5-Hydroxymethylcytosine is a predominantly stable DNA modification.

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

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.

5-Methylcytosine, Animals, Cell Cycle, Cell Proliferation, Cytosine, DNA, DNA Methylation, HCT116 Cells, Humans, MCF-7 Cells, Mice, Mice, Inbred C57BL
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Nat Chem
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Springer Science and Business Media LLC
Cancer Research Uk (None)
Wellcome Trust (099232/Z/12/Z)
We would like to acknowledge the CRUK CI Flow Cytometry and Histopathology/ISH core facilities for their contributions, David Oxley, Clive d’Santos and Donna Michelle-Smith for their support with mass spectrometry, Xiangang Zou for his help with mES cells and David Tannahill for critical reading of the manuscript. This work was funded by Cancer Research UK (all authors) and the Wellcome Trust Senior Investigator Award (S.B.).