Eukaryotic cells protect themselves against potentially deadly genotoxic insults with a network of DNA damage response (DDR) mechanisms, which are initiated by specific sensors that detect DNA damage or chromosome alterations. Signal transduction pathways that involve the ATR kinase and other phosphoinositol-3-kinase-like kinases then activate cell cycle checkpoints and other protective pathways to ensure that DNA lesions are fully repaired before cell division occurs. Here, Leon Mullenders and colleagues (p. 435) investigate how UV-induced DNA lesions activate the DDR in non-cycling human cells. They show that, in non-cycling cells proficient in nucleotide excision repair (NER; a repair pathway that removes UV-induced DNA lesions), DNA damage signalling is rapid and transient. By contrast, in non-cycling NER-deficient cells, DNA damage signalling is delayed and persistent, and UV-induced checkpoint activation coincides with the formation of single-stranded DNA breaks. Finally, the authors report that checkpoint activation in both NER-proficient and NER-deficient cells involves ATR-kinase-dependent signalling. Thus, in non-dividing mammalian cells, DNA damage signalling, which ultimately prevents the transition from G1 to S phase, involves both NER-dependent and NER-independent processing of UV-induced lesions.