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5-Formylcytosine does not change the global structure of DNA

Nature Structural & Molecular Biology volume 24, pages 544–552 (2017)Cite this article

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Abstract

The mechanism by which the recently identified DNA modification 5-formylcytosine (fC) is recognized by epigenetic writer and reader proteins is not known. Recently, an unusual DNA structure, F-DNA, has been proposed as the basis for enzyme recognition of clusters of fC. We used NMR and X-ray crystallography to compare several modified DNA duplexes with unmodified analogs and found that in the crystal state the duplexes all belong to the A family, whereas in solution they are all members of the B family. We found that, contrary to previous findings, fC does not significantly affect the structure of DNA, although there are modest local differences at the modification sites. Hence, global conformation changes are unlikely to account for the recognition of this modified base, and our structural data favor a mechanism that operates at base-pair resolution for the recognition of fC by epigenome-modifying enzymes.

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Figure 1: Similarities between the crystal structures of a DNA duplex containing fC and its unmodified counterpart.
Figure 2: Crystal-packing interactions exert a greater effect than fC on DNA conformation.
Figure 3: NMR analysis of the fC-containing DNA duplex, which features in the crystal structures PDB 5MVU and PDB 4QKK.
Figure 4: Site-dependent changes to the CD spectra of oligonucleotides containing fC and caC.

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Acknowledgements

This work was supported by a BBSRC sLoLa grant BB/J001694/2 (Extending the boundaries of nucleic acid chemistry), by an Oxford University/EPSRC Doctoral Training Partnership award (J.S.H.) and in part by a Carmen L. Buck endowment (to A.N.L.). NMR data were recorded at the Center for Environmental and Systems Biochemistry, which is supported by the University of Kentucky and NCI Cancer Center Support Grant (P30 CA177558).

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Author notes

  1. Jack S Hardwick and Denis Ptchelkine: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford, UK

    Jack S Hardwick, Afaf H El-Sagheer & Tom Brown

  2. Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK

    Denis Ptchelkine & Simon E V Phillips

  3. Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Denis Ptchelkine

  4. Department of Science and Mathematics, Chemistry Branch, Faculty of Petroleum and Mining Engineering, Suez University, Suez, Egypt

    Afaf H El-Sagheer

  5. ATDBio, School of Chemistry, University of Southampton, Southampton, UK

    Ian Tear & Daniel Singleton

  6. Department of Biochemistry, University of Oxford, Oxford, UK

    Simon E V Phillips

  7. Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, Kentucky, USA.,

    Andrew N Lane

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Contributions

T.B. and A.N.L. designed the project and wrote the manuscript, which includes contributions from all authors. J.S.H. carried out oligonucleotide synthesis and performed the crystallizations and biophysical experiments. A.H.E.-S. carried out oligonucleotide synthesis and biophysical experiments. A.N.L. recorded and interpreted the NMR spectra. I.T. and D.S. carried out large-scale oligonucleotide synthesis, purification and analysis. D.P. and J.S.H. acquired and analyzed X-ray crystallographic data and solved the structures. T.B., A.N.L. and S.E.V.P. supervised the project. All authors interpreted the data and read and approved the manuscript.

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Correspondence to Andrew N Lane or Tom Brown.

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T.B. is a consultant for ATDBio.

Integrated supplementary information

Supplementary Figure 1 A comparison of ideal A-DNA with crystal structures of a DNA duplex containing either 5-formylcytosine or cytosine.

(a) Overlay of the entire fC-containing crystal structure 4QKK (green) with ideal A-DNA of the same sequence (cyan). (b) Overlay of the central 6 bp of 4QKK, containing all fC residues, with ideal A-DNA. Significant deviation of 4QKK from ideal A-DNA occurs only at the duplex ends, which do not contain fC and results from differences in crystal packing. (c) Overlay of the entire fC-containing crystal structure 5MVU (magenta, an identical duplex to 4QKK, but crystallised at 10-fold lower salt concentration), its unmodified counterpart 5MVK (blue) and the ideal A-DNA model (cyan). (d) Overlay of the central 6 bp regions of the structures shown in (c).

Supplementary Figure 2 Structural parameters of DNA crystal structures containing either 5-formylcytosine or cytosine.

The comparison features two crystal structures of an identical fC-containing duplex, 4QKK (green) and 5MVU (magenta), which crystallized in the space groups P43212 and P3221, respectively, alongside their unmodified analogue 5MVK (blue), which also crystallized in the P3221 space group. The shaded region shows the maximum and minimum values exhibited at each step/base pair by the four unmodified crystal structures determined in this study, thereby illustrating the magnitude of variation observed for duplexes of similar sequence that crystallized in the same space group, under similar conditions. Also shown for comparison is the well-characterized B-DNA structure 1BNA (blue dots; Drew, H.R. et al. Proceedings of the National Academy of Sciences 78, 2179-2183, 1981). (a) Comparison of major and minor groove dimensions, which shows that all duplexes adopt similar groove geometry, especially 5MVU and 5MVK. Significant differences in groove geometry between the structures are related to the space group, rather than to the presence of fC. (b) Twist, slide, X-displacement, shift and roll, propeller twist, inclination and tilt. Twist angles of all three analogous structures are in close agreement, except the terminal steps of 4QKK, which exhibit values of just 10.4° and are responsible for the apparent underwinding of the duplex. Slide values are consistent with A-form geometry, again with the exception of the terminal steps of 4QKK. X-displacement for the three analogous structures is consistent with A-DNA. Shift and roll values between the three structures are also very similar. The periodic roll angles observed in 4QKK and 5MVU were also exhibited by the control 5MVK and 5MVT (not explicitly shown), which also retains pyrimidine-purine alternation; periodic roll angles have previously been observed in crystal structures of A form duplexes possessing alternating pyrimidine-purine repeats (Šponer, J. & Kypr, J. Journal of Molecular Biology 221, 761-764, 1991). With the exception of the duplex ends, propeller twist and inclination angles between 4QKK, 5MVK and 5MVU are similar, though there are modest differences between 5MVK and the fC-containing structures in the central region where fC is located. Tilt angles are also similar, with 5MVK possessing the most significant outlier. Overall, the data show that structural differences observed in the fC-containing structures are small, and comparable with A-DNA structures of similar sequence, crystallized under similar conditions. The largest differences observed are associated with the duplex ends of the fC-containing structure 4QKK, whose crystallization conditions contained a salt concentration an order of magnitude higher than the structures obtained in this study. Note that the minor groove depth values have been corrected by adding 0.3 Å to each data point to avoid negative depth values.

Supplementary Figure 3 Overlay of all six crystal structures determined in this study.

All structures crystallized in the P3221 space group exhibited a high degree of similarity (Supplementary Table 7), including the formylated structure 5MVU (magenta) and its unmodified analogue 5MVK (blue). Scrambling of the internal repeating CpG motif of the duplex d(CTACGCGCGTAG)2 (5MVP, 5MVT & 5MVQ, grey) did not have a significant effect on helical conformation.

Supplementary Figure 4 Models of junctions between ideal A- or B-DNA and crystal structures containing either 5-formylcytosine or cytosine.

Shown are 60-base-pair duplexes modelled with crystallographically-determined structures 4QKK (green), 5MVU (magenta) or 5MVK (light blue) inserted centrally, demonstrating the effect of junctions with flanking ideal A-DNA (a) or ideal B-DNA (b,c). To illustrate the influence of the outermost base pairs, which are most severely distorted by crystal-packing interactions and furthest from the formylated regions, on the curvature of ideal DNA, three models were constructed for each type of junction. (i) Includes the full 12-mer duplex d(CTAXGXGXGTAG)2 of the crystal structure, where X = fC for 4QKK/5MVU and C for 5MVK, flanked by ideal 24-mer A- or B-DNA duplexes; (ii) d(TAXGXGXGTA)2, flanked by ideal 25-mer A- or B-DNA; (iii) d(AXGXGXGT)2. As (iii) incorporates both the entire formylated region of structures 4QKK and 5MVU and the experimentally-derived stacking geometries with the surrounding unmodified base pairs, while also omitting the significant distortions associated with crystal packing at the duplex ends, it represents the most realistic model of each junction. The results show that the fC-containing structures 4QKK and 5MVU adopt the A-form, with any significant deviations occurring at the duplex ends. Furthermore, the fC-containing structures’ geometry is even closer to ideal A-form than that of the unmodified control, as also indicated by the RMSD comparison (Table 1 and Supplementary Table 7).

Supplementary Figure 5 DQF–COSY spectra of the unmodified duplex from crystal structure 5MVK and an analog containing a central diformylated CpG step.

(a) d(CTACGCGCGTAG)2 (b) d(CTACGfCGCGTAG)2. The spectra were recorded under identical conditions as that described for Figure 3 in the main text.

Supplementary Figure 6 NOESY spectra of the 5-formylcytosine-containing duplex from crystal structures 4QKK and 5MVU showing B-like helical conformation.

(a) NOESY spectrum in 93% H2O at 288 K with a mixing time of 250 ms, showing imino, amino and Ade H2 assignments. (b) NOESY spectrum in D2O at 293 K with a mixing time of 50 ms showing base-H2′/H2″ intra and internucleotide NOEs, and the much weaker base-H1′ and H3′ NOEs. These are typical of the B-family of conformations for all non-terminal nucleotides. Spectra were recorded at 14.1 T as described in the methods. (c) Base to H8/H6 region of a NOESY spectrum recorded with a mixing time of 50 ms, and acquisition times of 1 s in t2 and 0.05 s in t1 with spectral widths of 6 kHz in both dimensions. The data were apodized using an unshifted Gaussian function, with zero filling to 16 k by 2 k complex data points prior to Fourier transformation. The black lines connect the complete sequential base proton interactions characteristic of a right-handed duplex, including the modified C residues. The locations of the modified cytosine H6-formyl protons are also indicated.

Supplementary Figure 7 A comparison of chemical-shift and NOE differences for duplexes featuring varying degrees of formylation.

The duplexes compared are ODN1 [d(CTACGCGCGTAG)2], ODN6 [d(CTAfCGfCGfCGTAG)2] and ODN7 [d(CTACGfCGCGTAG)2]. (a) Chemical shift differences of base protons (H8 and H6) in the major groove and chemical shift differences of sugar protons in the minor groove (b) between ODN6 or ODN7 and their unmodified analogue ODN1. E.g. DH1′ ODN6 and DH1′ ODN7 are the chemical shift differences between the H1′ resonances of ODN6 and ODN1, and ODN7 and ODN1, respectively, and similarly for the H2′, H2″ and H3′ resonances. (c,d) Pairwise sum of squared NOE differences at the nucleotide level. (c) Intranucleotide NOEs H8/6 to H1′, H2′, H2″ and H3′. (d) Internucleotide NOEs H8/6 to preceding H2′ and H2″. Global rmsds are given in Supplementary Table 10. The intranucleotide NOE differences are largest for the bases, consistent with differences in the nucleotide conformations for the modified cytosines in terms of sugar pucker and glycosyl torsion angles. (e) 31P NMR spectra of duplexes d(CTACGCGCGTAG)2 (blue), d(CTACG fCGCGTAG)2 (green) and d(CTA fCG fCG fCGTAG)2 (brown), acquired at 20 °C on a 16.45 T (280 MHz for 31P) Bruker Avance III spectrometer. The 31P chemical shift range for all three duplexes is narrow, only 0.4 ppm, consistent with a simple duplex in which the backbone is in the BI conformation (Roongta, V.A. et al. Biochemistry 29, 5245-5258, 1990) and inconsistent with unusual backbone torsions (Lane, A.N. et al. Biochemistry 31, 12087-12095 1992) as seen in the A-form crystal structures (see Supplementary Table 4).

Supplementary Figure 8 Further spectroscopic and structural analysis of DNA duplexes containing either 5-formylcytosine, 5-carboxylcytosine or cytosine.

(a) UV absorption spectra of various duplexes containing fC and caC, whose CD spectra are shown in Fig. 4. (b) The duplex d(CTA fCG fCG fCGTAG)2 in PBS (green), PBS + 200 mM spermine (cyan) and PBS + 800 mM spermine (violet); the addition of spermine did not significantly affect the CD spectrum. (c) CD spectra of the duplexes d(CTA fCG fCG fCGTAG)2 (green) and d(CTACG fCGCGTAG)2 (cyan) in PBS (solid line) and the crystallization buffer of 4QKK (dotted line). (d) CD spectra of the duplex d(CTACGCGCGTAG)2 in PBS (solid line), the crystallization buffer of 4QKK (dotted line) and that of 5MVK (dashed line). The CD spectrum of the duplex in PBS is consistent with the NMR analysis. The crystallization buffer of 4QKK, which contains a high salt concentration (including 1.8 M lithium sulfate) effects the largest change in the CD spectrum of the unmodified duplex, while that of 5MVK reduces the intensity of both bands, which may be due to denaturation caused by the presence of a large proportion of the precipitant MPD. Note that for these spectra, a quartz cuvette with a 3-mm path length and a duplex concentration of 20 μM were used. (e) CD spectra of the duplexes d(CTA fCG fCG fCGTAG)2, d(CTACG fCGCGTAG)2, and d(CTACGCGCGTAG)2, in the buffer used for NMR analysis (80 mM KCl, 22 mM NaPi, pH 7.3). Despite the unusual CD spectra of the formylated duplexes, NMR analysis shows that they adopt the B-form conformation in solution. (f) Hydration of the CpG tract of 5MVK. Despite notable differences in the hydration of 5MVK and 4QKK, the respective (CpG)3 and (fCpG)3 regions of both structures were conformationally similar, thus suggesting that the hydration network does not play a dominant role in determining the observed base-stacking geometries. Only three water molecules were assigned in 5MVU, which may have been a consequence of twinning. Therefore, the hydration network of 5MVK could not be compared to that of the fC duplex crystallized under more similar conditions.

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Hardwick, J., Ptchelkine, D., El-Sagheer, A. et al. 5-Formylcytosine does not change the global structure of DNA. Nat Struct Mol Biol 24, 544–552 (2017). https://doi.org/10.1038/nsmb.3411

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