Exposure of eukaryotic cells to UV light induces a checkpoint response that delays cell-cycle progression after cells enter S phase. It has been hypothesized that this checkpoint response provides time for repair by signaling the presence of structures generated when the replication fork encounters UV-induced DNA damage. To gain insight into the nature of the signaling structures, we used time-lapse microscopy to determine the effects of deficiencies in translesion DNA polymerases on the checkpoint response of the fission yeast Schizosaccharomyces pombe. We found that disruption of the genes encoding translesion DNA polymerases Polκ and Polη significantly prolonged the checkpoint response, indicating that the substrates of these enzymes are signals for checkpoint activation. Surprisingly, we found no evidence that the translesion polymerases Rev1 and Polζ repair structures that are recognized by the checkpoint despite their role in maintaining viability after UV irradiation. Quantitative flow cytometry revealed that cells lacking translesion polymerases replicate UV-damaged DNA at the same rate at WT cells, indicating that the enhanced checkpoint response of cells lacking Polκ and Polη is not the result of stalled replication forks. These observations support a model in which postreplication DNA gaps with unrepaired UV lesions in the template strand act both as substrates for translesion polymerases and as signals for checkpoint activation.NA damage produced by the UV component of sunlight presents a constant challenge for the survival of organisms on the earth's surface. In response to this challenge, eukaryotic cells have evolved excision repair processes that remove the damage, postreplication repair processes that facilitate the replication of damaged DNA, and checkpoint mechanisms that delay cell-cycle progression to make time for repair (1-3). Because the genes that mediate these processes are conserved in eukaryotes, model systems such as yeast have provided valuable insights that are applicable to the DNA damage responses of higher organisms (4). In previous studies, we used time-lapse microscopy to measure the cell-cycle dynamics of UV-irradiated fission yeast cells and observed two distinct DNA damage checkpoint responses: the previously characterized G2/M checkpoint response that delays cell division when cells are irradiated in G2 phase and a postreplication checkpoint response that delays division when cells irradiated in any stage of the cell cycle carry lesions into S phase (5). Only the latter response occurs after moderate UV doses comparable to sunlight exposure, so it is likely to be particularly important in nature (3). The postreplication checkpoint response is activated following the encounter of replication forks with UV-induced DNA damage and requires the activity of checkpoint proteins that recognize structures containing transitions between double-stranded and single-stranded DNA (3, 6-11). The nature and origin of the signaling structures that determine the Schizosaccharomyces pombe po...

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