopentyl]amino}-5-formylamino-6-aminopyrimidine
  12. N-4-{[(1'R,3'S,4'R)-3'-[(tert-Butyldimethylsilyl)oxy]-4'-{[(tert-butyldimethylsilyl)oxy]methyl}cyclopentyl]amino}-5-formylamino-6-tritylaminopyrimidine
    Compound 10a N-4-{[(1'R,3'S,4'R)-3'-[(tert-Butyldimethylsilyl)oxy]-4'-{[(tert-butyldimethylsilyl)oxy]methyl}cyclopentyl]amino}-5-formylamino-6-tritylaminopyrimidine
  13. N-4-{[(1'R,3'S,4'R)-3'-Hydroxy-4'-(hydroxymethyl)cyclopentyl]amino}-5-formylamino-6-tritylaminopyrimidine
    Compound 11 N-4-{[(1'R,3'S,4'R)-3'-Hydroxy-4'-(hydroxymethyl)cyclopentyl]amino}-5-formylamino-6-tritylaminopyrimidine
  14. N-4-{[(1'R,3'S,4'R)-4'-{[(Dimethoxytrityl)oxy]methyl}-3'-hydroxycyclopentyl]amino}-5-(formylamino)-6-tritylaminopyrimidine
    Compound 11a N-4-{[(1'R,3'S,4'R)-4'-{[(Dimethoxytrityl)oxy]methyl}-3'-hydroxycyclopentyl]amino}-5-(formylamino)-6-tritylaminopyrimidine
  15. N-(5-(Formylamino)-4-{[(1'R,3'S,4'R)-3'-hydroxy-4'-(hydroxymethyl)cyclopentyl]amino}-6-aminopyrimidine
    Compound 12 N-(5-(Formylamino)-4-{[(1'R,3'S,4'R)-3'-hydroxy-4'-(hydroxymethyl)cyclopentyl]amino}-6-aminopyrimidine
  16. N-(4-(((1'R,3'S,4'R)-3-((tert-Butyldimethylsilyl)oxy)-4-(((tert-butyldimethylsilyl)oxy)methyl)cyclopentyl)amino)-5-nitro-6-oxo-1,6-dihydropyrimidin-2-yl)acetamide
    Compound 13 N-(4-(((1'R,3'S,4'R)-3-((tert-Butyldimethylsilyl)oxy)-4-(((tert-butyldimethylsilyl)oxy)methyl)cyclopentyl)amino)-5-nitro-6-oxo-1,6-dihydropyrimidin-2-yl)acetamide
  17. N-[9-[(1'R,3'S,4'R)-3'-[(tert-Butyldimethylsilyl)oxy]-4'-[[(tert-butyldimethylsilyl)oxy]methyl]cyclopentyl]-6-oxo-6,9-dihydro-1H-purin-2-yl]acetamide
    Compound 14 N-[9-[(1'R,3'S,4'R)-3'-[(tert-Butyldimethylsilyl)oxy]-4'-[[(tert-butyldimethylsilyl)oxy]methyl]cyclopentyl]-6-oxo-6,9-dihydro-1H-purin-2-yl]acetamide
  18. 2-Amino-9-[(1'R,3'S,4'R)-3'-[(tert-butyldimethylsilyl)oxy]-4'-[[(tert-butyldimethylsilyl)oxy]methyl]cyclopentyl]-1,9-dihydropurine-6-one
    Compound 15 2-Amino-9-[(1'R,3'S,4'R)-3'-[(tert-butyldimethylsilyl)oxy]-4'-[[(tert-butyldimethylsilyl)oxy]methyl]cyclopentyl]-1,9-dihydropurine-6-one
  19. 2-Amino-9-((1'R,3'S,4'R)-3-hydroxy-4-(hydroxymethyl)cyclopentyl)-1,9-dihydro-6H-purin-6-one
    Compound 16 2-Amino-9-((1'R,3'S,4'R)-3-hydroxy-4-(hydroxymethyl)cyclopentyl)-1,9-dihydro-6H-purin-6-one
  20. 2'-Desoxyguanosine
    Compound 17 2'-Desoxyguanosine
  21. N-(2-Amino-4-(((2'R,5'R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)amino)-6-oxo-1,6-dihydropyrimidin-5-yl)formamide)
    Compound 18 N-(2-Amino-4-(((2'R,5'R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)amino)-6-oxo-1,6-dihydropyrimidin-5-yl)formamide)
  22. N-(2-Amino-4-(((1'R,4'R)-3-hydroxy-4-(hydroxymethyl)cyclopentyl)amino)-6-oxo-1,6-dihydropyrimidin-5-yl)formamide
    Compound 19 N-(2-Amino-4-(((1'R,4'R)-3-hydroxy-4-(hydroxymethyl)cyclopentyl)amino)-6-oxo-1,6-dihydropyrimidin-5-yl)formamide

Accession codes

Primary accessions

Referenced accessions

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Author information

  1. These authors contributed equally to the work.

    • Tim H Gehrke,
    • Ulrike Lischke,
    • Karola L Gasteiger &
    • Sabine Schneider

Affiliations

  1. Center for Integrated Protein Science at the Department of Chemistry, Ludwig Maximilians University, Munich, Germany.

    • Tim H Gehrke,
    • Ulrike Lischke,
    • Karola L Gasteiger,
    • Sabine Schneider,
    • Simone Arnold,
    • Heiko C Müller,
    • David S Stephenson,
    • Hendrik Zipse &
    • Thomas Carell
  2. Present address: Department of Chemistry, Technical University, Munich, Germany.

    • Sabine Schneider

Contributions

T.C. conceived and directed the study. He wrote the manuscript and designed experiments. T.H.G. and U.L. designed experiments. T.H.G. performed the synthesis of the lesions and of the DNA strands. U.L. and T.H.G. performed the biochemical experiments. U.L. purified the protein. K.L.G. performed the synthesis of cdG. S.A. developed the synthesis of cFaPydA. H.C.M. developed the synthesis of cdG. S.S. conducted crystallographic data collection and solved the crystal structures. H.Z. performed the theoretical studies. D.S.S. performed the NMR studies.

Competing financial interests

The authors declare no competing financial interests.

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