Significance Solar UV generates abundant carcinogenic DNA lesions, and consequently, cells need to cope with these deleterious lesions to survive UV damage. This study used lesion-specific photolyases to isolate the biologically relevant effects of each major type of UV-induced DNA lesions (cyclobutane pyrimidine dimers [CPDs] and 6-4 photoproducts [6-4PPs]) on ATR activation, a crucial DNA damage response. A newly developed, flow cytometric single-cell analysis of UV-induced DNA lesions, thymidine analog incorporation, and DNA damage response enabled unprecedented determination of cellular events following UV irradiation. The striking finding is that only 6-4PPs, the shorter-lived and less abundant lesion type, cause replication blockage and activation of the ATR DNA damage response.