Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Genome-wide mapping reveals that deoxyuridine is enriched in the human centromeric DNA

Nature Chemical Biology volume 14, pages 680–687 (2018)Cite this article

Abstract

Uracil in DNA can be generated by cytosine deamination or dUMP misincorporation; however, its distribution in the human genome is poorly understood. Here we present a selective labeling and pull-down technology for genome-wide uracil profiling and identify thousands of uracil peaks in three different human cell lines. Surprisingly, uracil is highly enriched at the centromere of the human genome. Using mass spectrometry, we demonstrate that human centromeric DNA contains a higher level of uracil. We also directly verify the presence of uracil within two centromeric uracil peaks on chromosomes 6 and 11. Moreover, centromeric uracil is preferentially localized within the binding regions of the centromere-specific histone CENP-A and can be excised by human uracil-DNA glycosylase UNG. Collectively, our approaches allow comprehensive analysis of uracil in the human genome and provide robust tools for mapping and future functional studies of uracil in DNA.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: dU-seq is a selective labeling and pull-down technology for genome-wide uracil profiling.
Fig. 2: dU-seq reveals the genome-wide profile of uracil in K562, WPMY-1 and HEK293T cells, respectively.
Fig. 3: Uracil is enriched in the human centromeric DNA.
Fig. 4: Uracil peaks colocalize with the CENP-A binding regions at the centromere.
Fig. 5: Uracil at the centromere can be excised by UNG.

Similar content being viewed by others

References

  1. Suzuki, M. M. & Bird, A. DNA methylation landscapes: provocative insights from epigenomics. Nat. Rev. Genet. 9, 465–476 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 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 

  4. Luo, G. Z., Blanco, M. A., Greer, E. L., He, C. & Shi, Y. DNA N6-methyladenine: a new epigenetic mark in eukaryotes? Nat. Rev. Mol. Cell Biol. 16, 705–710 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. David, S. S., O’Shea, V. L. & Kundu, S. Base-excision repair of oxidative DNA damage. Nature 447, 941–950 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wyrick, J. J. & Roberts, S. A. Genomic approaches to DNA repair and mutagenesis. DNA Repair (Amst.) 36, 146–155 (2015).

    Article  CAS  Google Scholar 

  7. Krokan, H. E., Drabløs, F. & Slupphaug, G. Uracil in DNA—occurrence, consequences and repair. Oncogene 21, 8935–8948 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Kavli, B., Otterlei, M., Slupphaug, G. & Krokan, H. E. Uracil in DNA—general mutagen, but normal intermediate in acquired immunity. DNA Repair (Amst.) 6, 505–516 (2007).

    Article  CAS  Google Scholar 

  9. Stavnezer, J., Guikema, J. E. & Schrader, C. E. Mechanism and regulation of class switch recombination. Annu. Rev. Immunol. 26, 261–292 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Di Noia, J. M. & Neuberger, M. S. Molecular mechanisms of antibody somatic hypermutation. Annu. Rev. Biochem. 76, 1–22 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Harris, R. S. & Dudley, J. P. APOBECs and virus restriction. Virology 479-480, 131–145 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Siriwardena, S. U., Chen, K. & Bhagwat, A. S. Functions and malfunctions of mammalian DNA-cytosine deaminases. Chem. Rev. 116, 12688–12710 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Burns, M. B., Temiz, N. A. & Harris, R. S. Evidence for APOBEC3B mutagenesis in multiple human cancers. Nat. Genet. 45, 977–983 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Matsumoto, Y. et al. Up-regulation of activation-induced cytidine deaminase causes genetic aberrations at the CDKN2b-CDKN2a in gastric cancer. Gastroenterology 139, 1984–1994 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. David, S. S. & Williams, S. D. Chemistry of glycosylases and endonucleases involved in base-excision repair. Chem. Rev. 98, 1221–1262 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Krokan, H. E. & Bjørås, M. Base excision repair. Cold Spring Harb. Perspect. Biol. 5, a012583 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Nilsen, H. et al. Nuclear and mitochondrial uracil-DNA glycosylases are generated by alternative splicing and transcription from different positions in the UNG gene. Nucleic Acids Res. 25, 750–755 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Otterlei, M. et al. Post-replicative base excision repair in replication foci. EMBO J. 18, 3834–3844 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Vallabhaneni, H. et al. Defective repair of uracil causes telomere defects in mouse hematopoietic cells. J. Biol. Chem. 290, 5502–5511 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Muha, V. et al. Uracil-containing DNA in Drosophila: stability, stage-specific accumulation, and developmental involvement. PLoS Genet. 8, e1002738 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kavli, B. et al. hUNG2 is the major repair enzyme for removal of uracil from U:A matches, U:G mismatches, and U in single-stranded DNA, with hSMUG1 as a broad specificity backup. J. Biol. Chem. 277, 39926–39936 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Hendrich, B., Hardeland, U., Ng, H. H., Jiricny, J. & Bird, A. The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites. Nature 401, 301–304 (1999).

    Article  CAS  PubMed  Google Scholar 

  23. Neddermann, P. & Jiricny, J. Efficient removal of uracil from G.U mispairs by the mismatch-specific thymine DNA glycosylase from HeLa cells. Proc. Natl Acad. Sci. USA 91, 1642–1646 (1994).

    Article  CAS  PubMed  Google Scholar 

  24. Suspène, R., Henry, M., Guillot, S., Wain-Hobson, S. & Vartanian, J. P. Recovery of APOBEC3-edited human immunodeficiency virus G- A hypermutants by differential DNA denaturation PCR. J. Gen. Virol. 86, 125–129 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Stenglein, M. D., Burns, M. B., Li, M., Lengyel, J. & Harris, R. S. APOBEC3 proteins mediate the clearance of foreign DNA from human cells. Nat. Struct. Mol. Biol. 17, 222–229 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Maul, R. W. et al. Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions. Nat. Immunol. 12, 70–76 (2011).

    Article  CAS  PubMed  Google Scholar 

  27. Riedl, J., Fleming, A. M. & Burrows, C. J. Sequencing of DNA lesions facilitated by site-specific excision via base excision repair DNA glycosylases yielding ligatable gaps. J. Am. Chem. Soc. 138, 491–494 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Róna, G. et al. Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications. Nucleic Acids Res. 44, e28 (2016).

    Article  CAS  PubMed  Google Scholar 

  29. Bryan, D. S., Ransom, M., Adane, B., York, K. & Hesselberth, J. R. High resolution mapping of modified DNA nucleobases using excision repair enzymes. Genome Res. 24, 1534–1542 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Horváth, A. & Vértessy, B. G. A one-step method for quantitative determination of uracil in DNA by real-time PCR. Nucleic Acids Res. 38, e196 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hansen, E. C. et al. Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells. eLife 5, e18447 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Galashevskaya, A. et al. A robust, sensitive assay for genomic uracil determination by LC/MS/MS reveals lower levels than previously reported. DNA Repair (Amst.) 12, 699–706 (2013).

    Article  CAS  Google Scholar 

  33. Bulgar, A. D. et al. Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: overriding the limit with methoxyamine to inhibit base excision repair. Cell Death Dis. 3, e252 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Xiao, G. et al. Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: structure and glycosylase mechanism revisited. Proteins 35, 13–24 (1999).

    Article  CAS  PubMed  Google Scholar 

  35. Ali, M. H., Al-Saad, K. A. & Ali, C. M. Biophysical studies of the effect of high power ultrasound on the DNA solution. Phys. Med. 30, 221–227 (2014).

    Article  PubMed  Google Scholar 

  36. Costello, M. et al. Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation. Nucleic Acids Res. 41, e67 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Shepelev, V. A. et al. Annotation of suprachromosomal families reveals uncommon types of alpha satellite organization in pericentromeric regions of hg38 human genome assembly. Genom. Data 5, 139–146 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Pfaffeneder, T. et al. The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew. Chem. Int. Edn. Engl. 50, 7008–7012 (2011).

    Article  CAS  Google Scholar 

  39. Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Allshire, R. C. & Karpen, G. H. Epigenetic regulation of centromeric chromatin: old dogs, new tricks? Nat. Rev. Genet. 9, 923–937 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Nilsen, H. et al. Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication. Mol. Cell 5, 1059–1065 (2000).

    Article  CAS  PubMed  Google Scholar 

  42. Lloyd, R. S. Investigations of pyrimidine dimer glycosylases–a paradigm for DNA base excision repair enzymology. Mutat. Res. 577, 77–91 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. Boiteux, S., Coste, F. & Castaing, B. Repair of 8-oxo-7,8-dihydroguanine in prokaryotic and eukaryotic cells: properties and biological roles of the Fpg and OGG1 DNA N-glycosylases. Free Radic. Biol. Med. 107, 179–201 (2017).

    Article  CAS  PubMed  Google Scholar 

  44. Grogan, B. C., Parker, J. B., Guminski, A. F. & Stivers, J. T. Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA. Biochemistry 50, 618–627 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Müller, S. & Almouzni, G. Chromatin dynamics during the cell cycle at centromeres. Nat. Rev. Genet. 18, 192–208 (2017).

    Article  CAS  PubMed  Google Scholar 

  46. Zeitlin, S. G., Patel, S., Kavli, B. & Slupphaug, G. Xenopus CENP-A assembly into chromatin requires base excision repair proteins. DNA Repair (Amst.) 4, 760–772 (2005).

    Article  CAS  Google Scholar 

  47. Zeitlin, S. G. et al. Uracil DNA N-glycosylase promotes assembly of human centromere protein A. PLoS One 6, e17151 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Periyasamy, M. et al. APOBEC3B-mediated cytidine deamination is required for estrogen receptor action in breast cancer. Cell Reports 13, 108–121 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors would like to thank G. Liu and H. Li for measurements with LC–MS/MS; B. Xia, X. Li and X. Xiong for technical advice and discussions. This work was supported by the National Natural Science Foundation of China (nos. 21522201 and 21472009 to C.Y.), the National Basic Research Foundation of China (nos. 2016YFC0900301 and 2014CB964900 to C.Y.) and the Fok Ying Tung Education Foundation (no. 161018 to C.Y.).

Author information

Author notes

  1. These authors contributed equally: Xiaoting Shu, Menghao Liu, Zhike Lu.

Authors and Affiliations

  1. State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Xiaoting Shu, Menghao Liu, Zhike Lu, Chenxu Zhu, Haowei Meng, Sihao Huang & Chengqi Yi

  2. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Xiaoting Shu, Menghao Liu & Xiaoxue Zhang

  3. Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

    Chengqi Yi

Authors
  1. Xiaoting Shu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Menghao Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhike Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chenxu Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Haowei Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sihao Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaoxue Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Chengqi Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

X.S. and C.Y. conceived the project; X.S., M.L. and C.Y. designed the experiments and wrote the manuscript with the help of Z.L.; X.S., M.L., C.Z., S.H. and X.Z. performed the experiments; Z.L. and H.M. performed bioinformatics analysis. All authors commented on and approved the paper.

Corresponding author

Correspondence to Chengqi Yi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Notes 1 and 2, Supplementary Figures 1–16, Supplementary Tables 1–4

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shu, X., Liu, M., Lu, Z. et al. Genome-wide mapping reveals that deoxyuridine is enriched in the human centromeric DNA. Nat Chem Biol 14, 680–687 (2018). https://doi.org/10.1038/s41589-018-0065-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0065-9

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing