Nature 524, 114–118 (2015)

DNA nucleotides are produced either by de novo biosynthesis or by salvage pathways that recycle nucleobases from catabolized nucleic acids. As the genome contains epigenetically modified cytosine bases, including 5-hydroxymethyl- and 5-formyl cytosine (5hmC and 5fC), it remains an open question whether these modified bases, when released as nucleosides (5hmdC and 5fdC respectively), affect subsequent DNA metabolism. Zauri et al. now reveal how spurious genomic incorporation of these epigenetic nucleosides is avoided. HPLC analysis of DNA from cell lines transfected with 5hmdC triphosphate (5hmdCTP) demonstrates that 5hmdC is tolerated by the DNA replication apparatus and incorporated into genomic DNA. Biochemical studies showed that genomic integrity is instead maintained by the nucleotide salvage pathway: two kinases involved in the biosynthesis of deoxycytidine triphosphates exclude the modified cytidines as substrates, thereby preventing the synthesis of their dNTPs. Cancer cell line profiling revealed that 5hmdC treatment generally had no adverse effects on cells, but was cytotoxic in cell lines that overexpressed cytidine deaminase (CDA), which converts cytosine nucleotides to their corresponding uracil derivatives. Perturbation of cellular CDA levels by shRNA knockdown or overexpression validated the link between CDA activity and 5hmdC cytotoxicity. The authors further showed in vitro that CDA converts 5hmdC and 5fdC to 5hmdU and 5fdU; these analogs, after phosphorylation and genomic incorporation, induce a DNA damage response, cell cycle arrest and cell death. To test the therapeutic potential of CDA-mediated activation of 5hmdC and 5fdC, the authors demonstrated in a mouse xenograft model that CDA expression facilitated tumor reduction under 5hmdC and 5fdC treatment. Through clarification of the metabolic fate of epigenetic cytosine modifications, the authors have, in parallel, identified a potential nucleotide therapeutic approach for certain cancers.