Pluripotent embryonic stem cells (ES cells) are the precursors of all different cell types comprising the organism. Since persistent DNA damage in this cell type might lead to mutations that cause huge malformations in the developing organism, genome caretaking is of prime importance. We first compared the sensitivity of wild type mouse embryonic fibroblasts (MEFs) and ES cells for various genotoxic agents and show that ES cells are more sensitive to treatment with UV-light, gamma-rays and mitomycin C than MEFs. We next investigated the contribution of the transcription-coupled (TC-NER) and global genome (GG-NER) sub-pathways of nucleotide excision repair (NER) in protection of ES cells, using cells from mouse models for the NER disorders xeroderma pigmentosum (XP) and Cockayne syndrome (CS). TC-NER-deficient Csb(-/-) and GG-NER/TC-NER-defective Xpa(-/-) MEFs are hypersensitive to UV, whereas GG-NER-deficient Xpc(-/-) MEFs attribute intermediate UV sensitivity. The observed UV hypersensitivity in Csb(-/-) and Xpa(-/-) MEFs correlates with increased apoptosis. In contrast, Xpa(-/-) and Xpc(-/-) ES cells are highly UV-sensitive, while a Csb deficiency only causes a mild increase in UV-sensitivity. Surprisingly, a UV-induced hyperapoptotic response is mainly observed in Xpa(-/-) ES cells, suggesting a different mechanism of apoptosis induction in ES cells, mainly triggered by damage in the global genome rather than in transcribed genes (as in MEFs). Moreover, we show a pronounced S-phase delay in Xpa(-/-) and Xpc(-/-) ES cells, which might well function as a safeguard mechanism for heavily damaged ES cells in case the apoptotic response fails. Although Xpa-/- and Xpc(-/-) ES cells are totally NER-defective or GG-NER-deficient respectively, mutation induction upon UV is similar compared to wild type ES cells indicating that the observed apoptotic and cell cycle responses are indeed sufficient to protect against proliferation of damaged cells. In conclusion, we show a double safeguard mechanism in ES cells against NER-type of damages, which mainly relies on damage detection in the global genome. (c) 2008 Elsevier B.V All rights reserved.