Precocious S-Phase Entry in Budding Yeast Prolongs Replicative State and Increases Dependence Upon Rad53 for Viability

Corresponding author: Julia M. Sidorova, 1100 Fairview Ave. N, Seattle, WA 98109., jsidorov{at}fred.fhcrc.org (E-mail)

Communicating editor: P. RUSSELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 (FOIANI et al. 2000 ). In the absence of any genotoxic treatment, genomic stability can be negatively affected by relaxation of the control over the transition between G1 and S phases. For example, forced entry of some quiescent cells into S phase upon c-myc or cyclin E overproduction is known to result in genomic rearrangements (FELSHER and BISHOP 1999 ; MAI et al. 1999 ; SPRUCK et al. 1999 ). Loss of Rb or ectopic expression of cyclin D1 in tissue culture cells can increase gene amplification frequencies (DONEHOWER 1997 ). In budding yeast, inactivation of the B cyclin inhibitor SIC1, which causes earlier activation of the S-phase cyclin/CDK complexes and earlier entry into S phase (SCHWOB et al. 1994 ), leads to an elevated rate of chromosome loss (NUGROHO and MENDENHALL 1994 ). Overexpression of G1 cyclins CLN1 or CLN2, whose levels are rate limiting for the G1-to-S transition, is synthetically lethal with mutations in the DNA damage checkpoint kinase gene MEC1 and causes an increased level of chromosome loss (VALLEN and CROSS 1995 , VALLEN and CROSS 1999 ). The mechanisms of genomic instability induced by relaxation of control over the G1to-S transition remain largely unknown. We sought to investigate these mechanisms using budding yeast as a model system.

Swi4 is a late G1-specific transcriptional activator, which, in complex with Swi6, binds to SCB elements (MENDENHALL and HODGE 1998 ) in the promoters of numerous late G1-specific genes (IYER et al. 2001 ). The activity of the Swi4/Swi6 complex gives rise to the concerted burst of transcription of the G1 cyclin genes CLN1 and CLN2 and of a number of other genes, which promote the transition to S phase (NASMYTH and DIRICK 1991 ; OGAS et al. 1991 ). Swi4-t, a C-terminally truncated and stabilized form of Swi4, is unable to interact with Swi6 and cannot form the Swi4/Swi6 complex (ANDREWS and MOORE 1992 ; PRIMIG et al. 1992 ; SIDOROVA and BREEDEN 1993 ). However, unlike the full-length Swi4 (BAETZ and ANDREWS 1999 ), the truncated Swi4-t is capable of binding to promoters independently of Swi6 (ANDREWS and MOORE 1992 ; PRIMIG et al. 1992 ; SIDOROVA and BREEDEN 1993 ), and when overexpressed from the GAL promoter (GAL::SWI4-t), it deregulates the transcription of target genes (BREEDEN and MIKESELL 1994 ). Swi4-t can cause a precocious entry into S phase similar to the checkpoint mutant of Rad53 in cells that receive DNA damage in G1 (SIDOROVA and BREEDEN 1997 ).

Rad53, a kinase conserved from yeast to humans, is involved in coordinating DNA metabolism with cell cycle transitions (WEINERT 1998 ; FOIANI et al. 2000 ). In the presence of DNA damage in G1 and G2, Rad53 is most likely recruited to the damage recognition and processing complex (FOIANI et al. 2000 ) via Mec1-phosphorylated Rad9 (EMILI 1998 ; SUN et al. 1998 ; VIALARD et al. 1998 ; DUROCHER et al. 1999 ). Rad53 is phosphorylated and activated in a Mec1 kinase-dependent manner (NAVAS et al. 1996 ; SANCHEZ et al. 1996; SUN et al. 1996 ). Activated Rad53 and Mec1 phosphorylate specific targets, which can delay cell cycle transitions and induce DNA repair (ALLEN et al. 1994 ; SIDOROVA and BREEDEN 1997 ; HUANG et al. 1998 ; PELLICIOLI et al. 1999 ; SANCHEZ et al. 1999 ; BASHKIROV et al. 2000 ; KIHARA et al. 2000 ). Rad53 is also important for monitoring DNA damage and nucleotide shortages within S phase (PAULOVICH and HARTWELL 1995 ; NAVAS et al. 1996 ). Rad53 prevents firing of origins when DNA replication is encumbered by damage (SANTOCANALE and DIFFLEY 1998 ; SHIRAHIGE et al. 1998 ; TERSERO and DIFFLEY 2001 ) and potentially stabilizes stalled replication forks (LOPES et al. 2001 ). Rad53 also blocks premature metaphase-to-anaphase transition in cells with incompletely replicated DNA (ALLEN et al. 1994 ). Finally, Rad53 is an essential gene (ZHENG et al. 1993 ) whose role in the unperturbed cell cycle is most likely involved in S-phase progression control (DESANY et al. 1998 ; DOHRMANN et al. 1999 ). Along with Mec1, Rad53 may act during S phase to remove the Sml1-mediated inhibition of ribonucleotide reductase (ZHAO et al. 1998 ).

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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and plasmids:
The yeast strain BY2006 MATa ura3 leu2 trp1 his3 has been described (SIDOROVA and BREEDEN 1997 ). BY2390 is a ura3 derivative of BY2007 MATa ura3 leu2 trp1 his3 rad53-11::URA3 (SIDOROVA and BREEDEN 1997 ). BY2912 and BY2913 were derived from BY2006 and BY2390, respectively, by integration of Escherichia coli dam methylase on a pRS305 vector into the endogenous LEU2 locus (FRIEDMAN et al. 1995 ). BY2887 and BY2888 are isogenic to BY2006 and BY2390, respectively, except they carry a rad52::LEU2 disruption. BY2914 is MATa ura3 lys2 leu2 trp1 pep4::HIS3 prb11.6R with the endogenous RAD53 gene tagged with the HA tag (EMILI 1998 ). BY2226 MATa ura3 leu2 trp1 his3 mec1-1::HIS3 and BY2227 MATa ura3 leu2 trp1 his3 rad9::LEU2 were described before (SIDOROVA and BREEDEN 1997 ). BY479 is MATa dbf4-1 ura3 trp1 ade5. Mating tester strains BY26 and BY27 are MATa his1 and MAT his1, respectively.

The plasmid pBD1168 is a YCp50 vector with GAL::SWI4-t and was described previously (SIDOROVA and BREEDEN 1993 ). pBD1411 and pBD1948 were described before (TYERS et al. 1993 ; EPSTEIN and CROSS 1994 ). The plasmid pBD2385 has been described previously (SIDOROVA and BREEDEN 1993 ) and is a Ycp50 with the GAL promoter-driven SWI4 gene. The plasmid pBD2972 is a kind gift of Dr. S. Elledge and contains the RNR1 open reading frame under the control of the GAL promoter on a 2-µm TPR1 shuttle vector (DESANY et al. 1998 ).

Growth conditions:
All rich (YEP) and minimal (YC) media and growth conditions were as described before (BREEDEN and MIKESELL 1991 ). Cultures used for elutriation were grown in YC-ura glucose media and then inoculated into YEP media with 2% raffinose and grown to OD₆₆₀ = 1.5–1.8. To induce Swi4-t, galactose was added 80–100 min before the zero time point of the experiment (this induction time included the time in the elutriator). The zero time point was the time when the unbudded eluted cells were placed in the 30° incubator. This time was ample to fully induce Swi4-t overexpression, which takes 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 2.5–3 hr and released into the cell cycle by shifting back to 25°.

FACS analysis:
FACS analysis was done exactly as described before (SIDOROVA and BREEDEN 1997 ) except that cells were analyzed on a Calibur Analyser (Becton Dickinson, San Jose, CA).

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 (JACOBS et al. 1988 ). Cells that had passed this transition divided once and arrested in metaphase of the second cycle. Thus, the final cell concentration after the nocodazole treatment was dependent upon the proportion of cells that had traversed the first anaphase and become nocodazole resistant at the time point when the aliquot was taken. Percentage of nocodazole-resistant cells at each time point was calculated using these cell concentration data. In addition, aliquots of cells were taken every 10 min from the untreated growing culture. These samples were also arrested by 1% sodium azide, and cell concentration and proportion of budded cells were determined in each sample.

MAT locus heterozygosity loss measurements:
These measurements were done essentially as described (ROSE et al. 1990 ), taking advantage of the fact that diploid cells that lose one of the MAT alleles will mate as haploids. RAD/rad53-11 heterozygous diploids were constructed by mating BY2006 MATa ura3 leu2 trp1 his3 (SIDOROVA and BREEDEN 1997 ) and BY2918 MAT 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 (SIDOROVA and BREEDEN 1997 ) with 12CA5 mouse monoclonal antibodies to the HA epitope, except that no exogenous substrate was added to the kinase reaction.

Hemi-methylation analysis:
Hemi-methylation analysis was done as described (FRIEDMAN et al. 1995 , FRIEDMAN et al. 1997 ). A constitutively expressed E. coli dam methylase gene on a pRS305 plasmid (FRIEDMAN et al. 1995 ) was integrated into the endogenous LEU2 locus of yeast strains. These strains did not show a growth disadvantage compared to the parental strains, and GAL::SWI4-t overexpression had the same phenotype in the methylated strains as in the parental strains. DNA was digested overnight with DpnI and EcoRI and loaded onto 0.8% agarose gels. Southern blotting and hybridization with the ARS607 probe were performed on GeneScreen membranes (New England Nuclear, Boston) according to the manufacturer's recommendations. The probe for the ARS607 region maps to chromosome VI coordinates from 202,454 to 203,480 (http://www-genome.stanford.edu/Saccharomyces/) downstream of the ARS607 ARS consensus sequence. In a hemi-methylated or unmethylated DNA, this probe preferentially hybridizes to a DpnI-resistant EcoRI-digested fragment of 3.0 kb (DpnI^R). Full methylation of DNA allows this fragment to be further digested by DpnI. In this case, the probe hybridizes mostly to a DpnI-sensitive (DpnI^S), 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 (FRIEDMAN and BREWER 1995 ). The isolated DNAs were digested with NheI for rARS replicative intermediates and resolved as follows. The first dimension separation was through 0.4% agarose at 1 V/cm for 20 hr at room temperature, and the second dimension was run in 1% agarose at 5 V/cm for 5 hr at 4°. To visualize ARS608, DNAs were digested with XhoI and resolved in 0.4% agarose at 1 V/cm for 24 hr in the first dimension and in 0.8% agarose at 2.7 V/cm for 15 hr at 4°. The probe for rARS maps to chromosome XII coordinates 460,050–460,777. The probe for ARS608 maps to chromosome VI coordinates 213,306–214,317.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 (SIDOROVA and BREEDEN 1997 ). To explore whether combining these two genetic determinants in one background will result in a complete elimination of this delay, we introduced three different SWI4 alleles under control of the GAL1 promoter into a rad53-11 strain. These alleles encoded wild-type Swi4 and two of its truncated derivatives lacking 140 or 280 amino acids from the C terminus. We found that overexpression of the 280-amino-acid truncation Swi4-t from the GAL promoter (GAL::SWI4-t) severely abrogated the rad53-11 strain's viability even in the absence of exogenous DNA damage (Fig 1A). These cells typically formed micro-colonies of up to 20 to 40 cells and division was arrested within 48 hr of growth on galactose. Neither the full-length Swi4 nor a shorter truncation of 140 amino acids compromised the strain's viability (data not shown).

View larger version (92K): In this window In a new window Download PPT slide   Figure 1. Overexpression of Swi4-t is lethal in rad53-11 or mec1-1 mutants, but not in rad9 mutants. (A) Wild-type (BY2006), rad53-11 (BY2390), mec1-1 (BY2226), and rad9 (BY2224) strains were transformed with the empty vector (Ycp50), GAL::SWI4-t plasmid (pBD1168), GAL::CLN1 plasmid (pBD1141), or GAL::CLN2 plasmid (pBD1948) and streaked onto selective plates with galactose. (B) The rad53-11 strain carrying GAL::SWI4-t was transformed with either the empty vector p414GAL1 or the GAL::RNR1 plasmid (pBD2972). The strains were grown on selective media plates with galactose. (C) 105, 0.33 x 105, and 104 of cells of the wild-type (BY2006), rad52 (BY2887), rad53-11 (BY2390), and rad53-11 rad52 (BY2888) strains carrying GAL::SWI4-t plasmid (pBD1168) were spotted onto selective media plates with galactose.

VALLEN and CROSS 1995 , VALLEN and CROSS 1999 have previously reported that high levels of either of the G1 cyclins, Cln1 or Cln2, are lethal in combination with the checkpoint-deficient mec1 allele in the BF264-15D background. Since CLN1 and CLN2 levels increase upon induction of Swi4-t (BREEDEN and MIKESELL 1994 ), we examined whether these cyclins were sufficient to cause lethality in the A364a rad53-11 strain and whether GAL::SWI4-t was lethal to a mec1 strain in the A364a background. Overexpression of Swi4-t did lead to poor viability with mec1-1. However, GAL::CLN1 or GAL::CLN2 did not abolish colony formation in rad53-11 or mec1-1 strains (Fig 1A). These results suggest that the lethality caused by Swi4-t in the A364a background cannot be attributed entirely to G1 cyclin overproduction.

Mec1 and Rad53 are required for all three DNA damage checkpoints in G1, S, and G2 phases of the cell cycle (FOIANI et al. 2000 ). Rad9, on the contrary, is predominantly required for the G1 and G2 checkpoints (NAVAS et al. 1996 ). We found that Swi4-t overexpression had no effect on the viability of a rad9 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 (FOIANI et al. 2000 ).

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 (IYER et al. 2001 ) and RNR1 is transcribed at a higher level when Swi4 or Swi4-t is overexpressed (data not shown).

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 (SUNG et al. 2000 ), has a synthetic phenotype with rad53-11 GAL::SWI4-t. The rad52 deletion exacerbates Swi4-t-induced lethality in rad53-11 cells while having no measurable effect on RAD53 cells (Fig 1C). In addition, long-term Swi4-t overexpression results in an increase in chromosome instability as judged by the loss of heterozygosity of the MAT locus. A RAD/rad53-11 heterozygous diploid strain overexpressing Swi4-t shows about a threefold higher frequency of allele loss events at the MAT locus than cells carrying the vector alone (1.25 ± 0.33 x 10^-4 vs. 4.18 ± 0.66 x 10^-4).

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 (ALLEN et al. 1994 ; SANCHEZ et al. 1996; SUN et al. 1996 ). Fig 2 shows the state of Rad53 immunoprecipitated out of wild-type cells harboring vector control plasmid or GAL::SWI4-t and grown in galactose. An unmodified, high-mobility form of Rad53 remains the predominant form of this kinase in vector control cells throughout the time course. In contrast, after 2 hr of galactose induction of Swi4-t, there is a detectable accumulation of the low-mobility form of Rad53. Since typically only the unmodified form of Rad53 is readily detectable during a normal cell cycle (SUN et al. 1996 ; J. SIDOROVA, data not shown), this increased presence of the low-mobility, modified form of Rad53 upon induction of Swi4-t suggests that these cells are undergoing a Rad53-mediated checkpoint response.

View larger version (74K): In this window In a new window Download PPT slide   Figure 2. GAL::SWI4-t induces Rad53 kinase activity. A wild-type strain (BY2914) with a HA-tagged Rad53 was transformed with the empty vector pYES2 (top) or with the GAL::SWI4-t plasmid (pBD1168, bottom). The cells were grown in selective media with raffinose and then diluted into fresh media with galactose to induce Swi4-t expression. Aliquots were taken immediately before galactose was added and every hour for 6 hr. Rad53 was immunoprecipitated with HA antibodies and allowed to autophosphorylate in the presence of [-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.

Swi4 is a critical activator of the G1/S transition, and Swi4-t is a hyperactive form that deregulates transcription of G1 cyclins (BREEDEN and MIKESELL 1994 ). As such, Swi4-t overproduction is expected to affect the G-to-S transition. However, it is less evident that Swi4-t may affect S-phase progression. We used centrifugal elutriation to examine cell cycle progression in Swi4-t cells with or without functional Rad53. Swi4-t was induced 120 min before the first measurement was taken. This protocol enabled us to look at the immediate effects of GAL::SWI4-t on the cell cycle. The fractions collected from the elutriator were allowed to progress through the cell cycle and sampled at 10-min intervals. Overexpression of Swi4-t did not change the kinetics of cell growth in G1 (data not shown), but it caused cells to bud at a smaller size than controls. For example, GAL:SWI4-t cells achieved 50% budding at the average mean volume of 17.4 fl, while the vector control cells were 50% budded at the average mean volume of 24 fl. Direct comparison of fractions of cells with identical starting size distributions showed that there was a 25-min difference in the initiation of budding of the control vs. Swi4-t cells (Fig 3A). As with control cells, the budding induced by Swi4-t was accompanied by an earlier entry into S phase as determined by FACS (Fig 3B), indicating that these events remain coupled in the Swi4-t cells. rad53-11 cells carrying Swi4-t reproducibly exited G1 at a smaller size than control cells. However, the volume difference between GAL::SWI4-t and vector cells was less dramatic than in the case of the wild-type cells (20 fl vs. 23 fl; see also Fig 3A and Fig B). We also found that elutriated rad53-11 cells exhibited a less synchronous exit from G1 than their wild-type counterparts. Budding was slower and 1N DNA persisted for a longer time in these cells (Fig 3B).

View larger version (47K): In this window In a new window Download PPT slide   Figure 3. GAL::SWI4-t speeds up budding and S-phase onset in elutriated G1 cells. (A) Wild-type (BY2912) or rad53-11 (BY2913) cells were transformed with GAL::SWI4-t (pBD1168) or empty vector (Ycp50) and grown in rich medium with raffinose, galactose was added for 20–40 min, and small G1 cells were harvested by centrifugal elutriation in fresh YEP media with raffinose and galactose. Fractions of identical cell size distribution were collected and allowed to progress through the cell cycle. Samples were taken from these cultures every 10–15 min and the percentage of budding cells was determined. (B) Wild-type strain fractions and rad53-11 strain fractions obtained by elutriation were subjected to FACS analysis. Mean cell volumes at the corresponding time points are indicated to the right of the profiles.

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.

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 (FRIEDMAN et al. 1997 ). We used the strains that constitutively express E. coli dam methylase, which methylates both strands of yeast DNA in vivo. In strains carrying dam methylase, replication through a particular region can be followed as a transient state of hemi-methylation, because hemi-methylated DNA is resistant to cleavage by the DpnI restriction enzyme. To compare GAL::SWI4-t and vector cells, elutriated G1 cells of the two strains were followed as they progress through G1 and S, but were arrested in anaphase by adding microtubule inhibitor nocodazole (Fig 4A). Alternatively, we employed -factor synchronization (Fig 4, B–D). This method induces a higher degree of synchrony but the first G1-to-S transition is very rapid.

View larger version (41K): In this window In a new window Download PPT slide   Figure 4. GAL::SWI4-t extends the interval during which nascent DNA can be detected by hemi-methylation analysis. (A) The wild-type (BY2912) strain transformed with the empty vector (Ycp50) or with GAL::SWI4-t (pBD1168) was elutriated as described for Fig 3. Mean volumes of the starting cultures were 16.4 fl for both wild-type strains. These cultures were allowed to progress into S phase, and after 40 min nocodazole was added to the final concentration of 12 mg/liter to prevent escape into the second cell cycle. Samples were taken every 10 min to isolate DNA and methylation state of nascent DNA was followed by digesting with DpnI and EcoRI. The Southern blots of EcoRI, DpnI-digested DNA were hybridized with probes to the ARS607 region and quantified. The DpnI resistance was determined as a ratio of the DpnI-resistant (DpnIR) to the sum of the DpnI-resistant and DpnI-sensitive (DpnIS) DNA. The DpnI resistance at time point 0 was set equal to 1 unit, so that the fold induction of DpnI resistance could be compared for the indicated strains. (B–D) The wild-type (BY2912) strain transformed with the empty vector (pBD1129) or with GAL::SWI4-t (pBD1168) was synchronized in late G1 by incubating with -factor for 105 min. Galactose was added 30 min before the release. Cells were released from the arrest by filtration and resuspended in fresh media with raffinose and galactose. Aliquots of cultures were taken at 10-min intervals and analyzed by FACS (B) and hemi-methylation analysis (C). Southern blot hybridizations to ARS607 region DNA are shown (C), and quantitation of these data is shown in (D). Hemi-methylated DNA is resistant to DpnI digest, giving rise to a large EcoRI-EcoRI fragment (DpnIR), and fully methylated DNA is sensitive to DpnI digest, giving rise to a smaller EcoRI-DpnI-digested (DpnIS) fragment.

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; FRIEDMAN and BREWER 1995 ). In these gels, bubble-shaped intermediates arise from origin firing within the given DNA fragment. Y-shaped intermediates are generated either when forks are initiated distally and passively replicate through the monitored fragment or when the replication bubble is positioned asymmetrically within the fragment and one fork completes its replication before the other. To quantify replicative intermediates, signal intensities from bubbles or Ys can be normalized to that of the double-stranded DNA, which migrates as a spot in front of the replicating molecules (1N DNA, Fig 5A; IVESSA et al. 2000 ).

View larger version (63K): In this window In a new window Download PPT slide   Figure 5. GAL::SWI4-t extends the window of activity of the ribosomal ARS in wild-type cells. (A) Diagram depicting the migration patterns of replication intermediates of ribosomal DNA through 2D gels. (B) A wild-type (BY2006) strain transformed with the empty vector (Ycp50) or with GAL::SWI4-t (pBD1168) was synchronized with -factor. Galactose was added 30 min before the release. Cells were released from the arrest by adding Pronase E (Sigma) to the final concentration of 10 mg/liter and aliquots of cultures were taken at specified times for FACS analysis (B) and 2D gel analysis (C). The DNAs were digested with the NheI restriction enzyme and resolved in two dimensions. Gels were subjected to Southern blotting and hybridization and probed for the ribosomal ARS locus as specified in MATERIALS AND METHODS.

Using this assay, we first followed replication through the ribosomal gene cluster on chromosome XII, which spans 1000–2000 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 (LINSKENS and HUBERMAN 1988 ; BREWER et al. 1992 ). In addition, at later stages of replication, converging forks form X-shaped, Holliday junction-like intermediates, which migrate separately from simple Ys (Fig 5A and Fig C). Thus, the rDNA cluster provides an opportunity to monitor accumulation of the regular early (bubble) and late (Y) intermediates, including the stalled and converged replicative forks.

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.

View larger version (61K): In this window In a new window Download PPT slide   Figure 6. GAL::SWI4-t increases the levels of replicative intermediates detectable in rad53-11 cells. A rad53-11 (BY2390) strain transformed with the empty vector (Ycp50) or with GAL::SWI4-t (pBD1168) was treated as described for the wild-type strain in the legend to Fig 5. Samples of cells were taken every 10 min to determine DNA content by FACS. (A) Note that GAL::SWI4-t cells are slightly ahead of the vector controls in S-phase progression. (B) DNAs isolated from these cultures at the times specified were digested with the NheI restriction enzyme and resolved in two dimensions as in Fig 5. The arrow points to the X-shaped intermediates prominent in the rad53-11 GAL::SWI4-t cells. (C) A shorter exposure of the two-dimensional gel from an independent experiment is shown to compare the abundance of the RFB-stalled forks in the rad53-11 vector vs. rad53-11 GAL::SWI4-t cells. Positions of RFB-stalled forks and of 1N DNA are marked by arrows. (D and E) Autoradiograms of rARS replicative intermediates of the rad53-11 BY2390 strain with the empty vector or GAL::SWI4-t were quantified, and the signals in bubbles and Ys (D) or in the RFB-stalled fork (E) intermediates were normalized to the signal in 1N DNA and plotted. The average of two measurements is presented.

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 (FRIEDMAN et al. 1997 ; YAMASHITA et al. 1997 ). As seen in Fig 7A, both in RAD and in rad53-11 cells, GAL::SWI4-t extended the window of time during which the large bubble intermediates (marked by arrows) were detectable in ARS608 DNA by at least 10 min. Y intermediates in GAL::SWI4-t cells were also more prevalent late in S phase.

View larger version (104K): In this window In a new window Download PPT slide   Figure 7. GAL::SWI4-t extends the window of time during which ARS608 replicates. (A) The wild-type (BY2006) or rad53-11 (BY2390) strains with the empty vector or GAL::SWI4-t presented in Fig 5 and Fig 6 were used to examine ARS608 replication. DNAs from specified time points were digested with the XhoI restriction enzyme and resolved in two dimensions as specified in MATERIALS AND METHODS and probed with ARS608 locus DNA. (B) Three to four blots of ARS608 replicative intermediates, taken from the times when ARS608 showed the most activity, were quantified using PhosphoImager for each strain and the ratio of the signals in the full arc of bubble intermediates and the full arc of Y intermediates was averaged and plotted.

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 (FRIEDMAN et al. 1997 ; YAMASHITA et al. 1997 ). As Fig 7B shows, the maximal detectable firing frequency of ARS608 was reproducibly increased about twofold in rad53-11 cells. The quantification also showed that in both RAD and rad53-11 cells, Swi4-t overexpression did not lead to a significant increase in the ARS608 bubble-to-Y ratio during the peak activity of this ARS. Overall, the 2D analysis indicates that both rad53-11 and GAL::SWI4-t contribute to the increase in origin firing although these contributions are manifested differently. While rad53-11 shifts the distribution toward early intermediates, GAL::SWI4-t extends the window of time during which the intermediates are detectable.

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 (JOHNSTON and THOMAS 1982 ; BOUSSET and DIFFLEY 1998 ; DONALDSON et al. 1998 ). dbf4-1 is a temperature-sensitive mutation, which reduces the Dbf4/Cdc7 complex kinase activity (KIHARA et al. 2000 ) and prevents replication initiation at the nonpermissive temperature (JOHNSTON and THOMAS 1982 ). We found that Swi4-t, but not the full-length Swi4, improved the growth of the dbf4-1 strain at the semipermissive temperature of 33° (Fig 8A). We then arrested the GAL::SWI4-t and vector cells at the beginning of S phase by shifting them to 37°, induced Swi4-t expression, and then shifted them back to the permissive temperature. FACS analysis showed that the dbf4-1 cells that overexpressed Swi4-t completed S phase significantly faster than controls, suggesting that Swi4-t may increase initiation in dbf4-1 cells (Fig 8B).

View larger version (51K): In this window In a new window Download PPT slide   Figure 8. GAL::SWI4-t partially suppresses temperature sensitivity of the dbf4-1 strain. (A) A dbf4-1 (BY479) strain transformed with the empty vector (Ycp50), GAL::SWI4 (pBD2385), or GAL::SWI4-t (pBD1168) was streaked onto selective media plates with galactose and grown at 33°. (B) The dbf4-1 (BY479) strain with the empty vector (Ycp50) or with GAL::SWI4-t (pBD1168) was grown at 25° in YEP media with raffinose and arrested before S phase by incubation at 37° for 2.5 hr. Galactose was added 30 min prior to the release. The cultures were released from the arrest by shifting back to 25°. Aliquots of these cultures were taken every 15 min and subjected to FACS analysis.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 (JOHNSTON and THOMAS 1982 ), and one may expect that the consequences of partial Dbf4 inactivation could be counteracted by upregulating origin firing.

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 (SANTOCANALE and DIFFLEY 1998 ; SHIRAHIGE et al. 1998 ; TERSERO and DIFFLEY 2001 ). Swi4-t overexpression leads to the activation of the Rad53 kinase. rad53-11 checkpoint mutant cells are not able to tolerate overexpression of Swi4-t and lose viability within three to five divisions. These observations suggest that Swi4-t overexpression invokes a checkpoint response. Our data indicate that rad53-11 GAL::SWI4-t cells do not enter into or progress through S phase any faster than RAD GAL::SWI4-t cells. However, rad53-11 in combination with Swi4-t results in a further increase in the levels of both early (bubble) and late (stalled and converged forks) rDNA replicative intermediates compared to controls. rad53-11 also results in a significant increase in the firing frequency of ARS608. This latter observation is consistent with the previous report by SHIRAHIGE et al. 1998 made with a rad53-1 allele of RAD53, but differs from the study of ARS301 in a rad53-21 strain where no increased firing was found (SANTOCANALE et al. 1999 ). This discrepancy is probably due to differences between these RAD53 alleles. Only a subset of RAD53 mutations suppresses the temperature-sensitive initiation defect of dbf4-1, and rad53-21 is synthetically lethal with dbf4-1 at the permissive temperature (DESANY et al. 1998 ; DOHRMANN et al. 1999 ; KIHARA et al. 2000 ).

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 (MCINERNY et al. 1997 ). Elevating Swi4 activity with a stable, constitutively produced form of Swi4 may not only erroneously signal the cell to enter S, but may also signal its capacity to replicate at a high rate. This could lead to an increase in the number of replicative forks concurrently present on DNA. When the density of forks is elevated above what a given growth condition can support, this puts strain on the cell. Elevated fork density could deplete dNTP and/or histone pools, increase torsional stress on DNA, or make other key components of the replication machinery rate limiting. The consequences of these adversities may be transient stalling or slowing of fork movement.

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 (DESANY et al. 1998 ; LOPES et al. 2001 ). Stalled forks "marked" with activated Rad53 send a signal inhibiting further origin firing and thus prevent further accumulation of replicative intermediates and the unproductive spending of origins. Thus, the excessive firing promoted by GAL::SWI4-t is neutralized. In rad53-11 cells, this protective mechanism is deactivated, leading to the accumulation of many unstable replicative intermediates, which may require disassembly and processing through a recombination pathway to complete replication (ROTHSTEIN et al. 2000 ). In agreement with this, the defect in recombination caused by a rad52 disruption further exacerbates Swi4-t-induced lethality in rad53-11 cells, but has no detectable effect on RAD53 Swi4-t cells.

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 (IVESSA et al. 2000 ). Our work suggests the possibility that cells that experience precocious S phase due to a hyperactivation of a G1-to-S transition regulator acquire genomic instability precisely because these regulators promote an increase in the number of stalled replicative forks.

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 (VAN DE VLIET 1999 ). An interesting possibility is raised by the recent report by MASER et al. 2001 , which finds enhanced binding of mammalian E2F1 near active replication origins. This and other findings (ROYZMAN et al. 1999 ) imply that transcriptional activators important for the G1-to-S transition could have a direct role in DNA replication control.

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 (VALLEN and CROSS 1995 , VALLEN and CROSS 1999 ). However, CLN overproduction is not sufficient to kill either mec1-1 or rad53-11 cells in the A364a background. This difference may be attributed to the state of the SML1 locus (ZHAO et al. 1998 ); however, this locus is certainly not the only one that differs between these strains. In mec1 and rad53 cells, Sml1-mediated inhibition of the ribonucleotide reductase is not relieved (ZHAO et al. 1998 ) and thus dNTP levels may be limiting. In the VALLEN and CROSS study (1999), the lethality of G1 cyclin overexpression to the mec1 mutants could be explained by a concomitant decline in the mRNA level of the large subunit of the ribonucleotide reductase Rnr1 (VALLEN and CROSS 1999 ), which further depletes dNTP pools. In our case, however, Swi4-t overproduction is lethal to rad53-11 cells even though it upregulates RNR1 transcription (J. SIDOROVA, unpublished data), and SML1 is inactivated (PAULOVICH et al. 1997 ). Hence, Rnr1 is not likely to be the cause of the Swi4-t-induced lethality, even though it can mitigate the lethal consequences of Swi4-t overproduction, when it, too, is overexpressed. Moreover, suppression of the lethality of GAL::SWI4-t can also be achieved by overexpression of SRL1 (J. SIDOROVA, unpublished data), and thus it is not specific to RNR1. SRL1, whose function is unknown, is another Swi4-activated gene (IYER et al. 2001 ), and like RNR1, it can suppress the lethality of the rad53 deletion when highly overexpressed (DESANY et al. 1998 ). All these observations suggest that the Swi4-t-induced lethality cannot be tracked to one Swi4-t target gene and may instead result from altered expression of many targets.

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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