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

Journal name:
Nature Chemical Biology
Year published:
Published online


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


  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.


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


  1. Finkel, T. & Holbrook, N.J. Oxidants, oxidative stress and the biology of ageing. Nature 408, 239247 (2000).
  2. Cadet, J., Douki, T. & Ravanat, J.L. Oxidatively generated base damage to cellular DNA. Free Radic. Biol. Med. 49, 921 (2010).
  3. Steenken, S. & Jovanovic, S.V. How easily oxidizible is DNA? One-electron reduction potentials of adenosine and guanosine radicals in aqueous solution. J. Am. Chem. Soc. 119, 617618 (1997).
  4. Cadet, J., Douki, T. & Ravanat, J.L. Oxidatively generated damage to the guanine moiety of DNA: mechanistic aspects and formation in cells. Acc. Chem. Res. 41, 10751083 (2008).
  5. Jena, N.R. & Mishra, P.C. Formation of ring-opened and rearranged products of guanine: mechanisms and biological significance. Free Radic. Biol. Med. 53, 8194 (2012).
  6. Hsu, G.W., Ober, M., Carell, T. & Beese, L.S. Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase. Nature 431, 217221 (2004).
  7. Duarte, V., Muller, J.G. & Burrows, C.J. Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine. Nucleic Acids Res. 27, 496502 (1999).
  8. Brieba, L.G. et al. Structural basis for the dual coding potential of 8-oxoguanosine by a high-fidelity DNA polymerase. EMBO J. 23, 34523461 (2004).
  9. Greenberg, M.M. The formamidopyrimidines: purine lesions formed in competition with 8-oxopurines from oxidative stress. Acc. Chem. Res. 45, 588597 (2012).
  10. Brieba, L.G. & Ellenberger, T. Hold tight (but not too tight) to get it right: accurate bypass of an 8-oxoguanine lesion by DNA polymerase β. Structure 11, 12 (2003).
  11. Krahn, J.M., Beard, W.A., Miller, H., Grollman, A.P. & Wilson, S.H. Structure of DNA polymerase β with the mutagenic DNA lesion 8-oxodeoxyguanine reveals structural insights into its coding potential. Structure 11, 121127 (2003).
  12. Eoff, R.L., Irimia, A., Angel, K.C., Egli, M. & Guengerich, F.P. Hydrogen bonding of 7,8-dihydro-8-oxodeoxyguanosine with a charged residue in the little finger domain determines miscoding events in Sulfolobus solfataricus DNA polymerase Dpo4. J. Biol. Chem. 282, 1983119843 (2007).
  13. Vasquez-Del Carpio, R. et al. Structure of human DNA polymerase κ inserting dATP opposite an 8-OxoG DNA lesion. PLoS ONE 4, e5766 (2009).
  14. Batra, V.K. et al. Mutagenic conformation of 8-oxo-7,8-dihydro-2′-dGTP in the confines of a DNA polymerase active site. Nat. Struct. Mol. Biol. 17, 889890 (2010).
  15. Dizdaroglu, M., Kirkali, G. & Jaruga, P. Formamidopyrimidines in DNA: mechanisms of formation, repair, and biological effects. Free Radic. Biol. Med. 45, 16101621 (2008).
  16. Büsch, F. et al. Dissecting the differences between the α and β anomers of the oxidative DNA lesion FaPydG. Chemistry 14, 21252132 (2008).
  17. Graziewicz, M.A. et al. Fapyadenine is a moderately efficient chain terminator for prokaryotic DNA polymerases. Free Radic. Biol. Med. 28, 7583 (2000).
  18. Delaney, M.O., Wiederholt, C.J. & Greenberg, M.M. Fapy.dA induces nucleotide misincorporation translesionally by a DNA polymerase. Angew. Chem. Int. Ed. Engl. 41, 771773 (2002).
  19. Wiederholt, C.J. & Greenberg, M.M. Fapy.dG instructs Klenow exo to misincorporate deoxyadenosine. J. Am. Chem. Soc. 124, 72787279 (2002).
  20. Ober, M., Müller, H., Pieck, C., Gierlich, J. & Carell, T. Base pairing and replicative processing of the formamidopyrimidine-dG DNA lesion. J. Am. Chem. Soc. 127, 1814318149 (2005).
  21. Tudek, B. et al. Mutagenic specificity of imidazole ring-opened 7-methylpurines in M13mp18 phage DNA. Acta Biochim. Pol. 46, 785799 (1999).
  22. Kalam, M.A. et al. Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells. Nucleic Acids Res. 34, 23052315 (2006).
  23. Patro, J.N. et al. Studies on the replication of the ring opened formamidopyrimidine, Fapy.dG in Escherichia coli. Biochemistry 46, 1020210212 (2007).
  24. Asagoshi, K., Terato, H., Ohyama, Y. & Ide, H. Effects of a guanine-derived formamidopyrimidine lesion on DNA replication: translesion DNA synthesis, nucleotide insertion, and extension kinetics. J. Biol. Chem. 277, 1458914597 (2002).
  25. Christov, P.P., Angel, K.C., Guengerich, F.P. & Rizzo, C.J. Replication past the N5-methyl-formamidopyrimidine lesion of deoxyguanosine by DNA polymerases and an improved procedure for sequence analysis of in vitro bypass products by mass spectrometry. Chem. Res. Toxicol. 22, 10861095 (2009).
  26. Ober, M., Linne, U., Gierlich, J. & Carell, T. The two main DNA lesions 8-oxo-7,8-dihydroguanine and 2,6-diamino-5-formamido-4-hydroxypyrimidine exhibit strongly different pairing properties. Angew. Chem. Int. Ed. Engl. 42, 49474951 (2003).
  27. Ober, M., Marsch, M., Harms, K. & Carell, T. A carbocyclic analogue of a protected β-d-2-deoxyribosylamine. Acta Crystallogr. Sect. E Struct. Rep. Online 60, o1191o1192 (2004).
  28. Patro, J.N., Haraguchi, K., Delaney, M.O. & Greenberg, M.M. Probing the configurations of formamidopyrimidine lesions Fapy·dA and Fapy·dG in DNA using endonuclease IV. Biochemistry 43, 1339713403 (2004).
  29. Kiefer, J.R. et al. Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 Å resolution. Structure 5, 95108 (1997).
  30. Raoul, S., Bardet, M. & Cadet, J. Gamma irradiation of 2′-deoxyadenosine in oxygen-free aqueous solutions: identification and conformational features of formamidopyrimidine nucleoside derivatives. Chem. Res. Toxicol. 8, 924933 (1995).
  31. Lukin, M. et al. Novel post-synthetic generation, isomeric resolution, and characterization of Fapy-dG within oligodeoxynucleotides: differential anomeric impacts on DNA duplex properties. Nucleic Acids Res. 39, 57765789 (2011).
  32. Münzel, M. et al. Improved synthesis and mutagenicity of oligonucleotides containing 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. Chemistry 17, 1378213788 (2011).
  33. Stathis, D., Lischke, U., Koch, S.C., Deiml, C.A. & Carell, T. Discovery and mutagenicity of a guanidinoformimine lesion as a new intermediate of the oxidative deoxyguanosine degradation pathway. J. Am. Chem. Soc. 134, 49254930 (2012).
  34. Johnson, S.J., Taylor, J.S. & Beese, L.S. Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations. Proc. Natl. Acad. Sci. USA 100, 38953900 (2003).
  35. Topal, M.D. & Fresco, J.R. Complementary base pairing and the origin of substitution mutations. Nature 263, 285289 (1976).
  36. Wang, W., Hellinga, H.W. & Beese, L.S. Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis. Proc. Natl. Acad. Sci. USA 108, 1764417648 (2011).
  37. Johnson, S.J. & Beese, L.S. Structures of mismatch replication errors observed in a DNA polymerase. Cell 116, 803816 (2004).
  38. Creighton, S., Bloom, L.B. & Goodman, M.F. Gel fidelity assay measuring nucleotide misinsertion, exonucleolytic proofreading, and lesion bypass efficiencies. Methods Enzymol. 262, 232256 (1995).
  39. Münzel, M., Lercher, L., Müller, M. & Carell, T. Chemical discrimination between dC and 5MedC via their hydroxylamine adducts. Nucleic Acids Res. 38, e192 (2010).
  40. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125132 (2010).
  41. CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760763 (1994).
  42. Evans, P. Joint CCP4 and ESF-EACMB. Newsletter Prot. Crystallogr. 33, 2224 (1997).
  43. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658674 (2007).
  44. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486501 (2010).
  45. Adams, P.D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D Biol. Crystallogr. 58, 19481954 (2002).
  46. Winn, M.D., Murshudov, G.N. & Papiz, M.Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300321 (2003).
  47. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355367 (2011).
  48. Olson, W.K. et al. A standard reference frame for the description of nucleic acid base-pair geometry. J. Mol. Biol. 313, 229237 (2001).
  49. Lavery, R., Moakher, M., Maddocks, J.H., Petkeviciute, D. & Zakrzewska, K. Conformational analysis of nucleic acids revisited: Curves+. Nucleic Acids Res. 37, 59175929 (2009).

Download references

Author information

  1. These authors contributed equally to the work.

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


  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


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.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (3 MB)

    Supplementary Results, Supplementary Notes 1 and 2

Additional data