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Precocious S-Phase Entry in Budding Yeast Prolongs Replicative State and Increases Dependence Upon Rad53 for Viability
Julia M. Sidorovaa and Linda L. Breedenaa Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
Corresponding author: Julia M. Sidorova, 1100 Fairview Ave. N, Seattle, WA 98109., jsidorov{at}fred.fhcrc.org (E-mail)
Communicating editor: P. RUSSELL
| ABSTRACT |
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Precocious entry into S phase due to overproduction of G1 regulators can cause genomic instability. The mechanisms of this phenomenon are largely unknown. We explored the consequences of precocious S phase in yeast by overproducing a deregulated form of Swi4 (Swi4-t). Swi4 is a late G1-specific transcriptional activator that, in complex with Swi6, binds to SCB elements and activates late G1-specific genes, including G1 cyclins. We find that wild-type cells tolerate Swi4-t, whereas checkpoint-deficient rad53-11 cells lose viability within several divisions when Swi4-t is overproduced. Rad53 kinase activity is increased in cells overproducing Swi4-t, indicating activation of the checkpoint. We monitored the transition from G1 to S in cells with Swi4-t and found that there is precocious S-phase entry and that the length of S phase is extended. Moreover, there were more replication intermediates, and firing of at least a subset of origins may have been more extensive in the cells expressing Swi4-t. Our working hypothesis is that Rad53 modulates origin firing based upon growth conditions to optimize the rate of S-phase progression without adversely affecting fidelity. This regulation becomes essential when S phase is influenced by Swi4-t.
PROPER integration of cell cycle transitions with DNA metabolism is crucially important for cell survival and error-free propagation of a cell's genetic material. Cells that are unable to adjust the cell cycle clock upon receiving DNA damage are known to lose viability and/or compromise the fidelity of genetic transmission (![]()
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Swi4 is a late G1-specific transcriptional activator, which, in complex with Swi6, binds to SCB elements (![]()
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Rad53, a kinase conserved from yeast to humans, is involved in coordinating DNA metabolism with cell cycle transitions (![]()
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In this study, we demonstrate that Swi4-t overexpression can cause precocious entry into S phase in the absence of exogenous DNA damage. We also show that Swi4-t overexpression results in a loss of viability in combination with an allele of RAD53 that is defective in checkpoint function (rad53-11). Analysis of S phase in RAD and rad53-11 strains overexpressing Swi4-t suggests that Swi4-t prolongs the replicative state of cells and may increase the frequency of replication initiation. We propose that excessive origin firing can result in stalling of forks due to depletion of resources such as dNTPs or histones. In the absence of Rad53, stalled forks are not stable enough to resume replication when resources are replenished, and there is no signal to inhibit further origin firing. Combined, these deficiencies lead to lethality. In other words, during a normal S phase, Rad53 serves as a pacemaker, coordinating S-phase progression with growth conditions. By preventing excessive origin firing, it minimizes the effects of precocious S-phase entry imposed by hyperactive Swi4-t.
| MATERIALS AND METHODS |
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Strains and plasmids:
The yeast strain BY2006 MATa ura3 leu2 trp1 his3 has been described (![]()
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1.6R with the endogenous RAD53 gene tagged with the HA tag (![]()
::LEU2 were described before (![]()
his1, respectively.
The plasmid pBD1168 is a YCp50 vector with GAL::SWI4-t and was described previously (![]()
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Growth conditions:
All rich (YEP) and minimal (YC) media and growth conditions were as described before (![]()
30 min (J. SIDOROVA, unpublished results), and ensured that these cells had high levels of Swi4-t from the very beginning of G1 phase. For
-factor synchrony experiments, a culture at OD660 = 0.2 was typically arrested by incubation with 5 mg/liter of
-factor for 1 hr 45 min. Cells were released from the arrest by filtration or by addition of Pronase E (Sigma, St. Louis) to a final concentration of 10 mg/liter. dbf4-1 strains were arrested at 37° for 2.53 hr and released into the cell cycle by shifting back to 25°.
FACS analysis:
FACS analysis was done exactly as described before (![]()
Elutriation:
Elutriation was performed in a J-6B centrifuge (Beckman, Fullerton, CA) using a JE-5.0 rotor and a 40-ml chamber (Beckman). The chamber was loaded with cells at 3500 rpm and 28 ml/min flow rate. Flow rate was then increased to 35 ml/min and the chamber was equilibrated with fresh media. Small G1 cell fractions were harvested in fresh media by further increasing the flow rate from 35 ml/min to 55 to 60 ml/min in 3- to 5-ml/min increments. The size of cells in these fractions was determined using a Z2 Particle Count and Size Analyzer and the data were analyzed using the AccuComp version 2.01 software (Beckman Coulter, Miami).
Nocodazole execution point measurements:
To determine the nocodazole execution point, elutriated G1 cells were allowed to progress through the cell cycle. Aliquots (5 ml) of this culture were taken every 10 min, transferred to culture tubes with nocodazole (final concentration 12 mg/liter), and incubated at 30° for 2 hr on a roller drum. After 2 hr these cells were treated with 1% sodium azide and sonicated and the cell numbers were counted. As expected, cells treated with nocodazole before the first anaphase were arrested in the first cycle (![]()
MAT locus heterozygosity loss measurements:
These measurements were done essentially as described (![]()
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ura3 leu2 his3 ade rad53-11::ura3::LEU2. Diploids were transformed with the empty vector pYES2 or with the GAL::SWI4-t pBD1168 plasmid and were grown in YC-ura media with 2% raffinose and 2% galactose for about eight generations. Of these cells, 5 x 106 were mated with the equal number of cells of either one of the mating tester strains, BY26 or BY27. Upon plating, mated cells gave rise to prototrophic colonies. A separate control was included with only RAD/rad53-11 and no mating tester strains. Neither RAD/rad53-11 diploids nor mating tester strains alone gave rise to prototrophic colonies at a measurable frequency (>10-6). Loss of heterozygosity was calculated as the number of mating events per milliliter of culture divided by the number of viable cells per milliliter of culture, and it was typically
10-4. The average numbers and average deviation of five measurements are reported.
Rad53 kinase assay:
Immunoprecipitation of HA-Rad53 and kinase assays were done as described before (![]()
Hemi-methylation analysis:
Hemi-methylation analysis was done as described (![]()
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3.0 kb (DpnIR). Full methylation of DNA allows this fragment to be further digested by DpnI. In this case, the probe hybridizes mostly to a DpnI-sensitive (DpnIS), EcoRI/DpnI-digested fragment of
1.0 kb.
Two-dimensional gel electrophoresis:
We followed the previously described procedures for the DNA isolation and two-dimensional gel electrophoresis of replicative intermediates (![]()
| RESULTS |
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Cells overproducing Swi4-t require a functional S-phase checkpoint for viability:
We have shown before that the checkpoint-deficient allele of RAD53 (rad53-11) or overproduction of a C-terminally truncated form of Swi4 (Swi4-t) both significantly reduce but do not completely eliminate the methyl methanesulfonate-dependent delay of the G1/S transition (![]()
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Mec1 and Rad53 are required for all three DNA damage checkpoints in G1, S, and G2 phases of the cell cycle (![]()
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strain (Fig 1A). The fact that Swi4-t is lethal in the mec1-1 and rad53-11 but not in the rad9 background suggests that functions of Mec1 and Rad53 that do not overlap with Rad9's function are critical for protection against Swi4-t. Response to disruptions in DNA replication is one such function (![]()
If the lethal effect of Swi4-t on rad53 cells is associated with the way the S phase progresses in these cells, we would anticipate that gene products important for proper S-phase progression might have synthetic phenotypes when overproduced in a GAL::SWI4-t rad53-11 strain. RNR1, the large subunit of ribonucleotide reductase that is an important and perhaps limiting component of an S-phase cell, rescues the lethality of GAL::SWI4-t rad53-11 cells when overexpressed (Fig 1B). Interestingly, RNR1 is a target of Swi4 in vivo (![]()
The presence of Swi4-t could generate some kind of damage during replication, which could evoke the Rad53-dependent checkpoint and necessitate repair. Indeed, RAD52, which is critical for DNA recombination and repair (![]()
Swi4-t induces Rad53 kinase activity and speeds up the G1/S transition:
DNA damage or stalled replication forks cause modification and activation of the Rad53 kinase. This modification results in the appearance of a low-mobility form of Rad53 on protein gels (![]()
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Swi4 is a critical activator of the G1/S transition, and Swi4-t is a hyperactive form that deregulates transcription of G1 cyclins (![]()
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Comparison of budding indices of the synchronized GAL::SWI4-t and vector cells indicates that GAL::SWI4-t cells may spend more time as budded cells (Fig 3A). Moreover, the FACS data (Fig 3B) suggest that while GAL::SWI4-t cells traverse the G1-to-S transition earlier than controls, they spend a longer time completing S phase. In GAL::SWI4-t cells, the fraction of cells with 2N DNA content gradually rises between 60 and 140 min with no indication of progression into the next cell cycle. In contrast, the control cells show no pausing at the 2N or near 2N DNA stage and proceed rapidly into the next cycle. To address this in more detail, we obtained elutriated populations of cells with identical starting size and monitored their progression between G1 and G2 phases. Swi4-t cells budded 23 min earlier than controls (Table 1). However, the execution of anaphase, as measured by the acquisition of 50% resistance to the microtubule-destabilizing drug nocodazole, occurred only 14 min earlier in Swi4-t cells compared to controls (Table 1). Hence, it appears that Swi4-t cells spend more time between the end of G1 phase and anaphase. rad53-11 cells overexpressing Swi4-t also experienced an extension of this interval (Table 1). rad53-11 GAL::SWI4-t cells were less efficient in completing the cell cycle, since the number of nocodazole-resistant cells never exceeded 50%. This suggests that these GAL::SWI4-t cells do not undergo premature mitosis due to mutation in RAD53.
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Extended replicative state in cells expressing Swi4-t:
The extension of the interval between G1 and anaphase in cells overexpressing Swi4-t suggests that Swi4-t may slow or impair DNA replication. To follow the replication more directly, we employed hemi-methylation analysis (![]()
-factor synchronization (Fig 4, BD). This method induces a higher degree of synchrony but the first G1-to-S transition is very rapid.
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Consistent with previous observations, in elutriated wild-type G1 cells arrested before anaphase by nocodazole, the onset of S phase and replication through the ARS607 region as assayed by DpnI resistance occurred earlier in GAL::SWI4-t cells than in controls and just as efficiently, as indicated by the sharp increase of hemi-methylation level (Fig 4A). However, despite the earlier onset of replication, the hemi-methylated state of the ARS607 locus persisted at a high level for a longer time in GAL::SWI4-t cells than in controls.
Using the
-factor synchronization we observed a rapid G1-to-S transition in both GAL::SWI4-t and vector cells (Fig 4B). This was expected because
-factor-arrested cells exceed the critical size needed for the transition into S phase and thus exit G1 very quickly upon release. Nonetheless, the hemi-methylation assay showed that while both GAL::SWI4-t and vector control cells started to accumulate newly replicated, hemi-methylated DNA in the early ARS607 locus at about the same time, GAL::SWI4-t cells carried hemi-methylated DNA for a longer time than vector controls (Fig 4C and Fig D). In rad53-11 cells, induction of hemi-methylation was low due to a low basal level of methylation, precluding a definitive interpretation of results.
The hemi-methylation studies suggest that Swi4-t overexpression can result in both an earlier appearance and a prolonged presence of nascent DNA in the ARS607 region in wild-type cells. However, it is also possible that the observed differences in hemi-methylation profiles are caused by an altered accessibility to dam methylase after one round of replication, for example, due to a change in the nascent chromatin structure.
Swi4-t overexpressing cells display more replicative intermediates during S phase:
To test whether extension of the hemi-methylated state in DNA upon Swi4-t overexpression correlates with the prolonged replicative state and to assess replication in rad53-11 cells, we employed two-dimensional (2D) gel electrophoresis (Fig 5A; ![]()
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Using this assay, we first followed replication through the ribosomal gene cluster on chromosome XII, which spans 10002000 kb of 9-kb repeats of rDNA each containing an ARS sequence (rARS). Replication intermediates of the rDNA cluster are detectable throughout S phase (Fig 5C). In addition to bubble- and Y-shaped molecules, two other types of replicative intermediates can be detected in rDNA during replication. Leftward-moving forks that stall at the replication fork barrier (RFB) show up as a dot of increased intensity on the Y arc (![]()
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To follow replication, cells were synchronized in late G1 by
-factor. The highest levels of replicative intermediates in the wild-type cells were detectable at 50 min (Fig 5B and Fig C). At this time and later, we could detect higher levels of Y intermediates in GAL::SWI4-t cells compared to controls, suggesting that replicative intermediates persist for a longer time in these cells. In rad53-11 cells, the effect of Swi4-t overexpression was more pronounced (Fig 6). The rad53-11 GAL::SWI4-t cells maintained higher levels of bubbles, Ys, and stalled and converged forks than did the rad53-11 vector controls (Fig 6B). Fig 6C shows a repeat of this experiment with a lighter exposure so that the levels of the RFB-stalled forks in rad53-11 GAL::SWI4-t vs. rad53-11 vector cells can be compared (marked by arrows). Quantitation of these gels confirms the observation that the presence of GAL::SWI4-t results in about twice as many Ys and RFB-stalled forks (Fig 6D and Fig E). Thus, the two-dimensional gel analysis suggests that replicative forks are present on rDNA for an extended time in GAL::SWI4-t cells. In addition, the levels of bubble intermediates evident in a fixed amount of DNA are two- to threefold higher in rad53-11 GAL::SWI4-t cells than in controls at the earliest time point (see Fig 6B and Fig D). These data suggest that the ribosomal ARS fires more frequently when GAL::SWI4-t is present.
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rad53-11 and GAL::SWI4-t may influence origin firing frequency:
We next followed replication of a single copy origin, ARS608, whose firing frequency is variable and limited to a narrow window of time in the first half of S phase (![]()
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Interestingly, we also observed a difference between the relative amounts of Y and bubble intermediates in RAD and rad53-11 cells. An important difference between the rARS and ARS608 is the fact that the latter is a single-copy ARS and it is replicated either actively, if it fires, or passively from the nearby ARS607. Thus, the bubble-to-Y ratio is a good indicator of the firing efficiency of ARS608 (![]()
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To further address the effects of Swi4-t, we asked whether it can suppress replication initiation mutations. DBF4 is an essential gene required for initiation but not elongation of replication throughout S phase (![]()
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| DISCUSSION |
|---|
Swi4-t is a hyperactive derivative of the late G1-specific transcriptional activator Swi4. When overexpressed, it causes Swi4 target genes to be ectopically expressed at all stages of the cell cycle. In this study, we demonstrated that it can cause precocious entry into S phase. In addition, we observed that even though GAL::SWI4-t cells can enter S phase earlier than normal cells, they spend more time between the end of G1 and anaphase. Hemi-methylation analysis suggests that Swi4-t overexpression leads to an extension of the time during which nascent DNA is generated in a given region. If that is the case, it could result from DNA reduplication or from gap filling or strand break repair, or any combination thereof. 2D gel analysis specifically suggests that Swi4-t overexpression correlates with a prolonged presence and/or increased abundance of replicative intermediates. A higher level of replicative forks could be detected in rDNA in GAL::SWI4-t cells as compared to vector cells. Also, bubble and Y intermediates were detectable for a longer time in ARS608 DNA in GAL::SWI4-t cells.
The fact that we could detect an extended window of time during which bubbles and Ys were present in rARS and ARS608 DNA in cells carrying GAL::SWI4-t suggests that some replicative forks in these regions were slowing or stalling. Another interpretation of this result is that Swi4-t may cause an extension of the window of time within which origins fire and/or an increase in the frequency with which they fire. Support for the notion that Swi4-t may increase efficiency of at least some origins is rendered by the fact that Swi4-t overexpression partially suppresses the temperature sensitivity of the dbf4-1 strain and speeds up the course of S phase in dbf4-1 cells. Dbf4 is involved in initiation of DNA replication (![]()
The cellular response to Swi4-t involves Rad53. Rad53 is required for viability, and when hyperactivated in response to DNA damage, it slows down S-phase progression by inhibiting origin firing (![]()
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We propose a simple interpretation of the data presented in this study, which is that both GAL::SWI4-t and rad53-11 affect initiation of replication, albeit to a different extent. In a normal cell, Swi4 activity is rate limiting for S-phase entry (![]()
Rad53 plays a critical role in mitigating the effects of Swi4-t on replication. In a RAD cell, forks that stall or slow down may recruit Rad53. By stabilizing these forks with the replicative machinery assembled on them, Rad53 allows most of them to resume replication when supplies are replenished (![]()
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There is growing evidence that abundance of stalled forks during replication may contribute to genome destabilization. For example, mutations in the rDNA-specific helicase, Pif1, decrease the number of stalled forks, and this is correlated with a decrease in the amount of rDNA breakage and a reduction in the number of rDNA circles (![]()
Finally, a number of hypotheses can be entertained to explain the mechanism by which hyperactive Swi4-t signals S-phase entry. Similar to other transcription factors, a DNA-bound Swi4-t could promote initiation of replication in cis (![]()
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It is also possible that the effect of Swi4-t on S-phase progression is mediated by the altered expression of Swi4 target genes. Two critical targets of the Swi4 activator, which are rate limiting for the G1-to-S transition, are the G1 cylins CLN1 and CLN2. Overproduction of CLN1 or CLN2 is lethal in combination with mec1 and, to a lesser degree, rad53 mutations in some backgrounds (![]()
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A dividing eukaryotic cell faces intrinsic challenges that arise from its ability to initiate DNA replication from many loci throughout the genome. It seems that for an efficient S phase, it may be critically important to adjust origin-firing efficiency depending on the cellular resources as well as to have a negative feedback constantly monitoring the progress of replication.
| ACKNOWLEDGMENTS |
|---|
We thank members of the Breeden lab for support and discussions and Bonny Brewer for critical reading of the manuscript. Thanks are due to Steve Elledge, Andrew Emili, and Bonny Brewer for strains and plasmids. This work was funded by grant GM-41073 from the National Institutes of Health to L.B. J.S. was supported by the Leukemia and Lymphoma Society Fellowship.
Manuscript received August 30, 2001; Accepted for publication November 5, 2001.
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-32PO4] ATP. Products of the kinase reactions were resolved on an SDS polyacrylamide gel and autoradiographed. Positions of the modified and unmodified forms of Rad53 are marked by lines on the right.








