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Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, California
Received 3 August 2004/ Returned for modification 1 September 2004/ Accepted 8 September 2004
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ABSTRACT |
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cells is not due to checkpoint regulation, but instead, directly reflects the decreased abundance of S-phase CDK, as Clb6 activates late origins when its dosage is increased. Moreover, the viability of clb5 cells depends on Rad53. Activation of Rad53 by either Mrc1 or Rad9 contributes to the survival of clb5 cells, suggesting that both DNA replication and damage pathways are responsive to the decreased origin usage. These results suggest that reduced origin usage leads to stress or DNA damage at replication forks, necessitating the function of Rad53 in fork stabilization. Consistent with the notion that decreased S-CDK function creates stress at replication forks, deletion of RRM3 helicase, which facilitates replisome progression, greatly diminished the growth of clb5 cells. Together, our findings indicate that deregulation of S-CDK function has the potential to exacerbate genomic instability by reducing replication origin usage. |
INTRODUCTION |
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The pre-replicative complex (pre-RC) assembles at and governs the function of each replication origin (reviewed in reference 4). Activation of the pre-RC results in its conversion into two, divergent, replication fork complexes (replisomes) through the recruitment of additional DNA synthesis factors, such as Cdc45 and DNA polymerases. Pre-RC activation requires the function of cyclin-dependent kinase (CDK), which consists of a catalytic subunit (Cdk1) controlled by a cyclin subunit whose expression and stability are cell cycle regulated. In Saccharomyces cerevisiae, two B-type cyclins, Clb5 and Clb6, accumulate at the beginning of each cell cycle and stimulate the origin activation function of Cdk1, commencing S phase (9, 15, 30).
Clb5-Cdk1 appears to account for the majority of S-phase CDK function as mutation of CLB5, but not CLB6, results in a lengthened S phase, suggesting that fewer pre-RCs are activated in CLB5-deficient cells (9, 30). Consistent with this, late origin activation is specifically deficient in the absence of CLB5, whereas deletion of CLB6 has no apparent effect on DNA synthesis or late origin firing (8). Thus, Clb5 appears capable of activating a full complement of origins. In the absence of both Clb5 and Clb6, S phase becomes dependent on later-expressed cyclins Clb1 to -4 and begins significantly later than in either single mutant (30). Together, these results suggest that Clb6 can drive pre-RC activation, but only early in S phase. Whether this reflects absence of Clb6 during late S phase, a specific ability of Clb5 to target late origins for activation (perhaps due to the local chromatin structure or chromatin-specific substrates), or a requirement for a higher threshold of Clb-Cdk1 activity for late origin firing remains unresolved.
An implied consequence of decreased late origin function in clb5 cells is an increase in average replicon size, particularly in later-replicating regions of the genome. This apparently has the effect of delaying completion of chromosomal DNA replication, which may elicit a checkpoint-mediated mitotic delay (S-M checkpoint). A second possible consequence of increased replicon size is an increased propensity for replisome defects, which may result in replication stress or DNA damage, either of which may elicit an intra-S checkpoint response (reviewed in reference 24). These cell cycle checkpoints in response to replication defects or DNA damage involve signaling pathways that sense the presence of a replication defect or damaged DNA and initiate cellular responses aimed at rectifying the problem. The Mec1 kinase plays a major role in sensing sites of DNA damage or replication stress, apparently through recruitment by Ddc2 to such sites and activating the effector kinases, Rad53 and Chk1 (reviewed in reference 19). The activation of these effector kinases appears to involve their phosphorylation by Mec1, which is mediated by one of two adapter proteins, Rad9 and Mrc1. Rad9 mediates the activation of Rad53 and Chk1 in response to DNA damage, whereas Mrc1 mediates the activation of Rad53 in response to replication inhibition. Activated Chk1 and Rad53 together delay mitotic progression, providing time for the cell to cope with the insult, while Rad53 also induces DNA repair genes, increases deoxyribonucleotide concentrations, inhibits late origin activation, and stabilizes replisomes. In combination, these responses are critical for the viability of cells subjected to replicative stress or DNA damage.
Recent studies argue that replisome stabilization by Rad53 is of paramount importance in replicating cells exposed to a replication inhibitor (e.g., hydroxyurea [HU]) or DNA-damaging agent (e.g., methyl methanesulfonate [MMS]) (5, 18, 34, 37). In the absence of MEC1 or RAD53, aberrant replication fork structures are detected at stalled replisomes; these cells rapidly lose viability and are unable to resume DNA synthesis, even after the inhibitory compound is removed. Mrc1 appears to function exclusively during S phase and is the primary mediator of Rad53 function in cells blocked in replication by HU, whereas Rad9 functions in DNA damage responses throughout the cell cycle. However, overlap between these two pathways exists, as deletion of both MRC1 and RAD9 creates cells that are much more sensitive to HU than either single mutant and have comparable sensitivity to cells lacking RAD53 (1). This indicates either that Rad9 can substitute for Mrc1 in relaying a replication fork stress signal from Mec1 or, more likely, that replication stress creates DNA damage detectable by Mec1 and Rad9, thereby activating Rad53's replisome stabilization function. Neither the mechanism(s) nor a target through which Rad53 acts to stabilize replication forks is known.
The similar effects on late origin firing caused by CLB5 deletion or by intra-S checkpoint activation suggested the possibility that loss of late origin firing in clb5 cells is the result of checkpoint activation. Decreased origin activation due to decreased S-CDK levels should increase the average length of DNA synthesized by each replisome, possibly creating a requirement for Rad53's stabilization function. Alternatively, lack of Clb5 could cause an inherent defect in replisome function (independent of replicon size) that stimulates checkpoint activation. To determine whether the loss of Clb5 function is associated with a checkpoint, we have examined the effects of CLB5 deletion on checkpoint signaling and the effects on origin function and cell viability of disrupting different checkpoint functions in clb5 mutant cells.
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MATERIALS AND METHODS |
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::kanMx locus. To construct pclb5-CLB6, the CLB6 open reading frame was amplified by PCR, sequenced to confirm that no mutations were introduced, and inserted into plasmid pC5C2-3NF (6). For replacement of CLB5 by CLB6 at the native CLB5 locus, pclb5-CLB6 was digested with StuI and KpnI and transformed into clb5::URA3 cells; transformants were selected on 5-fluoroorotic acid and confirmed by PCR.
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Analysis of replication structures. Two-dimensional (2D) gel analysis was performed as described at http://fangman-brewer.genetics.washington.edu, except that replication structures were enriched on benzoylated naphthoylated DEAE-cellulose (Sigma). Fifty micrograms of DNA was digested with NcoI and BamHI for analysis of ARS603 (3.6 kb), ARS603.5 (2.6 kb), and ARS1011 (2.4 kb) or with EcoRI for analysis of ARS305 (5.8 kb) and ARS1413 (5.3 kb). Samples being compared were run in a single large gel and transferred together to a single membrane for hybridizations. DNA probes were labeled with the MegaPrime DNA labeling kit (Amersham Pharmacia) and detected on a PhosphorImager (Storm 860; Molecular Dynamics).
Analysis of Rad53. Protein extracts were prepared by trichloroacetic acid precipitation as described previously (10) and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 10% polyacrylamide (77:1) gels. 16B12 (Covance-BabCo) anti-hemagglutinin (HA) antibody was used at 1:5,000 in conjunction with SuperSignal Elisa Femto (Pierce) for quantitative chemiluminescent detection of protein with a ChemiDoc XRS 170-8070 (Bio-Rad) and Quantity One Analysis software (Bio-Rad). Quantification of Rad53 phosphorylation appears to indicate a higher proportion of phosphorylated Rad53 than visual inspection of the images suggests; however, this is apparently due to the presence of various Rad53 species with reduced mobility that do not migrate as distinct bands because quantification of multiple subsaturated exposures of the blots yielded identical results.
Microscopy. An Olympus IX71 microscope with a x60, 1.4-NA PlanApo oil immersion objective was used. Green fluorescent protein (GFP) fluorescence was detected using a Chroma fluorescein isothiocyanate filter set (excitation, 485/20 nm; emission, 515/30 nm), and the images were captured with a Roper Scientific DV42059 camera. The images were visualized using SoftWoRx software (Applied Precision). Fields of cells were photographed in several focal planes through the nuclei, until the foci were visible. Exposure time, gain, and binning functions were held constant. The percentage of cells with Ddc2 foci and cellular morphologies were determined from analysis of at least 100 cells at each time point.
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RESULTS |
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cells is not due to checkpoint inhibition.
In a previous study of replication origin function in clb5 cells, initiation of late origins was greatly diminished, whereas initiation of early origins was essentially normal (8). We hypothesized that the failure of late origin activation in clb5 cells results from checkpoint inhibition of late origins, which is dependent on Mec1, Rad53, and Mrc1 (1, 29, 33). To test this idea, we analyzed initiation of three late (ARS603, ARS1011, and ARS1413) and two early (ARS305 and ARS603.5) replication origins of wild-type and clb5 strains and checkpoint-defective derivatives of these strains. We analyzed origin firing by examining replication initiation structures using 2D agarose gel (2D gel) electrophoresis of unsynchronized, cycling cells. This allows a determination of the overall frequency (efficiency) of origin usage because all replication structures occurring at the origin are represented as a composite (11).
Analysis of wild-type cells showed efficient activation of all of these origins based on the strong signals for "bubble" and "large Y" structures (Fig. 1A, indicated in the first ARS603 panel by filled and empty arrowheads, respectively). In clb5 mutants, initiations of ARS1011 and ARS603 were impaired severely as shown by the reduction in the "bubble" and "large Y" structures and the corresponding increase in the "small Y" structures (indicative of passive replication of the origin by a replication fork emanating from a flanking origin), particularly apparent at the apex of the "Y" arc (Fig. 1A, double arrowhead in the second ARS603 panel). In contrast to ARS603 and ARS1011, we observed that initiation from late origin ARS1413 (and ARS501; data not shown) was reduced less in clb5 cells. Unexpectedly, we also observed reduced initiation of the early origin ARS603.5 in clb5 cells (Fig. 1A). However, as expected, initiation of the very-early-firing origin, ARS305 (and ARS607; data not shown), was unaffected by CLB5 deletion. Thus, in agreement with the results of the previous study, late origins initiate replication inefficiently in the absence of CLB5. However, in contrast to the previous study, we observed that loss of CLB5 has a more modest effect on some late origins and reduces initiation of at least one relatively early origin (Fig. 1A).
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cells. This allele, which contains mutations in all of its potential Mec1 phosphoacceptor sites, significantly reduces or eliminates Mrc1-mediated phosphorylation of Rad53, but does not have the replication defect associated with a null mutation of MRC1 (24). Elimination of the replication checkpoint did not increase activation efficiency of late origins in clb5 cells (Fig. 1A). The presence of Mrc1AQ did not appear to alter initiation of either early or late origins in clb5 or wild-type cells. This strongly suggests that the failure of late origin firing in clb5 cells is not due to inhibition by the intra-S replication checkpoint.
DNA-damaging agents (e.g., MMS) also can lead to inhibition of late origin firing (33). Thus, if loss of Clb5 function were creating DNA damage, Rad9 might be involved in relaying DNA damage signals from Mec1, leading to late origin inhibition. However, deletion of RAD9 did not increase late origin firing in clb5 cells (Fig. 1A). To eliminate the possible redundancy of Mrc1 and Rad9 in relaying signals from Mec1, we tested the effect of deleting MEC1. Deletion of SML1 suppresses the lethality of cells lacking MEC1 or RAD53 (45) and was used to enable analysis of these strains; its deletion did not otherwise affect the results. Analysis of late origin firing in clb5 mec1 sml1 cells showed that Mec1 does not block late origin firing in clb5 cells (Fig. 1A). All the origins initiated replication with normal frequencies in sml1 and mec1 sml1 cells (Fig. 1A). We also tested the effect of deleting TEL1 in addition to MEC1, because Tel1 can substitute, at least partially, for Mec1 in the activation of Rad53 (28, 39). As discussed in detail below, elimination of Mec1 and Tel1 severely compromised the proliferation of clb5 cells. Nevertheless, we were able to grow cultures of these cells and analyze replication structures (Fig. 1A). The results show that Tel1 does not inhibit late origin firing in clb5 mec1 cells. Thus, neither the DNA replication nor DNA damage checkpoints are involved in suppressing the activity of late origins in clb5 cells.
The DNA content of unsynchronized cells shows the distribution of the population of cells in different periods of the cell cycle, providing an indication of the relative durations spent in each cell cycle phase. Comparison of clb5 with wild-type cells shows an increase in the length of S phase in clb5 cells, as more cells have a DNA content between 1C and 2C (Fig. 1B). The proportions of S phase cells in cultures of mrc1AQ, rad9, mec1, and mec1 tel1 cells were similar to those in wild-type cells, suggesting no increase in the length of S phase in these checkpoint mutant cells (Fig. 1B). In contrast, the proportions of S-phase cells in cultures of clb5 mrc1AQ, clb5 rad9, clb5 mec1, and clb5 mec1 tel1 cells were similar to those in clb5 cells, indicating that the lengthened S phase of clb5 cells is not due to replication inhibition by these checkpoint functions (Fig. 1B). Together with the analyses of late origin function, these data demonstrate that neither replication nor damage checkpoints are responsible for the observed replication defects of clb5 cells.
Decreased S-CDK activity triggers Rad9-mediated activation of Rad53.
We had wished to directly address the role of Rad53 in origin regulation in clb5 cells; however, we found that the combined deletion of CLB5 and RAD53 was lethal (see below). This suggested that Rad53 was active in clb5 cells, possibly contributing to replisome stabilization, which is critical when cells experience replicative stress. One hallmark of Rad53 kinase activation is its phosphorylation in a Mec1-dependent manner (25, 28, 35). To determine whether Rad53 was activated in clb5 cells, we monitored Rad53 phosphorylation, which decreases its mobility in SDS-PAGE. Cells were synchronized in G1 with 
-factor and released into S phase. Every 12 min, we analyzed Rad53 mobility by SDS-PAGE and measured the proportion of all slower-migrating forms of Rad53 using quantitative luminescence detection, while monitoring S-phase progression by DNA content analysis.
In wild-type cells, Rad53 migrated as the unmodified form throughout the cell cycle, indicating no checkpoint signaling in these cells (Fig. 2A). However, we observed slower-migrating forms of Rad53 in cells lacking CLB5, indicating that checkpoint activation of Rad53 had occurred (Fig. 2A). Phosphatase treatment eliminated the slower-migrating forms, demonstrating they were phosphorylated forms of Rad53, and the slower-migrating forms were associated with increased autokinase activity (data not shown). Also as expected, the slower-migrating forms of Rad53 were dependent on Mec1, whereas deletion of SML1 did not affect the level of Rad53 phosphorylation in wild-type or clb5 cells (Fig. 2B). The proportion of Rad53 that was modified peaked during late S/G2 phase. For example, in clb5 cells, Rad53 phosphorylation peaked at 72 min (Fig. 2A), which was after the bulk of chromosomal DNA had been replicated (Fig. 2E). In clb5 sml1 cells, the pattern was similar: Rad53 phosphorylation peaked at 60 min (Fig. 2B), after the bulk of DNA synthesis had been completed (Fig. 2E). Although the absolute level of Rad53 phosphorylation in cells lacking Clb5 is significantly below that caused by treatment with high doses of a replication inhibitor or DNA-damaging compound typically used, their effects on chromosomal replication are also much more profound.
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clb6 cells, Rad53 phosphorylation peaked at 84 min (Fig. 2A), by which time DNA replication was complete or nearly so (Fig. 2E). Rad53 activation declined as these cells divided and entered G1 (Fig. 2A and E, 108 to 132 min). Together, these results suggest that the activation of Rad53 is a direct result of undergoing DNA replication in the absence of Clb5 function.
Because the completion of DNA synthesis is delayed in cells lacking CLB5, we considered the possibility that checkpoint activation might have resulted from attempted anaphase entry with incompletely replicated DNA, causing DNA damage. To determine whether Rad53 activation resulted from mitosis, we monitored Rad53 phosphorylation in the presence of nocodazole, which blocks spindle assembly and mitosis. Nocodazole arrest results in a modification of Rad53 that reduces its gel mobility, even in wild-type cells (Fig. 2C). Nevertheless, additional slower-migrating forms of Rad53 were observed in nocodazole-arrested clb5 cells (Fig. 2C, asterisk). Thus, Rad53 phosphorylation was observed in the presence of nocodazole, indicating that checkpoint activation did not result from anaphase entry. On the contrary, checkpoint activation appeared to delay cell division briefly in clb5 cells as large-budded cells persisted in the population at 96 and 108 min when most wild-type cells had divided (Fig. 3C); the emergence of unbudded and small-budded cells in the second cell cycle also was delayed relative to the wild type (Fig. 3C). Careful inspection of the DNA content profiles also points to a delay in cell division of clb5 cells (Fig. 2E). Whereas most wild-type cells had completed the second S phase by 120 min (note the 4C peak, in addition to the 2C peak and the lack of newly emerging cells with 1C DNA content), a significant number of clb5 cells continued to undergo cytokinesis at 120 to 132 min (note the populations of cells with <2C DNA content). Furthermore, this delay in cell division of clb5 cells was abolished by deletion of MEC1 (Fig. 2E).
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mrc1AQ cells was not reduced compared to that in clb5 cells (Fig. 2D). Instead, Rad9 is responsible for the majority of Rad53 activation in clb5 cells, because the proportion of phosphorylated Rad53 was diminished greatly in clb5 rad9 cells (Fig. 2D). These data suggest that the replication defect of clb5 cells produces aberrant DNA, which is detected by Mec1 and Rad9, leading to Rad53 activation.
Ddc2 localization suggests the presence of DNA damage in clb5 cells.
Cellular recognition of damaged DNA involves binding of Ddc2 to sites of DNA damage (20, 27). In cells expressing Ddc2 fused to GFP, DNA damage causes the accumulation of Ddc2-GFP into one or more brightly fluorescent foci (20). To address directly whether DNA damage results from loss of Clb5 function, we examined the cellular localization of Ddc2 in wild-type and clb5 cells released synchronously from G1. In wild-type cells, Ddc2-GFP was present throughout the nucleus and did not accumulate into distinct foci, regardless of cell cycle stage (Fig. 3A); the percentage of cells with at least one Ddc2-GFP focus was quantified (Fig. 3B). In clb5 cells, very few brightly fluorescent foci were observed prior to S phase or during early S (Fig. 3A and B, 0 and 24 min). As cells progressed farther into S, one or more foci of Ddc2-GFP were observed, suggesting that Ddc2-GFP recognized and associated with sites of DNA damage (Fig. 3A and B, 48 and 72; e.g., arrowhead). At 72 min, when the majority of cells were large budded, about half of the cells showed one or more Ddc2-GFP foci (Fig. 3B and C); fewer than 10% of large-budded wild-type cells showed a focus (Fig. 3B and C), and no such foci were observed in cells lacking Ddc2-GFP (data not shown). Ddc2-GFP foci persisted in most clb5 cells at 96 and 120 min, when DNA content analysis suggested that DNA replication was complete (Fig. 3A and B, and see Fig. 2E for DNA content). Ddc2-GFP foci were observed very infrequently in unbudded cells, indicating that Ddc2-GFP disperses prior to cell division. This is consistent with repair of DNA damage prior to mitosis and cell division, as expected if a checkpoint were delaying mitotic progression of clb5 cells. Analysis of unsynchronized cells confirmed the correspondence between cell cycle stage and Ddc2-GFP focus formation (data not shown). Together with the analysis of Rad53 phosphorylation, these results provide strong evidence that DNA damage occurs during S phase and peaks during late S and G2 in clb5 cells.
Viability of clb5 mutants depends on Rad53 activation.
As mentioned previously, deletion of Rad53 in clb5 cells is lethal, which contrasted sharply with the viability of clb5 mec1 cells. In our initial analysis at 23°C, dissection of a diploid heterozygous for clb5 and rad53 (homozygous for sml1) yielded no viable clb5 rad53 spores, whereas each of the single mutants was obtained at normal frequencies (Table 2). In contrast, dissection of a diploid heterozygous for clb5 and mec1 (homozygous for sml1) yielded similar numbers of clb5 mec1 segregants to those of either single mutant (Table 2). This result suggested that the function of Rad53 in sustaining the viability of clb5 cells could function independently of the Mec1-dependent signaling pathways, which normally lead to stimulation of Rad53's kinase activity. In subsequent analyses of spore viabilities at 30°C, we again found lethality of combined CLB5 and RAD53 deletion (Table 2 and Fig. 4A). However, we found a significant requirement for Mec1 in the viability of clb5 cells at 30°C; spore viability of clb5 mec1 cells was about 40% that of either single mutant or the wild type (Table 2). Furthermore, the very small colony size of clb5 mec1 cells indicated a growth defect at 30°C (Fig. 4B). Together, these results suggest a greater requirement for Mec1 in sustaining the viability of clb5 cells at the higher temperature.
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rad53-11 (mec2-1) cells from tetrad dissections performed at 23°C, although at only about 25% of the frequency of either single mutant or wild-type cells (Table 2). We recovered no viable clb5 rad53-11 cells at 30°C (Table 2). Rad53-11 appears to be defective in undergoing Mec1-dependent phosphorylation (35). Thus, the viability (albeit poor) of clb5 rad53-11 cells at 23°C and the much better viability of clb5 mec1 cells at 23°C than at 30°C together support the idea that Mec1-mediated activation of Rad53 is required at 30°C, but less critical for viability at 23°C.
To address further the requirement for Rad53 activation by upstream kinase signaling, we analyzed the contributions of Rad9 and Mrc1 to the viability of clb5 cells. Individually, neither Rad9 nor Mrc1 was required for maintaining the viability of clb5 cells. Dissection at 30°C of a diploid heterozygous for clb5, rad9, and mrc1AQ (homozygous for sml1) yielded similar numbers of clb5 mrc1AQ and clb5 rad9 haploid segregants as any of the single mutant strains (Table 2). And the growth rate of the clb5 mrc1AQ and clb5 rad9 double mutants was similar to that of clb5 cells (Fig. 4C). However, elimination of both Rad9 and Mrc1 checkpoint functions significantly reduced the growth rate of clb5 cells compared with any of the single or double mutant strains, although spore viability was not decreased (Table 2 and Fig. 4C). These results suggest that Mrc1- and/or Rad9-dependent Rad53 activation pathways function in response to loss of Clb5 function.
In view of the lethality of clb5 rad53 cells and poor viability of clb5 mec1 cells at 30°C, we found the spore viability of clb5 rad9 mrc1AQ surprising. However, it appears that Mrc1AQ retains some ability to activate Rad53 (because rad9 mrc1AQ SML1 cells are viable) (23), so we tested the effect of MRC1 deletion. At 30°C, deletion of MRC1 reduced the growth rate of clb5 cells, suggesting that checkpoint signaling through Mrc1 occurs in clb5 cells (Fig. 4D). Although the spore viability of clb5 mrc1 cells was not reduced, we did not recover viable clb5 rad9 mrc1 spores, suggesting that residual function of mrc1AQ maintained the viability of clb5 rad9 mrc1AQ cells (Table 2 and Fig. 4D). At 23°C, we recovered viable clb5 rad9 mrc1 spores; however, their viability was reduced significantly as only about half as many were recovered as the double mutant strains (Table 2). Together, these results strongly suggest that the function of Rad53 in sustaining the viability of clb5 cells requires activation through a checkpoint-signaling pathway that involves Rad9 and/or Mrc1. Cells lacking Mrc1 have a replication defect, which is manifested by a slower progression through S phase (1), so we cannot rule out the possibility that the lethality of clb5 rad9 mrc1 cells derives at least in part from an enhancement of the clb5 replication defect by the mrc1 replication defect. Nevertheless, the poor growth of clb5 rad9 mrc1AQ cells and lethality of clb5 rad9 mrc1 cells are consistent with the effect of MEC1 deletion, in that the viability of clb5 cells at 30°C depends on an intact Rad53 activation pathway.
Tel1 contributes to the viability of clb5 cells in the absence of Mec1.
The reduced dependence on Mec1 of clb5 cells at 23°C might indicate a less-severe replication defect due to loss of CLB5 at 23°C and as a consequence a diminished checkpoint signal. However, Mec1-mediated phosphorylation of Rad53 occurred in clb5 cells to a similar degree at 23 and 30°C (data not shown), indicating that the replication defect(s) of clb5 cells elicited a checkpoint response under both conditions. As deletion of RAD53 but not MEC1 was lethal at 23°C, the results argue that Rad53 can sustain the viability of clb5 cells even without stimulation by Mec1. However, the decreased viability of clb5 rad9 mrc1 cells suggests that stimulation of Rad53 kinase activity is important. Although Mec1 plays a predominant role in the activation of Rad53 in DNA damage and replication checkpoints in S. cerevisiae, yeast cells contain the closely related protein Tel1, which can partially substitute for Mec1 in its absence, suggesting that these two proteins share some overlapping functions in checkpoint responses (19). Thus, it was possible that in the absence of Mec1, Tel1 was responsible for stimulating the critical function of Rad53 in maintaining the viability of clb5 cells. If correct, elimination of TEL1 should reduce the viability of clb5 mec1 cells at 23°C.
Dissection at 23°C of a diploid heterozygous for clb5, mec1, and tel1 (homozygous for sml1) yielded similar numbers of clb5 tel1 haploid cells to those by either single mutant (Table 2). Thus, like Mec1, Tel1 alone is not required for the viability of clb5 cells. As predicted, the clb5 mec1 tel1 triple mutant strain showed significantly reduced viability; only about 60% as many clb5 mec1 tel1 spores were recovered as compared with mec1 tel1 spores (Table 2 and Fig. 4E). Also, the viable clb5 mec1 tel1 spores formed much smaller colonies than either the clb5 mec1 or clb5 tel1 strains. The mec1 tel1 cells formed very small colonies even in the presence of CLB5, so it is not possible to unambiguously ascribe the reduced growth rate of clb5 mec1 tel1 cells to loss of checkpoint function. Nevertheless, the results are consistent with Tel1 contributing to the viability and robust growth of clb5 mec1 cells at 23°C. The reduced viability at 30°C of clb5 mec1 cells suggests that Tel1 can substitute only partially for Mec1 at 30°C. Tel1 likely contributes to the viability of clb5 mec1 cells by stimulating the activity of Rad53 through phosphorylation, as overproduction of Tel1 partially restores Rad53 phosphorylation (in HU- or MMS-treated cells) in the absence of Mec1 (28, 39). Although we did not detect phosphorylation of Rad53 (based on a mobility shift) in clb5 mec1 cells (Fig. 2B), it is possible that Tel1-mediated activation of Rad53 can occur without affecting the mobility of Rad53 or that the amount of phosphorylated Rad53 in clb5 mec1 cells is below the level of detection in our assay, but sufficient for viability.
In summary, the analyses of clb5 cells lacking both Rad53 activation pathways (Mec1 and Tel1 or Mrc1 and Rad9) support the idea that Rad53 activation contributes to the viability of clb5 cells. However, none of these mutant combinations completely phenocopies the effect of RAD53 deletion, suggesting that a Mec1- and Tel1-independent function of Rad53 also is important for the viability of clb5 cells. A Mec1- and Tel1-independent role of Rad53 in the regulation of histone levels has been described recently (13). As deletion of one of the two copies of histone H3 and H4 genes partially suppressed the slow growth, DNA damage sensitivity, and elevated chromosome loss phenotypes of rad53 strains in the previous study, we tested whether the more severe phenotype of clb5 rad53 cells (compared with clb5 cells completely lacking factors that activate Rad53) derived from defective regulation of histone levels. We dissected spores of a diploid strain heterozygous for clb5, rad53, and hht2-hhf2 (one copy of the histone H3 and H4 genes). As before, we recovered no clb5 rad53 cells; however, we did recover viable clb5 rad53 hht2-hhf2 cells, although only at about one-third the frequency of wild-type or most single mutant cells (Table 2). In addition, the clb5 rad53 hht2-hhf2 cells grew significantly more slowly than wild-type cells (data not shown). These data are consistent with the conclusion that the lethality of clb5 rad53 cells is due in significant part to loss of Rad53 checkpoint function (as in the clb5 cells lacking the Rad53 activation pathways). Furthermore, the more severe phenotype of clb5 rad53 cells appears to be related to defective regulation of histone levels associated with loss of Rad53 function.
Rrm3 helicase is required for robust growth of clb5 cells.
Replication forks pause at numerous sites throughout the genome, such as tRNA genes, ribosomal DNA (rDNA), and centromeres (7, 14). Replication pause sites appear to be susceptible to DNA damage. Although its mechanism is not fully understood, Rrm3 helicase appears to facilitate the progression of replisomes through potential pause sites, as deletion of RRM3 increases fork pausing at many sites throughout the genome (14). Apparently as a consequence of increased fork stalling, DNA damage occurs and generates a checkpoint response that is required for the viability of rrm3 cells (14, 38). The increased replicon size of clb5 cells may enhance replicative stress by increasing the number of pause sites encountered by the average replisome because of the reduced number of converging replication forks. If true, clb5 cells might be expected to show a critical dependence on Rrm3 to facilitate progression of these replisomes and minimize DNA damage. To address whether Rrm3 contributes to the stability of replication forks in cells lacking Clb5 function, we determined the viability and growth characteristics of clb5 cells lacking RRM3 by dissection of a diploid heterozygous for clb5 and rrm3. Cells lacking Clb5 and Rrm3 grew very poorly, forming only microcolonies after 3 days of growth at 30°C, whereas each single mutant formed robust colonies only slightly smaller than wild type (Fig. 5). The very poor growth of clb5 rrm3 cells is consistent with the idea that replication fork stress results from the loss of Clb5 activity and suggests that Rrm3 plays an important role in stabilizing replisomes in clb5 cells.
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mutant cells.
The replication defect(s) and checkpoint activation resulting from loss of Clb5 function may be due to a decrease of total Clb-Cdk activity present during S phase or due to loss of a Clb5-specific function. To determine whether low abundance of Clb6-Cdk limits its ability to drive late origin activation, we introduced an extra copy of CLB6 under control of the CLB5 promoter into clb5 cells (clb5::CLB6); the CLB5 and CLB6 genes are normally expressed with indistinguishable cell cycle timing (30). In the clb5 cells, expression of the extra copy of CLB6 significantly increased late origin firing (Fig. 6A). Interestingly, cells expressing only the single copy of CLB6 from the CLB5 promoter (clb5::CLB6 clb6 cells) exhibited more efficient late origin activation than clb5 cells, which have CLB6 under its own promoter (Fig. 6A). This result suggests that expression from the CLB5 promoter produces more Clb6 protein. The increased late origin activation resulting from increased CLB6 dosage argues that a low abundance of Clb6, rather than a Clb5-specific function, explains the defective late origin firing of clb5 cells.
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cells in S phase was reduced significantly compared with clb5 cells and was similar to that of wild-type cells (Fig. 6B). Increased Clb6 dosage also reduced the level of Rad53 activation. Particularly striking is the lack of increase in Rad53 phosphorylation that occurs during late S phase in clb5 cells (Fig. 6D and E). In clb5 cells expressing an extra copy of CLB6, there is a low basal level of Rad53 phosphorylation throughout the cell cycle, but no increase during S phase. Expression of only the CLB5 promoter-driven copy of CLB6 also reduced Rad53 activation, though less so (Fig. 6E). Together, these data are consistent with the notion that loss of CLB5 results in decreased S-CDK function during mid- to late S phase, impairing the activation of late-firing replication origins and timely genome duplication; as a result, the DNA damage checkpoint is activated. The DNA damage response is a direct result of decreased origin activation as increased S-CDK levels are accompanied by increased late origin firing and decreased Rad53 activation. |
DISCUSSION |
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cells. Rad9-dependent Rad53 phosphorylation and the localization of Ddc2 into foci occur as a result of undergoing S phase in the absence of Clb5 (Fig. 2 and 3). Furthermore, robust viability of clb5 cells requires Rad53 and at least one functional Rad53 activation pathway (Fig. 4 and Table 2). In spite of this essential checkpoint response, the lack of late origin firing in clb5 cells is not caused by either a DNA replication or DNA damage checkpoint (Fig. 1). Instead, checkpoint activation appears to result from the lack of late origin firing, because Rad53 and Ddc2 activation increases during late S and G2 after most genome replication is complete (Fig. 2 and 3). In addition, increasing the dosage of Clb6 suppressed the replication defects of clb5 cells, as well as the checkpoint activation, demonstrating that both are the direct result of decreased S-CDK function (Fig. 6).
Clb5 and Clb6 have similar replication origin specificities.
The original observation that clb5 and clb6 mutants exhibit different origin firing characteristics gave rise to at least two models postulating differences in the functions of Clb5 and Clb6 (8). In the first, Clb5-Cdk1 and Clb6-Cdk1 have equivalent substrate specificities, but different levels of associated kinase activity, either due to lower abundance of Clb6 during late S or to a weaker ability of Clb6 to stimulate Cdk1, in which case late origins are postulated to require a higher threshold of Cdk activity due to late-determining factors. In the second model, Clb5-Cdk1 and Clb6-Cdk1 have distinct substrate specificities such that Clb5-Cdk1, but not Clb6-Cdk1, is able to phosphorylate and stimulate the replication function of a late-origin-specific substrate. We proposed a third alternative in which intra-S checkpoint activation caused inhibition of late origin firing.
Our results clearly eliminated the hypothesis that checkpoint regulation blocks late origin firing in clb5 cells. Mutation of factors required to block late origin firing in response to replication defects or DNA damage did not enable late origin firing in clb5 cells (Fig. 1A). However, increased dosage of CLB6 did enhance late origin firing in clb5 cells, demonstrating that Clb6 is capable of activating late origins (Fig. 6A). Importantly, increased CLB6 dosage also restored the wild-type replication rate (Fig. 6B and C) and significantly reduced Rad53 activation (Fig. 6D and E). Together, these results argue that the replication defect(s) and resulting checkpoint response of clb5 cells are due to a general decrease in S-CDK activity, rather than loss of a Clb5-Cdk1-specific function. We note, however, that increased CLB6 dosage did not fully restore late origin firing (compare Fig. 6A with 1A) or eliminate Rad53 activation (Fig. 6D and E). Although this may reflect still insufficient CLB6 dosage, we cannot exclude the possibility that one or more Clb5-specific substrates exist, whose lack of phosphorylation in clb5 cells is at least partly responsible for these residual defects.
Reduced S-CDK function causes DNA damage.
In the absence of CLB5, late origins initiate replication inefficiently, increasing average replicon size. This increase is likely to be particularly dramatic in large, late-replicating regions of the genome, some of which are hundreds of kilobases in length (26). Unusually large replicons may have an increased propensity for defects in replication fork progression due to normal fork impediments or limited processivity, leading to DNA damage. Hundreds of sites in the genome present obstacles or potential obstacles to fork progression, including tRNA genes, rDNA repeats, and unfired replication origins (7, 14, 41). A reduction in replication origin usage not only creates more potential pause sites at each unfired origin but also reduces the number of replication forks relative to the number of pause sites. Hence, the amount of time a fork spends at pause sites before a converging fork arrives may increase, which in turn may increase the likelihood of replication fork collapse and associated DNA damage. The effect of RRM3 deletion is consistent with the notion of increased replisome stress in the absence of Clb5; deletion of RRM3 seriously impaired the growth of clb5 cells (Fig. 5). Because loss of Rrm3 significantly increases replisome pausing and causes DNA damage, its loss probably exacerbates replication stress and/or resulting DNA damage of clb5 cells.
The proposition that decreased S-CDK function creates replication stress at forks is also supported by the critical importance of Rad53-activating pathways in the proliferation of clb5 cells (Table 2 and Fig. 4). As Rad53 stabilizes stressed replication forks, the loss of viability resulting from RAD53 disruption in clb5 cells is consistent with replisome stress or instability. Because Rad53 activation primarily occurs late in S phase and is dependent on Rad9, which mediates DNA damage signals, DNA damage appears to be the primary signal for Rad53 activation in clb5 cells (Fig. 2). The formation of Ddc2-containing foci in late S and G2 also directly points to the presence of DNA damage in cells that undergo S phase in the absence of Clb5 function (Fig. 3). We do observe a low level of Rad53 phosphorylation in G1-synchronized cells lacking Clb5 that might reflect a low level of Rad53 phosphorylation in all clb5 cells in a population. However, we think it is more likely to reflect a persistent DNA damage signal in a fraction of cells, because Ddc2 foci were observed only in a small fraction of unbudded cells.
In addition to stabilizing replication forks through stimulation of Rad53, DNA replication and damage checkpoints delay cell cycle progression through Mec1- and Rad9-dependent activation of Chk1. Hence, the viability of clb5 cells might depend on Chk1-dependent delay of mitotic entry preventing the lethal segregation of incompletely replicated DNA. There appears to be a delay in mitotic progression in clb5 cells, as cell division is delayed (Fig. 2E and 3C). This mitotic delay may allow the accumulated mitotic cyclins to activate remaining unfired origins and hasten completion of genome replication, while also permitting time for repair of any DNA damage. Nevertheless, mitotic delay does not appear to constitute the vital checkpoint function in clb5 cells, because deletion of RAD9 or RAD9 and CHK1 (Table 2 and Fig. 4C and D; data not shown for CHK1) did not affect the growth or viability of clb5 cells to the same extent as RAD53 deletion, which does not eliminate the Chk1-mediated mitotic delay (18). Thus, the vital function of checkpoint signaling in clb5 cells appears to be fork stabilization by Rad53, although a supplemental role by Chk1 and Rad53 in mitotic delay seems likely.
Indeed, a previous study analyzing the replication of a yeast artificial chromosome (YAC) containing a relatively large (170 kbp) region devoid of replication origins found evidence of a Rad9-dependent delay in cell division (40). Replication of the origin-free region caused an extension in the length of S phase, and loss of Rad9 led to mitotic instability of the YAC and DNA deletions within the origin-free region of the YAC. The apparently more critical role of Rad9 in replication of the YAC, which could be replicated only by a single replisome, compared to chromosomal replication in clb5 cells may be due to the ability of mitotic cyclins to enable completion of S phase by activating any unfired origins prior to mitosis, reducing the length of mitotic delay. The reduced requirement of Rad9 in clb5 cells may also reflect the involvement of Mrc1 because more replication forks likely are under stress in clb5 cells than in wild-type cells carrying the YAC.
Although the failure of late origin firing in clb5 cells cannot be attributed to checkpoint regulation, there appears to be a replication checkpoint response in clb5 cells, which probably occurs too late to inhibit origin function. Evidence for a replication checkpoint is based on the decreased growth rate of clb5 mrc1 cells and the strong effect on cell growth and viability of a combined MRC1 and RAD9 mutation compared with the effect of deleting only RAD9 (Fig. 4D and Table 2). Interestingly, mrc1AQ (and mrc1) appeared to increase the level of Rad53 phosphorylation in clb5 cells (Fig. 2D; data not shown for mrc1). Perhaps loss of Mrc1 checkpoint signaling at stressed forks allows for more DNA damage accumulation, resulting in a stronger Rad9-dependent Rad53 activation. Also, deletion of RAD9 did not entirely eliminate Rad53 phosphorylation, indicating that Mrc1 mediated Rad53 activation in clb5 rad9 cells (Fig. 2D). Indeed, the idea of replication fork stress due to increased replicon size might be expected to generate both a replication stress response through Mrc1, followed by a Rad9-mediated damage response, as DNA damage might result from replication stress or attempts to repair destabilized replication fork complexes. The occurrence of replication fork stress likely to elicit a replication checkpoint is supported by the critical role of Rrm3 in maintaining robust proliferation of clb5 cells (Fig. 5), as Rrm3 facilitates the progression of replication forks through sites of potential fork pausing.
Overlapping functions of Mec1 and Tel1 in response to replication stress.
A dual contribution by Mec1 and Tel1 may be involved in the checkpoint response to loss of Clb5 function. Deletion of MEC1 or TEL1 in clb5 cells had no effect on spore viability at 23°C; however, deletion of both significantly reduced viability and growth rate (Table 2 and Fig. 4E). Because telomeres are late replicating, loss of Clb5 function may have particularly strong effects on telomere replication. Considering the critical function of Tel1 in telomere maintenance, a role for Tel1 in sensing defective telomere replication in clb5 cells and activating Rad53 is plausible.
A related explanation for the dual involvement of Mec1 and Tel1 lies in the types of DNA damage that they may primarily sense and respond to. Association of Mec1 with chromatin involves interactions with the single-stranded DNA binding protein RPA, suggesting that single-stranded DNA at sites of DNA damage and/or excess unwound DNA at stressed replication forks is sensed by Mec1 (46). In contrast, Tel1 association with chromatin appears to occur at sites of double-strand DNA breaks (22, 39). The replication defects associated with decreased S-CDK activity may give rise to both types of DNA damage, explaining the involvement of both proteins. Nevertheless, Mec1 and Tel1 are able to effectively substitute for each other, as the deletion of both reduces the viability and growth rate of clb5 cells compared with clb5 mec1 or clb5 tel1 cells (Table 2 and Fig. 4E). The ability to substitute may occur because either pathway can lead to Rad53 activation, which is critical for viability, rather than an ability of Mec1 to sense DNA damage normally sensed by Tel1, and vice versa. For example, Rad53 activated at Tel1-sensed sites of damage may act to stabilize stressed replication forks or sites of DNA damage that may go undetected in the absence of Mec1. The function of Tel1 in clb5 cells probably involves Mrc1 and/or Rad9 as a mediator, as their combined elimination causes lethality (Table 2 and Fig. 4D). In accord with this, a recent study with Schizosaccharomyces pombe indicates that Tel1 and Rad3 (Mec1) can each play a role in Mrc1-mediated activation of Cds1 (Rad53) (44).
Significance of S-CDK regulation to tumorigenesis. Proper regulation of CDK activity is critical for normal cell division and organismal development, whereas deregulation of CDK activity is frequently associated with tumorigenesis (reviewed in reference 32). The finding that diminished S-CDK function elicits DNA damage and replicative stress in S. cerevisiae suggests that a similar effect of decreased S-CDK function in metazoan cells is likely. The inability to inhibit CDK activity in G1 or G0 cells contributes significantly to the inappropriate proliferation of many cancer cells. Similarly, deregulation of S-CDK functions may contribute to the genesis of cancer cells by affecting the accurate execution of S phase, thus creating DNA damage and enhancing genomic instability. For example, recent studies in yeast have shown that deregulation of G1-CDK suppresses the assembly of functional pre-RCs, which increases replicon size and increases genomic instability (16, 36). Although the checkpoint status in these previous studies was not reported, diminished pre-RC activity as a result of mutations in ORC subunits does result in a DNA damage checkpoint response (12, 42; D. G. Gibson, F. Hu, and O. M. Aparicio, unpublished observation) Thus, mutations that affect pre-RC assembly are comparable to diminished S-CDK activity in that both have the effect of reducing origin usage and creating DNA damage. However, cells with a severely defective replication protein(s) (e.g., ORC) are unlikely to proliferate enough to contribute to tumorigenesis, whereas cells with deregulated CDK may retain strong replicative capacity, perhaps damage prone. Hence, reduction of S-CDK function may present another potential mechanism of genomic instability and tumorigenesis.
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ACKNOWLEDGMENTS |
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This work was supported by a Burroughs-Wellcome Career Award (992834) and NIH grant (1RO1GM-CA65494-01A1) to O.M.A.
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FOOTNOTES |
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