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5-Hydroxymethylcytosine is a predominantly stable DNA modification

Nature Chemistry volume 6, pages 1049–1055 (2014)Cite this article

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Abstract

5-Hydroxymethylcytosine (hmC) is an oxidation product of 5-methylcytosine which is present in the deoxyribonucleic acid (DNA) of most mammalian cells. Reduction of hmC levels in DNA is a hallmark of cancers. Elucidating the dynamics of this oxidation reaction and the lifetime of hmC in DNA is fundamental to understanding hmC function. Using stable isotope labelling of cytosine derivatives in the DNA of mammalian cells and ultrasensitive tandem liquid–chromatography mass spectrometry, we show that the majority of hmC is a stable modification, as opposed to a transient intermediate. In contrast with DNA methylation, which occurs immediately during replication, hmC forms slowly during the first 30 hours following DNA synthesis. Isotopic labelling of DNA in mouse tissues confirmed the stability of hmC in vivo and demonstrated a relationship between global levels of hmC and cell proliferation. These insights have important implications for understanding the states of chemically modified DNA bases in health and disease.

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Figure 1: Global levels of mC and hmC do not change during the cell cycle.
Figure 2: DNA methylation and mC oxidation activities occur with a marked time difference.
Figure 3: The majority of genomic hmC is stable.
Figure 4: Isotopic labelling of DNA in vivo confirms the stability of hmC and reveals a relationship between global hmC levels and proliferation.

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Acknowledgements

We acknowledge the CRUK CI Flow Cytometry and Histopathology/In Situ Hybridization core facilities for their contributions, D. Oxley, C. d'Santos and D. Michelle-Smith for their support with mass spectrometry, X. Zou for his help with mES cells and D. Tannahill for a critical reading of the manuscript. This work was funded by CRUK (all authors) and the Wellcome Trust Senior Investigator Award (S.B.).

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Authors and Affiliations

  1. Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, UK

    Martin Bachman & Shankar Balasubramanian

  2. Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, CB2 0RE, UK

    Martin Bachman, Santiago Uribe-Lewis, Xiaoping Yang, Michael Williams, Adele Murrell & Shankar Balasubramanian

  3. Department of Biology and Biochemistry, Centre for Regenerative Medicine, University of Bath, Bath, BA2 7AY, UK

    Adele Murrell

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Contributions

M.B., S.U-L., A.M. and S.B. conceived and designed the experiments; M.B. and S.U-L. performed the experiments; X.Y., M.W. and M.B. developed the mass spectrometry methods; M.B. and S.U-L. analysed the data with the help of X.Y. and M.W.; M.B., S.U-L. and S.B. co-wrote the manuscript with contributions from all authors.

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Correspondence to Shankar Balasubramanian.

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The authors declare no competing financial interests.

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Bachman, M., Uribe-Lewis, S., Yang, X. et al. 5-Hydroxymethylcytosine is a predominantly stable DNA modification. Nature Chem 6, 1049–1055 (2014). https://doi.org/10.1038/nchem.2064

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