title: 5-Hydroxymethylcytosine is a predominantly stable DNA modification.
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.
authors.affiliation: 1] Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK [2] Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK.
authors.forename: Martin
authors.initials: M
authors.lastname: Bachman
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK.
authors.forename: Santiago
authors.initials: S
authors.lastname: Uribe-Lewis
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Yang
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Williams
authors.affiliation: 1] Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK [2] Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
authors.forename: Adele
authors.initials: A
authors.lastname: Murrell
authors.affiliation: 1] Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK [2] Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK.
authors.forename: Shankar
authors.initials: S
authors.lastname: Balasubramanian
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D002453:Cell Cycle; D049109:Cell Proliferation; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation; D045325:HCT116 Cells; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D008810:Mice, Inbred C57BL
chemical_list: C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA