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Molecular basis of the plant ROS1-mediated active DNA demethylation

Nature Plants volume 9, pages 271–279 (2023)Cite this article

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Abstract

Active DNA demethylation plays a crucial role in eukaryotic gene imprinting and antagonizing DNA methylation. The plant-specific REPRESSOR OF SILENCING 1/DEMETER (ROS1/DME) family of enzymes directly excise 5-methyl-cytosine (5mC), representing an efficient DNA demethylation pathway distinct from that of animals. Here, we report the cryo-electron microscopy structures of an Arabidopsis ROS1 catalytic fragment in complex with substrate DNA, mismatch DNA and reaction intermediate, respectively. The substrate 5mC is flipped-out from the DNA duplex and subsequently recognized by the ROS1 base-binding pocket through hydrophobic and hydrogen-bonding interactions towards the 5-methyl group and Watson–Crick edge respectively, while the different protonation states of the bases determine the substrate preference for 5mC over T:G mismatch. Together with the structure of the reaction intermediate complex, our structural and biochemical studies revealed the molecular basis for substrate specificity, as well as the reaction mechanism underlying 5mC demethylation by the ROS1/DME family of plant-specific DNA demethylases.

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Fig. 1: Structure of the ROS1–5mC DNA complex.
Fig. 2: Substrate recognition by ROS1.
Fig. 3: Structure of ROS1 in complex with T:G mismatch DNA.
Fig. 4: Molecular basis for the discrimination of other bases by ROS1.
Fig. 5: Structure of ROS1 in complex with a covalent-linked reaction intermediate.

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Data availability

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) with accession codes EMD-33832, EMD-33835 and EMD-33836. The coordinates have been deposited in the Protein Data Bank with accession codes 7YHO, 7YHP and 7YHQ. Protein sequences used in this study and discussion can be found in UniProt with accession codes of Q9SJQ6 (Arabidopsis ROS1), Q05066 (human SRY), Q8LK56 (Arabidopsis DME), Q9SR66 (Arabidopsis DML2) and O49498 (Arabidopsis DML3). Source data are provided with this paper.

References

  1. Zhang, H., Lang, Z. & Zhu, J.-K. Dynamics and function of DNA methylation in plants. Nat. Rev. Mol. Cell Biol. 19, 489–506 (2018).

    Article  CAS  PubMed  Google Scholar 

  2. Law, J. A. & Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Goll, M. G. & Bestor, T. H. Eukaryotic cytosine methyltransferases. Annu. Rev. Biochem. 74, 481–514 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Liu, R. & Lang, Z. The mechanism and function of active DNA demethylation in plants. J. Integr. Plant Biol. 62, 148–159 (2020).

    Article  CAS  PubMed  Google Scholar 

  5. Zhu, J.-K. Active DNA demethylation mediated by DNA glycosylases. Annu. Rev. Genet. 43, 143–166 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shen, L., Song, C.-X., He, C. & Zhang, Y. Mechanism and function of oxidative reversal of DNA and RNA methylation. Annu. Rev. Biochem. 83, 585–614 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Choi, Y. et al. DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Cell 110, 33–42 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Gong, Z. et al. ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111, 803–814 (2002).

    Article  CAS  PubMed  Google Scholar 

  9. Gehring, M. et al. DEMETER DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation. Cell 124, 495–506 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Penterman, J. et al. DNA demethylation in the Arabidopsis genome. Proc. Natl Acad. Sci. USA 104, 6752–6757 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ortega-Galisteo, A. P., Morales-Ruiz, T., Ariza, R. R. & Roldán-Arjona, T. Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks. Plant Mol. Biol. 67, 671–681 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Hong, S., Hashimoto, H., Kow, Y. W., Zhang, X. & Cheng, X. The carboxy-terminal domain of ROS1 is essential for 5-methylcytosine DNA glycosylase activity. J. Mol. Biol. 426, 3703–3712 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Buchan, D. W. A. & Jones, D. T. The PSIPRED protein analysis workbench: 20 years on. Nucleic Acids Res. 47, W402–W407 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Brooks, S. C., Fischer, R. L., Huh, J. H. & Eichman, B. F. 5-Methylcytosine recognition by Arabidopsis thaliana DNA glycosylases DEMETER and DML3. Biochemistry 53, 2525–2532 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Xie, W. et al. Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation. Proc. Natl Acad. Sci. USA 116, 11946–11955 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Werner, M. H., Huth, J. R., Gronenborn, A. M. & Marius Clore, G. Molecular basis of human 46X,Y sex reversal revealed from the three-dimensional solution structure of the human SRY–DNA complex. Cell 81, 705–714 (1995).

    Article  CAS  PubMed  Google Scholar 

  17. Ponferrada-Marín, M. I., Parrilla-Doblas, J. T., Roldán-Arjona, T. & Ariza, R. R. A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine. Nucleic Acids Res. 39, 1473–1484 (2011).

    Article  PubMed  Google Scholar 

  18. Kuo, C.-F. et al. Atomic structure of the DNA repair [4Fe–4S] enzyme endonuclease III. Science 258, 434–440 (1992).

    Article  CAS  PubMed  Google Scholar 

  19. Yang, Z. et al. EBS is a bivalent histone reader that regulates floral phase transition in Arabidopsis. Nat. Genet. 50, 1247–1253 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Holm, L. DALI and the persistence of protein shape. Protein Sci. 29, 128–140 (2020).

    Article  CAS  PubMed  Google Scholar 

  21. Mok, Y. G. et al. Domain structure of the DEMETER 5-methylcytosine DNA glycosylase. Proc. Natl Acad. Sci. USA 107, 19225–19230 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fromme, J. C. & Verdine, G. L. Structure of a trapped endonuclease III–DNA covalent intermediate. EMBO J. 22, 3461–3471 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Parrilla-Doblas, J. T., Ponferrada-Marín, M. I., Roldán-Arjona, T. & Ariza, R. R. Early steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residues. Nucleic Acids Res. 41, 8654–8664 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ponferrada-Marín, M. I., Roldán-Arjona, T. & Ariza, R. R. ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion. Nucleic Acids Res. 37, 4264–4274 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Norman, D. P. G., Chung, S. J. & Verdine, G. L. Structural and biochemical exploration of a critical amino acid in human 8-oxoguanine glycosylase. Biochemistry 42, 1564–1572 (2003).

    Article  CAS  PubMed  Google Scholar 

  26. Fromme, J. C., Banerjee, A., Huang, S. J. & Verdine, G. L. Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase. Nature 427, 652–656 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Norman, D. P. G., Bruner, S. D. & Verdine, G. L. Coupling of substrate recognition and catalysis by a human base-excision DNA repair protein. J. Am. Chem. Soc. 123, 359–360 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Morales-Ruiz, T. et al. DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases. Proc. Natl Acad. Sci. USA 103, 6853–6858 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cokus, S. J. et al. Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452, 215–219 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lister, R. et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133, 523–536 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Agius, F., Kapoor, A. & Zhu, J.-K. Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation. Proc. Natl Acad. Sci. USA 103, 11796–11801 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li, S. & Hong, M. Protonation, tautomerization, and rotameric structure of histidine: a comprehensive study by magic-angle-spinning solid-state NMR. J. Am. Chem. Soc. 133, 1534–1544 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Dodson, M. L., Michaels, M. L. & Lloyd, R. S. Unified catalytic mechanism for DNA glycosylases. J. Biol. Chem. 269, 32709–32712 (1994).

    Article  CAS  PubMed  Google Scholar 

  34. Sun, B., Latham, K. A., Dodson, M. L. & Lloyd, R. S. Studies on the catalytic mechanism of five DNA glycosylases. Probing for enzyme–DNA imino intermediates. J. Biol. Chem. 270, 19501–19508 (1995).

    Article  CAS  PubMed  Google Scholar 

  35. Mullins, E. A., Rodriguez, A. A., Bradley, N. P. & Eichman, B. F. Emerging roles of DNA glycosylases and the base excision repair pathway. Trends Biochem. Sci. 44, 765–781 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Gjaltema, R. A. F. & Rots, M. G. Advances of epigenetic editing. Curr. Opin. Chem. Biol. 57, 75–81 (2020).

    Article  CAS  PubMed  Google Scholar 

  37. de Groote, M. L., Verschure, P. J. & Rots, M. G. Epigenetic editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes. Nucleic Acids Res. 40, 10596–10613 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Devesa-Guerra, I. et al. DNA methylation editing by CRISPR-guided excision of 5-methylcytosine. J. Mol. Biol. 432, 2204–2216 (2020).

    Article  CAS  PubMed  Google Scholar 

  39. Parrilla-Doblas, J. T., Ariza, R. R. & Roldán-Arjona, T. Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain. Epigenetics 12, 296–303 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    Article  CAS  PubMed  Google Scholar 

  43. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Scheres, S. H. W. Amyloid structure determination in RELION-3.1. Acta Crystallogr D 76, 94–101 (2020).

    Article  CAS  Google Scholar 

  45. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  46. Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr D 74, 531–544 (2018).

    Article  CAS  Google Scholar 

  47. Pettersen, E. F. et al. UCSF chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Lavery, R., Moakher, M., Maddocks, J. H., Petkeviciute, D. & Zakrzewska, K. Conformational analysis of nucleic acids revisited: curves. Nucleic Acids Res. 37, 5917–5929 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Di Tommaso, P. et al. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 39, W13–W17 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Hu, L. et al. Crystal structure of TET2–DNA complex: insight into TET-mediated 5mC oxidation. Cell 155, 1545–1555 (2013).

    Article  CAS  PubMed  Google Scholar 

  53. Pidugu, L. S. et al. Structural basis for excision of 5-formylcytosine by thymine DNA glycosylase. Biochemistry 55, 6205–6208 (2016).

    Article  CAS  PubMed  Google Scholar 

  54. Hu, L. et al. Structural insight into substrate preference for TET-mediated oxidation. Nature 527, 118–122 (2015).

    Article  CAS  PubMed  Google Scholar 

  55. Zhang, L. et al. Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA. Nat. Chem. Biol. 8, 328–330 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank D. J. Patel and C. He for critical reading and the staff at Southern University of Science and Technology (SUSTech) Cryo-Electron Microscopy Center for assistance during data collection. This work was supported by Shenzhen Science and Technology Program (JCYJ20200109110403829 and KQTD20190929173906742) and Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes (2019KSYS006) to J.D., China Postdoctoral Science Foundation (2022M712173) to X.D. and National Natural Science Foundation of China (32188102) to J.-K.Z. Z.Y. was supported by National Institute of General Medical Sciences grant (R35GM127018) to E. Nogales.

Author information

Authors and Affiliations

  1. Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes, Institute of Plant and Food Science, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Xuan Du, Guohui Xie, Changshi Wang, Kaige Yan, Jian-Kang Zhu & Jiamu Du

  2. Department of Biochemistry and Molecular Biology, International Cancer Center, Shenzhen University Medical School, Shenzhen, China

    Xuan Du & Sisi Li

  3. California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, CA, USA

    Zhenlin Yang

  4. Howard Hughes Medical Institute, University of California, Berkeley, CA, USA

    Zhenlin Yang

  5. Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China

    Laixing Zhang & Maojun Yang

  6. Institute of Advanced Biotechnology, Southern University of Science and Technology, Shenzhen, China

    Jian-Kang Zhu

  7. Center for Advanced Bioindustry Technologies, Chinese Academy of Agricultural Sciences, Beijing, China

    Jian-Kang Zhu

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Contributions

X.D. performed the biochemical and structural experiments. Z.Y., G.X., C.W. and L.Z. helped with the cryo-EM data collection and processing. M.Y., K.Y., S.L. and J.-K.Z. contributed to data analysis and discussion. J.D. conceived the project and wrote the manuscript.

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Correspondence to Jiamu Du.

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Nature Plants thanks Ping Yin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Active DNA demethylation pathways in animals and plants.

a. In animals, the DNA demethylation process requires the TET family DNA oxygenases to oxidize the 5mC into 5-hydroxymethyl-cytosine (5hmC), 5-formyl-cytosine (5fC), and 5-carboxyl-cytosine (5caC), and the TDG to excise the latter two bases, yielding an AP site and allowing the DNA repair system to recover the AP site back to regular C. b. In contrast, plants utilize a different DNA demethylation system, which employs the bifunctional DNA glycosylase/lyase ROS1 (or DMEs) to specifically excise the 5mC and incise the deoxyribose ring, leaving a β-elimination product 3’-phosphor-α,β-unsaturated aldehyde (PUA) or δ-elimination product 3’-phosphate for the DNA repair system to recover the gap back to regular C. Generally, the DNA repair system are conserved in animals and plants, including AP endonuclease (APE1) or zinc finger DNA 3’-phosphoesterase (ZDP1), DNA polymerase (POL), and DNA ligase (LIG) etc. The PDB codes for important structures capturing various steps of DNA demethylation process are listed in the panel52,53,54,55.

Extended Data Fig. 2 The cryo-EM structure analysis of the ROS1-5mC DNA complex.

a. The purification of SRY-ROS1 protein. b. Gel-filtration profile of SRY-ROS1-5mC DNA complex. c. A representative electron micrograph. d. Representative 2D class average images. e. Flowchart of cryo-EM data processing. f. Gold standard FSC curves for the 3D reconstruction by RELION. g. Local resolution map. h. Fourier shell curves between the refined coordinate model with independent cryo-EM half-maps (black) and with full map (red). i. Orientation distribution of particles for the final 3D reconstruction. j-k. Global FSC (j) and the directional FSC (k) of the last iteration of the 3D auto-refinement by 3D FSC server https://3dfsc.salk.edu.

Source data

Extended Data Fig. 3 Structural analysis of the ROS1 in complex with 5mC-containing DNA.

a. The superimposition of the ROS1 CTD (in cyan) with EBS BAH domain (in magenta, PDB code: 5Z8L) showed a similar folding and suggest that the ROS1 CTD is a BAH domain. The EBS bounded H3K27me3 peptide is highlighted in black, which makes steric conflict with a loop of ROS1 BAH domain, suggesting a different function of ROS1 BAH other than H3K27me3 binding. CTD, C-terminal domain; BAH, bromo adjacent homology domain. b. The electron density map of the ROS1-bounded DNA with the flipped-out 5mC marked. c. The electron density map of key ROS1 residues involving in 5mC binding. d. The positive charged residues of ROS1 interact with the backbone of the 5mC-flanking region of the DNA.

Extended Data Fig. 4 A structure-based sequence alignment of the ROS1/DME family plant DNA demethylases.

The key residues involved in 5mC base flipping, 5mC binding, and catalysis are marked by stars, triangles, and hexagons, respectively. Most of the key residues are strictly conserved within the family.

Extended Data Fig. 5 The molecular structures of the modified bases discussed in this paper.

C, cytosine; T, thymine, which equals 5-methyl-uracil (5mU); 5mC, 5-methyl-cytosine; 5hmC, 5-hydroxymethyl-cytosine; 5fC, 5-formyl-cytosine; 5caC, 5-carboxyl-cytosine; 5hU, 5-hydroxy-uracil; 5FU, 5-fluor-uracil; 5BrU, 5-bromo-uracil.

Extended Data Fig. 6 The cryo-EM structure determination of the ROS1 in complex with a T:G mismatch-containing DNA.

a. A representative electron micrograph for ROS1-TG DNA complex. b. Representative 2D class average. c. Flowchart of cryo-EM data processing. d. Orientation distribution of particles for the final 3D reconstruction. e. Local resolution map. f. Gold standard FSC curves for the final reconstruction by RELION. g. Fourier shell curves between the refined coordinate model with independent cryo-EM half-maps (black) and with full map (red). h-i. Global FSC (h) and the directional FSC (i) in the last iteration of the 3D auto-refinement by 3D FSC server. j-l. Representative cryo-EM map for the flipped-out base T surrounding region (j-k) and an α-helix (l).

Extended Data Fig. 7 Covalent complex formation between ROS1 and reaction-intermediate.

The NaBH4 trapped ROS1-5mC DNA reaction product for the cryo-EM sample preparation was subjected to the SDS-PAGE with ROS1 protein and substrate DNA as control. While the Coomassie brilliant blue staining in the left panel showed the reaction product has significant shift compared to the free ROS1 protein, the nucleic acid dye SYBR Gold staining of the gel in the right panel suggested that the shifted band contained the DNA, confirming protein-DNA covalent complex formation. An aggregation of ROS1 (marked by *) was also shown to be covalently linked to DNA with significant shift (marked by **). The experiment was repeated 3 times independently with similar results.

Source data

Extended Data Fig. 8 The cryo-EM structure determination of the ROS1 in complex with a covalent-linked reaction intermediate.

a. A representative electron micrograph of covalent ROS1-DNA complex. b. Representative 2D class average. c. Flowchart of cryo-EM data processing. d. Orientation distribution of particles for the final 3D reconstruction. e. Local resolution map. f. Gold standard FSC curves for the final 3D reconstruction by RELION. g. Fourier shell curves between the refined coordinate model with independent cryo-EM half-maps (black) and with full map (red). h-i. Global FSC (h) and the directional FSC (i) of the covalent ROS1-DNA complex in the last iteration of the 3D auto-refinement by 3D FSC server. j-k. Representative cryo-EM map of the Lys953-PED linkage region (j) and an α-helix region (k).

Supplementary information

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Source Data Fig. 5

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Source Data Extended Data Fig. 2

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Source Data Extended Data Fig. 7

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Du, X., Yang, Z., Xie, G. et al. Molecular basis of the plant ROS1-mediated active DNA demethylation. Nat. Plants 9, 271–279 (2023). https://doi.org/10.1038/s41477-022-01322-8

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