title: EdU induces DNA damage response and cell death in mESC in culture.
abstract: Recently, a novel DNA replication precursor analogue called 5-ethynyl-2'-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.
authors: University of Manchester, Faculty of Life Sciences, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK.
authors: Fanni
authors: F
authors: Kohlmeier
authors: Apolinar
authors: A
authors: Maya-Mendoza
authors: Dean A
authors: DA
authors: Jackson
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D057930:Click Chemistry; D004247:DNA / Q000187:drug effects / Q000235:genetics*; D004249:DNA Damage / Q000187:drug effects* / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects* / Q000235:genetics; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005434:Flow Cytometry; D051379:Mice
chemical_list: D004247:DNA; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine