Activation of signaling pathways by UV radiation is a key event in the DNA damage response and initiated by different cellular processes. Here we show that non-cycling cells proficient in nucleotide excision repair (NER) initiate a rapid but transient activation of the damage response proteins p53 and H2AX; by contrast, NER-deficient cells display delayed but persistent signaling and inhibition of cell cycle progression upon release from G0 phase. In the absence of repair, UV-induced checkpoint activation coincides with the formation of single-strand DNA breaks by the action of the endonuclease Ape1. Although temporally distinct, activation of checkpoint proteins in NER-proficient and NER-deficient cells depends on a common pathway involving the ATR kinase. These data reveal that damage signaling in non-dividing cells proceeds via NER-dependent and NER-independent processing of UV photolesions through generation of DNA strand breaks, ultimately preventing the transition from G1 to S phase.