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Proc Natl Acad Sci U S A. 2006 Oct 24; 103(43): 15877–15882.
Published online 2006 Oct 16. doi:  10.1073/pnas.0607343103
PMCID: PMC1613229
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Cell Biology

UV irradiation induces a postreplication DNA damage checkpoint


Eukaryotic cells irradiated with high doses of UV exhibit cell-cycle responses referred to as G1/S, intraS, and G2/M checkpoints. After a moderate UV dose that approximates sunlight exposure and is lethal to fission yeast checkpoint mutants, we found unexpectedly that these cell-cycle responses do not occur. Instead, cells at all stages of the cell cycle carry lesions into S phase and delay cell-cycle progression for hours after the completion of bulk DNA synthesis. Both DNA replication and the checkpoint kinase, Chk1, are required to generate this cell-cycle response. UV-irradiation of Δchk1 cells causes chromosome damage and loss of viability only after cells have replicated irradiated DNA and entered mitosis. These data suggest that an important physiological role of the cell-cycle response to UV is to provide time for postreplication repair.

Keywords: cell cycle, chk1, nucleotide excision repair, yeast

UV radiation from sunlight exposure induces DNA damage that is potentially lethal to cells and is carcinogenic to animals (1). Studies in fission and budding yeasts have demonstrated that genes that promote survival in UV light encode both DNA repair factors (2, 3) and checkpoint proteins that regulate the cell cycle in response to DNA damage (4, 5). These genes are conserved among eukaryotes, so diverse model systems may be used to address questions of molecular mechanism (6).

The pyrimidine dimer is the most abundant form of DNA damage known to be induced by UV (7). This lesion is removed from duplex DNA by nucleotide excision repair (NER). When present during S phase, pyrimidine dimers cause gaps in the daughter DNA strand (812). The repair of these gaps is referred to as postreplication repair and involves bypass polymerases, homologous recombination, and other processes (13).

Several eukaryotic cell-cycle responses have been observed after high-dose UV irradiation. Fission yeast UV-irradiated in G2 delay the onset of mitosis, a response conserved in human cells that has been termed a G2/M DNA damage checkpoint (5, 1416). It is posited that the UV sensitivity of fission yeast G2/M checkpoint mutants results from a failure to make time for the repair of UV-induced DNA lesions before mitosis (5). Budding yeast exhibit a G1/S checkpoint response that delays S phase entry after UV irradiation in G1 (17). Because activation of the G1/S checkpoint requires NER genes, it has been suggested that the checkpoint monitors the presence of NER intermediates (12, 1820). Mammalian cells exhibit a p53-dependent G1/S DNA damage checkpoint response that can eliminate damaged cells through apoptosis, delay entry into S phase, and facilitate NER (21). Like the G1/S checkpoint of yeast, the induction of p53 in G1 requires NER and is independent of DNA replication (22). A NER-independent checkpoint is activated when UV lesions are present during S phase in Xenopus egg extracts and budding yeast NER mutants (18, 23). In both model systems, UV lesions cause an arrest in S phase; however, this arrest appears to be checkpoint-mediated only in the case of yeast.

We developed a method for extended time-lapse microscopy that allowed us to track UV-irradiated yeast cells through multiple divisions. Using this method, we detected a previously uncharacterized cell-cycle response to UV that occurs during the second cycle after irradiation. Fission yeast irradiated in G2 phase carry lesions through mitosis and S phase and delay the subsequent division, and budding yeast irradiated in G1 phase carry lesions into S phase and delay emergence of the second bud. When exposed to a UV dose comparable to sunlight exposure, we found that cells do not delay the cell cycle at the stage in which they were irradiated. Instead, budding and fission yeast cells irradiated at every stage in the cell cycle undergo a single checkpoint response after carrying lesions through S phase. We propose a model in which the DNA damage checkpoint provides time for repair by monitoring the presence of postreplication gaps generated by the replication of DNA containing unrepaired UV photoproducts.


Fission Yeast Delay Mitosis During the Second Cell Cycle After UV Irradiation.

An algorithm was developed to hold cells in focus, allowing routine observation of four sequential cleavages in an initial population of 100 fission yeast cells. To measure UV response, a field of asynchronous cells was observed for 3 h and then irradiated. The same field was observed for the subsequent three cleavages, and the resulting time-lapse series was examined to determine the times at which the progeny divided. From the cleavage times, we calculated the cycle times as well as the percentage of cells that had undergone cleavage as a function of time (Fig. 1). When exposed to a UV dose of 25 J/m2, the youngest two-thirds of the population at time of irradiation delayed the first cleavage (Fig. 1A). Because this population corresponds to cells irradiated in G2 phase, the response is consistent with a G2/M checkpoint. The remaining third of the population did not delay at all. Our calculations suggest that cells irradiated in the last 10 min of G2 and beyond did not undergo a cell-cycle checkpoint response before the first division (Fig. 6, which is published as supporting information on the PNAS web site). Because a full third of the population did not make time for repair on the first cycle, we investigated the fate of cells on the second cycle. Unexpectedly, cells underwent a greater UV-induced delay during the second cycle after irradiation than the first (Fig. 1B). When cycle times of individual cells were plotted, it was evident that the greatest second-cycle delays occurred in cells that failed to delay the first cycle (Fig. 1C), but many cells irradiated in G2 delayed on both the first and second cycles.

Fig. 1.
Fission yeast delay during the second cell cycle after UV irradiation. Asynchronous WT S. pombe cells grown in YE6s-rich medium were observed by time-lapse microscopy for 3 h and then exposed to UV. The same cells were observed for three subsequent divisions. ...

G2/M Checkpoint Is Absent After Irradiation with Moderate UV Doses.

DNA damage induced by sunlight is equivalent to a UV dose of ≈0.1 J·m−2·min−1 delivered from mercury vapor bulbs like those used in our experiments (24). Therefore, we wondered whether some of the observed second-cycle delays after 25 J/m2 resulted from the introduction of an unphysiologic burden of lesions. Based on the known half-life of UV lesions in fission and budding yeasts and the fluence of sunlight, we calculated that the steady-state lesion burden of yeast continually exposed to sunlight would be roughly that induced in the laboratory by a pulse of 5 J/m2 (25, 26). When the UV dose was reduced to 5 J/m2, the vast majority of cells did not delay at all on the first cycle but long delays still occurred on the second cycle (Fig. 1A). We conclude from this observation that the second-cycle delays are neither an artifact of high-dose UV nor the result of residual checkpoint activation from the first cell cycle. An analysis of second cycle times revealed that cells irradiated at 5 J/m2 exhibited significant second-cycle delays regardless of the stage of the cell cycle in which they were irradiated (Fig. 1D). Therefore, cells irradiated with a physiological UV dose do not mount a G2/M checkpoint response and do not remove all of the lesions capable of causing cell-cycle delay during the first cell cycle after irradiation. Rather, cells irradiated in G2 carry unrepaired lesions through mitosis and cell division into the second cycle where they induce a delay. In contrast, the third cell cycle after irradiation was not delayed at all and was actually accelerated in many of the cells (Fig. 1F). The accelerated divisions are presumably the result of a size control mechanism operating to reduce cell mass accumulated during the first two cell cycles.

Second-Cycle Delay Occurs After S Phase.

Duration of the second cycle was strongly correlated with the stage in the cell cycle at which the cells were irradiated (Fig. 1D). Fig. 1E shows a running average of second-cycle times as a function of the time of first cleavage after irradiation at 5 J/m2. This curve exhibits two distinct phases. During the first phase, the cycle times increase to a maximum value, and during the second phase, they decrease progressively, approaching the average cell-cycle time for unperturbed Schizosaccharomyces pombe. Most of the cells fell into the latter category. The rate of decrease in second-cycle times in this phase, determined by fitting an exponential curve to the data, is similar to the rate at which fission yeast excise pyrimidine dimers (half time of 20 min; ref. 25). Thus, the progressive reduction in second-cycle times can be explained by a model in which the duration of second-cycle delay is related to the number of lesions left unexcised at the end of the first cycle. The first phase of the curve in Fig. 1E cannot be explained by a repair process, because the delay increases with the time available for repair. More than 80% of the cells in the first phase were septated when irradiated and therefore were likely to be actively undergoing DNA replication (27). Thus, the data indicate that the further the cells had progressed through S phase at the time of irradiation, the shorter were the second-cycle delays. This response is consistent with the hypothesis that DNA lesions introduced in front of the replication fork induce delay, but those behind the fork do not.

To determine the stage at which second-cycle delay occurs, we made use of flow cytometry to measure the DNA contents and sizes of cells at hourly intervals after irradiation with a UV dose of 5 J/m2 (Fig. 2). The distribution of cells remained relatively unchanged for the first 2 h after irradiation. Because the time-lapse data indicate that an asynchronous population undergoes cleavage by 2.5 h after irradiation (Fig. 1A), the absence of cells with DNA contents <2C indicate that no significant cell-cycle delay occurred in G1 or early S phase. Therefore, neither a G1/S nor an intraS checkpoint is evident at this UV dose. Between 3 and 7 h after irradiation, we detected a population of elongated cells with 2C DNA content, indicating that second-cycle delay occurred after the completion of bulk DNA synthesis. Indeed, the presence of lesions does not appear to measurably slow DNA synthesis, even though a dose of 5 J/m2 is expected to induce >400 pyrimidine dimers (28, 29).

Fig. 2.
Second-cycle delay in response to 5 J/m2 occurs after the completion of bulk DNA synthesis. Asynchronous WT fission yeast cells were plated on agar dishes under conditions similar to Fig. 1. After a 3-h acclimation period, the cells were mock-irradiated ...

Postreplication Delay Requires DNA Replication and Checkpoint Kinase (Chk1).

The foregoing observations suggested that second-cycle delay might be a consequence of the encounter of replication forks with unrepaired UV lesions. To test this hypothesis, we asked whether the delay requires the initiation of DNA replication. For this purpose, we studied a S. pombe strain in which the transcription of the Cdc18 replication initiation factor is under the control of the thiamine-repressible nmt1 promoter (Fig. 3A). In the presence of thiamine, this strain does not initiate DNA replication and undergoes cell division with a 1C DNA content (30). Cells synchronized in G2 were UV-irradiated and followed for the subsequent two divisions in the presence or absence of thiamine. When cells expressing cdc18 were irradiated with a UV dose of 15 J/m2, both the first and second divisions were delayed (Fig. 3A). When cdc18 was repressed, the UV-induced first-division delay was largely unaffected, whereas the second-division delay was completely lost. Therefore, UV-induced second-cycle delay depends entirely on DNA replication. Cdc18-dependent second-cycle delays were also seen after a dose of 5 J/m2 (Fig. 7, which is published as supporting information on the PNAS web site). The role of DNA replication in generating a cell-cycle response to UV was confirmed by using cells synchronized in early S phase by treatment with hydroxyurea (HU). When such cells were irradiated with 5 J/m2 and released from the block, they delayed on the first cycle but not the second, indicating that the introduction of lesions in S phase is sufficient to generate a cell-cycle delay (Fig. 8, which is published as supporting information on the PNAS web site).

Fig. 3.
Second-cycle delay in response to UV requires DNA replication and chk1 in S. pombe. (A) A strain in which expression of cdc18 is under the control of a thiamine-repressible nmt1 promoter (h-cdc18::REP41x-cdc18-leu1+ cdc25–22 leu1–32 ura4 ...

Fission yeast excise UV lesions through NER and an alternative pathway initiated by the UV damage endonuclease (UVDE) (31). The latter pathway is thought to involve incision and nick translation and may not generate structures similar to NER intermediates (32). To determine the effect of NER on the cell cycle in the absence of the alternative pathway, we characterized a UVDE-null strain in which all detectable photoproduct removal occurs by NER (31). Like WT cells, the UVDE mutant cells did not exhibit a G2/M checkpoint response but delayed on the second cycle after 5 J/m2 (Fig. 3B and Fig. 9, which is published as supporting information on the PNAS web site). However, second-cycle delays were longer in the UVDE mutant than in WT cells. We conclude that NER intermediates are not sufficient to activate a G2/M checkpoint, and that UV lesions normally repaired by UVDE can induce second-cycle delays when carried into S phase.

Like human cells, fission yeast have two protein kinases that respond to DNA damage and delay mitosis. Chk1 is required for the G2/M DNA damage checkpoint response (33), and the Cds1 kinase is activated by DNA damage in S phase and can induce mitotic delay in response to nucleotide starvation (3437). To determine whether one of these kinases is required for second-cycle delay, we analyzed the responses of cds1 and chk1 null mutants to 5 J/m2 of UV. As shown in Fig. 3B, second-cycle delay in response to UV depends completely on chk1. In contrast, second-cycle delay is enhanced in the Δcds1 mutant. It is known that Cds1 contributes to survival in excision repair mutants (38). The longer second-cycle delays suggest that Cds1 functions after UV lesions are encountered in S phase.

Chk1 Mutants Die After Postreplication Checkpoint Failure.

Under the conditions of our experiments, the viability of Δchk1 cells in a colony-forming assay after 5 J/m2 (53 ± 4%) was considerably lower than that of irradiated WT cells (98 ± 4%). Because WT cells do not exhibit a G2/M checkpoint response at this dose, cell death cannot be explained by a G2/M checkpoint defect. Although 98.5% of Δchk1 cells completed the second cleavage after irradiation, only 52.5% completed the third cleavage (Fig. 4A). The percentage of cells able to complete a third cleavage after irradiation agreed closely with colony formation in several different experiments (Fig. 10, which is published as supporting information on the PNAS web site). Therefore, it appears that Δchk1 cells lose viability between the second and third cleavages postirradiation. Fission yeast chk1 mutants exhibit chromosomal abnormalities when DNA ligase function is compromised (33), and chk1 null mouse embryos exhibit gross chromosomal abnormalities in the absence of external perturbation (39, 40). To determine whether the death of UV-irradiated Δchk1 cells can be explained by chromosome damage, we examined the nuclear morphology of DAPI-stained cells (Fig. 4B). A number of abnormal nuclear morphologies were observed several hours after irradiation of Δchk1 cells, and these were classified into three groups (Fig. 4B and Fig. 11, which is published as supporting information on the PNAS web site). Cells with fragmented nuclei, unequal nuclear partition, or nuclei located near the division septum or cleavage furrow were scored as “cut cells.” This morphology has previously been observed in topoisomerase II mutants (41) and replication checkpoint mutants treated with HU (42). Cells with nuclear fragments located between two larger nuclear bodies were scored as containing “lagging chromosomes” (43). This phenotype has been observed after endonuclease-induced double-strand breaks and may reflect failed separation of synapsed sister chromatids or the presence of broken acentric chromosomes (44). Cells with the nucleus at the extreme end of the cell were observed only after irradiation and were scored as “tip cells.” These cells most likely represent cut cells that have completed cytokinesis. Samples from an asynchronous population were fixed at 20-min intervals after UV exposure and the morphologies scored. Comparison of Fig. 4 A and B shows that the nuclear abnormalities of Δchk1 cells first become evident at ≈3 h after 5 J/m2, as cells in the second cycle undergo division. Lagging chromosomes appear first, followed shortly by cut and tip cells. Most of the cut cells failed to complete nuclear separation, suggesting a possible topological linkage between sister chromatids (10/13 cells at 4 h and 13/15 at 5 h). Notably, UV did not cause nuclear abnormalities during the first division after 5 J/m2. After 25 J/m2, lagging chromosomes appeared transiently during the first division and reappeared at a later time along with cut and tip cells. These data indicate that both the loss of viability and the formation of gross chromosome damage in Δchk1 cells occur only after UV lesions have been propagated through S phase and are not the result of a G2/M checkpoint failure.

Fig. 4.
Chk1 maintains viability and prevents gross chromosome damage after lesions are carried into S phase. (A) The percentage of Δchk1 cells that had undergone cleavage is plotted as a function of time after irradiation with a UV dose of 5 J/m2. The ...

UV-Irradiated Budding Yeast Cells Exhibit a Postreplication Checkpoint.

To determine whether postreplication delay is evolutionarily conserved, we carried out time-lapse experiments with the budding yeast Saccharomyces cerevisiae. An asynchronous population of cells was UV-irradiated, and cell-cycle progression was followed using bud emergence as a landmark. After a dose of 5 J/m2, emergence of the first bud is relatively unaffected, and emergence of the second bud is delayed (Fig. 5 and Fig. 12, which is published as supporting information on the PNAS web site). Because budding is normally initiated at the beginning of S phase, this finding suggests that cell-cycle delay does not occur until after the onset of DNA replication. A UV dose of 5 J/m2 does not slow the progress of WT budding yeast cells from G1 through S phase (18). Thus, our data are most consistent with a postreplication delay similar to that observed in fission yeast. We were able to verify that most of the cells in the population, not just a susceptible subset, delayed emergence of the second bud (Figs. 13 and 14, which are published as supporting information on the PNAS web site). As expected, some late-budding cells expected to be in S phase at the time of irradiation delayed emergence of the first bud (Fig. 14). We found that cell-cycle delay after 5 J/m2 requires the checkpoint gene mec3 (data not shown). Thus, postreplication UV delay is conserved in an organism that diverged from fission yeast >300 million years ago.

Fig. 5.
The postreplication checkpoint is conserved in budding yeast. Asynchronous haploid budding yeast (ATCC 201389, MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) were irradiated and observed by time-lapse microscopy for two subsequent ...


The UV-induced cell-cycle response of yeast cells is typically studied using short (<5-min) UV doses of ≥25 J/m2. These conditions result in fluxes in vast excess of sunlight exposure (0.1 J/m2 per min). There are little published data at UV doses <25 J/m2, and it has been suggested that there may be a lack of cell-cycle delay in yeast at these doses because of the rapid completion of DNA repair (45, 46). We have found that this is not the case, and that there is a previously undetected checkpoint response that persists for hours after 5 J/m2, a dose expected to introduce a lesion burden comparable to continual sunlight exposure. At this dose, cell-cycle progression is delayed only after lesions are carried through S phase. Thus, fission yeast irradiated in G2 phase do not trigger a checkpoint response until the second cycle after irradiation. G1 and S phase cells also delay on the second cycle, because these stages occur immediately before cytokinesis in fission yeast. The second-cycle delay of fission yeast irradiated in G1 and S phase would occur on the first cycle in organisms in which S phase occurs after cytokinesis (e.g., most eukaryotes).

The term “G2/M checkpoint” is frequently used to describe a mitotic delay that occurs upon the introduction of DNA damage in G2 phase (5, 21), whereas we observed that fission yeast irradiated in G2 phase do not delay the cell cycle until passing the subsequent S phase. Although the delays we observed require the same genes as the G2/M checkpoint response (chk1, as well as rad3, rad26, crb2, rad9, and rad17; data not shown), it is clearly not a G2/M checkpoint response. Because there was no cell-cycle delay within G1 in either budding or fission yeast, the response cannot be termed a G1/S checkpoint. The response shares the most similarities to an intraS checkpoint, like that observed in budding yeast upon exposure to reactive oxygen species and alkylating agents, in that it requires DNA replication and does not occur in G1 or G2 phases (18, 47, 48). However, the intraS checkpoint is marked by a delay in early or mid-S phase, whereas we found that the checkpoint response to UV occurs after bulk DNA synthesis is completed, and that the rate of genome duplication is unaffected by the presence of UV lesions. This distinction has important consequences when considering the physiological role of the response as discussed below. We favor the term “postreplication checkpoint” to describe the response, because, like the repair pathway of the same name, it occurs after the replication of DNA containing UV lesions.

Although there are many studies on the eukaryotic checkpoint response to UV, the function of cell-cycle delay has remained somewhat obscure. It has been suggested that the DNA damage checkpoint monitors the presence of NER intermediates in UV-irradiated G1 and G2 cells (5, 18–22, 49). However, other studies have suggested that the DNA damage checkpoint monitors pyrimidine dimers in the absence of NER (50, 51). After a UV dose of 5 J/m2, which is expected to introduce ≈400 pyrimidine dimers per cell, we found that the cell-cycle response of budding yeast and ΔUVDE fission yeast, both of which excise UV lesions exclusively through NER, occurs only after passage through S phase. We have also shown that the fission yeast cell-cycle response requires the initiation of DNA replication at this dose, and that phosphorylation of chk1 is minimal in G2 phase (Fig. 15, which is published as supporting information on the PNAS web site). These data indicate that neither UV lesions themselves nor NER intermediates are sufficient to elicit a cell-cycle response. Nonetheless, long cell-cycle delays are seen at 5 J/m2 after the completion of bulk DNA synthesis, suggesting that the DNA damage checkpoint recognizes structures that arise after the encounter of replication forks with UV lesions.

Replication of UV-irradiated DNA in NER-defective budding yeast and Escherichia coli cells generates nascent DNA with an average size comparable to the distance between pyrimidine dimers (8, 10). This suggests that replication forks leave gaps across from lesions, a model recently supported by electron microscopy of DNA from UV-irradiated budding yeast cells (12). Like the postreplication checkpoint response we observed (Fig. 16, which is published as supporting information on the PNAS web site), UV-induced postreplication gaps are detectable after a UV dose as low as 1 J/m2 and have been shown to persist for hours after S phase (10, 11). It is thought that the checkpoint proteins required to activate Chk1 directly recognize gapped DNA structures (5256). Therefore, our data are most consistent with a model in which a postreplication checkpoint monitors the presence of postreplication gaps (Fig. 17, which is published as supporting information on the PNAS web site). Cells exhibiting a postreplication checkpoint do not delay on the subsequent cell cycle even after a dose of 25 J/m2 (Fig. 1F), so the checkpoint may be sufficiently sensitive to prevent postreplication gaps from being propagated through mitosis and into the subsequent S phase.

It has been suggested that the unwound DNA at stalled replication forks causes Chk1 activation when UV lesions are introduced in S phase. For example, Chk1 phosphorylation occurs when UV-irradiated plasmid DNA is incubated in Xenopus egg extracts at doses that cause an accumulation of unwound plasmid (23, 57). In this system, a UV dose of 500 J/m2 is required to activate Chk1, whereas the postreplication checkpoint response we report is detectable after 1 J/m2. This disparity suggests that the structures activating Chk1 in Xenopus extracts may be different from those in yeast cells. Fork stalling is not expected to play a major role under the conditions of our experiments. A UV dose of 5 J/m2 should induce about one pyrimidine dimer per fission yeast replicon, and the checkpoint response is detectable at one-fifth of this dose. As such, replication fork stalling would not be expected to prevent the completion of bulk DNA synthesis, because the fork initiated at the neighboring replication origin could complete the replicon within a matter of minutes to generate a postreplication gap.

It has generally been assumed that cell-cycle progression into mitosis and S phase in the presence of UV lesions is toxic to the cell; however, our data indicate this is not the case in WT or Δchk1 fission yeast. Virtually 100% of WT cells are viable after 5 J/m2, even though they carry UV lesions into S phase and mitosis (Fig. 10). ΔChk1 cells lose viability and exhibit chromosome damage only after they carry lesions into S phase and subsequently undergo mitosis. These data indicate that cell-cycle progression in the presence of UV lesions is not dangerous to the cells until they have been propagated into S phase. The morphology of some damaged Δchk1 cells indicates a failure of nuclear separation like that reported for topoisomerase II mutants, suggesting that a topological linkage between sister chromatids may persist as cells undergo mitosis. Although ≈50% of Δchk1 cells are inviable after a dose of 5 J/m2, gross chromosome damage is evident in only 6–8% of the population. This discrepancy suggests the presence of other forms of lethal damage to the cell, perhaps reflecting the loss of essential genetic information when a chromosome containing a postreplication gap is separated from its undamaged sister.

The appearance of G1/S and G2/M DNA damage checkpoint responses at high UV doses may reflect a double-strand break response, because excision of UV lesions at doses of 20 J/m2 and above has been shown to cause double-strand breaks (DSBs) in stationary phase fission yeast cells and E. coli (58, 59). Alternatively, it is possible that a threshold level of NER intermediates is needed to activate Chk1, or that a large number of lesions causes a depletion of nucleotides, giving time for an exonuclease to process gaps into larger structures or DSBs. Regardless of the mechanism of checkpoint activation at high UV doses, the physiological relevance of these responses remains unclear. An allele of the budding yeast Mec1 checkpoint gene has been found that lacks G1/S and intraS checkpoint responses but has almost no UV sensitivity (60). We have shown that 5 J/m2 does not elicit a G2/M checkpoint response but results in ≈50% lethality in Δchk1 cells, and that most of the gross chromosomal damage after a high dose of UV does not form until cells have propagated lesions through S phase. These results suggest that the UV sensitivity of fission yeast G2/M DNA damage checkpoint mutants is primarily the result of their postreplication checkpoint defect.

The postreplication checkpoint occurs in both budding and fission yeast. In addition, the E. coli SOS response is analogous in several ways to the eukaryotic DNA damage checkpoint response and also requires DNA replication for induction by UV (61). A cell-cycle response consistent with a postreplication checkpoint has been reported to occur after exposure of mammalian cell lines to low UV doses (62, 63). Given this degree of conservation, we suggest that postreplication delay may be an important cell-cycle checkpoint response to UV irradiation in higher eukaryotes as well.

Materials and Methods

Standard Yeast Methods.

Fission yeast strains were grown in YE6s medium, with the exception of the REP41x-cdc18 strain, which was grown in EMM6s minimal medium (64). Thiamine was added at a concentration of 5 μg/ml to repress cdc18. Budding yeast were grown in YPD rich medium (65). Flow cytometry was performed as described (30). Fission yeast mutants were isogenic to the WT strain (h-, leu1–32, and ura4-D18).

Time-Lapse Microscopy.

A detailed protocol is available in Supporting Text, which is published as supporting information on the PNAS web site. Fission and budding yeast cells were grown to log phase in liquid medium and plated on 1% agarose Petri dishes containing the same batch of medium. Cells were covered with a quartz slide and imaged at 2-min intervals by using standard light microscopy and a custom algorithm that holds cells in focus. Temperature was maintained at 30°C unless otherwise indicated. Time-lapse series were analyzed by using the Openlab software (Improvision, Lexington, MA).


UV irradiation (254 nm) was delivered from a Stratalinker by using mercury vapor bulbs and attenuated at all times with a neutral density filter (5% transmittance).

Supplementary Material

Supporting Information:


We thank Drs. Akira Yasui and Susan Forsburg for sharing fission yeast strains.


NERnucleotide excision repair.


The authors declare no conflict of interest.


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