Replication-independent MCB Gene Induction and Deoxyribonucleotide Accumulation at G1/S in Saccharomyces cerevisiae*

Ahmet KoçDagger , Linda J. Wheeler§, Christopher K. MathewsDagger §, and Gary F. MerrillDagger §

From the Dagger  Genetics Program and § Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331

Received for publication, December 20, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In Saccharomyces cerevisiae, many genes encoding enzymes involved in deoxyribonucleotide synthesis are expressed preferentially near the G1/S boundary of the cell cycle. The relationship between the induction of deoxyribonucleotide-synthesizing genes, deoxyribonucleoside triphosphate levels, and replication initiation was investigated using alpha  factor-synchronized wild-type yeast or dbf4 yeast that are temperature-sensitive for replication initiation. Neither the timing nor extent of gene induction was inhibited when alpha  factor-arrested dbf4 cells were released into medium containing the ribonucleotide reductase inhibitor hydroxyurea, which blocks replication fork progression, or were released at 37 °C, which blocks replication origin firing. Thus, the induction of deoxyribonucleotide-synthesizing genes at G1/S was fully independent of DNA chain elongation or initiation. Deoxyribonucleoside triphosphate levels increased severalfold at G1/S in wild-type cells and in dbf4 mutants incubated at the non-permissive temperature. Thus, deoxyribonucleoside triphosphate accumulation, like the induction of deoxyribonucleotide-synthesizing genes, was not dependent on replication initiation. Deoxyribonucleoside triphosphate accumulation at G1/S was suppressed in cells lacking Swi6, a transcription factor required for normal cell cycle regulation of deoxyribonucleotide-synthesizing genes. The results suggest that cells use gene induction at G1/S as a mechanism to pre-emptively, rather than reflexively, increase the synthesis of DNA precursors to meet the demand of the replication forks for deoxyribonucleotides.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In all eukaryotic cells many of the genes encoding enzymes involved in DNA precursor synthesis are induced as cells reach the G1/S boundary of the cell cycle. In budding yeast, this class of genes contains an upstream element termed a Mlu1 cell cycle box (MCB)1 that binds a multimeric complex containing the proteins Swi6 and either Swi4 or Mbp1 (1-4). Mutation of the MCB elements in the upstream region of the TMP1 (CDC21) gene encoding thymidylate synthase strongly inhibits promoter activity (5). Deletion of the SWI6 gene disrupts normal cell cycle-regulated expression of several MCB element-containing genes (1, 2). In mammalian cells, genes preferentially expressed at G1/S also contain an identifiable upstream element, termed an E2F site, that resembles the yeast MCB element and that binds heterodimers of the E2F and DP1 family of proteins (6).

Experiments described herein address two questions regarding MCB gene induction at G1/S. First, we ask whether MCB gene induction is in part dependent on DNA replication. In a screen for yeast mutations that result in rescued expression of MCB-dependent reporter genes in Delta swi6 null yeast, thioredoxin reductase was identified as a negative regulator of MCB element activity (7). Because thioredoxin is a donor of electrons in the reaction catalyzed by ribonucleotide reductase (RNR), and increased flux through RNR would be expected to occur as cells entered S phase and began consuming dNTPs, we speculated that a transient episode of RNR-mediated thioredoxin oxidation may contribute to MCB gene induction at G1/S (7). Implicit in this model is the prediction that MCB gene induction should be suppressed under conditions in which replication initiation is inhibited. In higher eukaryotes, the timing of replication initiation can be determined accurately by assaying for incorporation of bromodeoxyuridine or radiolabeled thymidine. The absence of a thymidine kinase enzyme precludes similar determination in yeast. The transition from G1 to S in yeast is generally assayed by flow cytometry. However, flow cytometric analysis of bulk DNA content is not a sensitive measure of the time at which cells begin synthesizing DNA. Thus, the relative timing of MCB gene induction with respect to replication origin firing is unclear. Genetic experiments have established that other events that occur at about the time of the G1/S transition occur independently of DNA replication. For example, bud emergence and spindle pole body duplication occur on schedule after START when DNA chain elongation or initiation is inhibited by using the ribonucleotide reductase inhibitor hydroxyurea (HU) or various cdc mutations (8). Similar experiments on MCB gene induction have either not been done or yielded equivocal results. In asynchronous yeast, HU has been shown to induce several MCB-containing genes, including RNR1, RNR2, and TMP1 (9, 10), suggesting that replication was not necessary for MCB gene induction. However, HU triggers the replication-arrest checkpoint (11) and induction of MCB genes due to triggering of this checkpoint may mask whether replication is required for MCB gene induction at G1/S during a normal cell cycle. A more definitive experiment was done by White and colleagues (12), who showed that the induction of three MCB genes (CDC8, CDC9, and TMP1) occurred on schedule in synchronized cdc4 mutants that were incubated at the non-permissive temperature for S phase entry. Although the result implies that DNA replication is not required for the timing of MCB gene induction, the level of MCB gene induction was significantly reduced at the non-permissive temperature. CDC4 encodes a ubiquitin ligase needed for destruction of the Cdc28/Clb inhibitor Sic1. Full induction of MCB gene expression at G1/S may thus require either DNA synthesis or some other process downstream from Cdc28/Clb activation. Analysis of MCB gene induction in a temperature-sensitive dbf4 mutant, in which replication origin firing is specifically inhibited at the non-permissive temperature (13), would address this issue.

A second question concerning MCB gene induction at G1/S is whether the increased levels of mRNAs encoding DNA precursor-synthesizing enzymes are associated with changes in dNTP levels. Ribonucleotide reductase, thymidylate synthase, and thymidylate kinase are essential enzymes dedicated to synthesizing DNA precursors. It is generally assumed that a mechanism for inducing these genes at G1/S evolved to meet the demands of the replication forks for DNA precursors. However, this assumption has not been tested. Also untested is whether replication initiation is associated with a decrease in dNTP levels (through their consumption at replication forks), constant dNTP levels (through allosteric regulation of dNTP-synthesizing enzymes), or an increase in dNTP levels (through non-allosteric mechanisms such as MCB gene induction).

To address the first of these questions, we measured MCB gene mRNA levels in alpha  factor-synchronized dbf4 yeast that were incubated with HU to block DNA chain elongation or were incubated at the non-permissive temperature to block DNA chain initiation. To address the second question we measured dNTP levels in alpha  factor-synchronized wild-type yeast and dbf4 yeast at the permissive and non-permissive temperature, and in Delta swi6 yeast that have previously been shown to express MCB genes throughout the cell cycle (1, 2). Our results show that MCB gene induction and dNTP accumulation occur at G1/S in the absence of replication initiation and that Swi6 protein is required for a normal pattern of dNTP accumulation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Media, Strains, and General Methods

Cells were grown in YEPD medium (1% bacto-yeast peptone, 2% bacto-peptone, 2% dextrose). Yeast strains are listed in Table I. MY273 and SY2626 are derivatives of W303-1 that carry a bar1 mutation to facilitate alpha  factor synchronization. To derive the dbf4-1 bar1 strain MY317, MY273 was mated to L128-20 (14) and random spores from the resulting diploid were screened for alpha  factor and temperature sensitivity. G1-arrested cells were obtained by treating exponentially growing bar1 cells with 100 ng/ml alpha  factor for 3 h at 25 °C. To achieve synchrony, arrested cells were collected on Whatman #1 filters, washed once with fresh YEPD and once with 100% conditioned medium, resuspended in 25% conditioned medium, and incubated at either 25 °C or 37 °C. Conditioned medium was prepared by growing W303-1a cells to saturation in YEPD and removing the cells by filtration. HU was used at a final concentration of 0.1 M.

                              
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Table I
Yeast strains

Plasmids

The plasmid pBS-TRT1 was constructed by inserting a 1400-bp HindIII fragment of pTRT1 containing the H2A and ADK1 genes (15) into the EcoRV site of pBS-KS(+). The plasmids pBS-H2A and pBS-ADK1 were constructed from pBS-TRT1 by dropping out a 750-bp SacII fragment containing the ADK1 gene or a 1143-bp HincII fragment containing the H2A gene, respectively. Plasmid pBS-TMP1 was described previously (7).

Flow Cytometry

About 5 × 106 cells were collected by centrifugation and fixed in 70% ethanol overnight. After microcentrifugation (10,000 rpm, 5 s), the cell pellet was washed with 100 µl of 50 mM sodium citrate buffer (pH 7.0), resuspended in 100 µl of sodium citrate buffer, and sonicated for 6 s. After a 1-h incubation at 55 °C with RNase A (0.25 mg/ml) and a 1-h incubation with proteinase K (1 mg/ml), 100 µl of sodium citrate buffer containing 16 µg/ml propidium iodide was added and samples were incubated at 4 °C overnight in the dark. A Beckman Coulter Epics XL flow cytometer was used to analyze the samples. For each histogram, 25,000 cells were analyzed.

Messenger RNA Analysis

Northern Blot Analyses-- Total cellular RNA was isolated from yeast by using glass beads and hot phenol (16) and quantitated by A260, assuming 1 OD = 40 µg/ml. Using a NorthernMax kit (Ambion Inc., Austin, TX), 8 µg of RNA from each sample was fractionated by formaldehyde/agarose gel electrophoresis and blotted to a positively charged BrightStar Plus nylon membrane (Ambion Inc.). After UV cross-linking at 1200 J using a Stratalinker (Stratagene, La Jolla, CA), membranes were prehybridized and hybridized with probes as described by the kit manufacturer. Probes (>2 × 108 cpm/µg) were made using [alpha -32P]dCTP (3000 Ci/mM, Amersham Biosciences, Piscataway, NJ) and a random priming DNA labeling system kit (Invitrogen, Gaithersburg, MD) as described by the manufacturer, except that incorporated radioactivity was isolated by using a QIAquick nucleotide removal kit (Qiagen, Valencia, CA). Hybridization probes were: RNR1, 2.6-kb EcoR1 fragment from pSE738 (S. Elledge, Baylor College of Medicine, Houston, TX); CDC9, 2.7-kb SstI fragment from pR12SeLig2 (L. Johnston, National Institute for Medical Research, London, UK); H2A/ADK1, 2.3-kb SstI fragment from pTRT1 (15). Blots were exposed to PhosphorImager plates for 3 days and analyzed using a PSI486 PhosphorImager and ImageQuaNT software (Amersham Biosciences).

Ribonuclease Protection Assay-- The plasmid pBS-TMP1 was cleaved with EcoR1 and transcribed with T3 RNA polymerase to generate a 1314-base TMP1 pseudo-RNA, or cleaved with SalI and transcribed with T7 RNA polymerase to generate a radiolabeled 269-base riboprobe that was complementary to a 200-base sequence near the 3'-end of the TMP1 mRNA. The plasmid pBS-H2A was cleaved with PvuII and transcribed with T3 RNA polymerase to make an 860-base H2A pseudo-RNA, or cleaved with HindIII and transcribed with T7 RNA polymerase to generate a 735-base riboprobe that was complementary to a 356-base segment of the H2A mRNA. The plasmid pBS-ADK1 was cleaved with HindIII and transcribed with T3 RNA polymerase to make a 311-base ADK1 pseudo-RNA or was cleaved with BglII and transcribed with T7 RNA polymerase to give a 129-base riboprobe that was complementary to a 48-base segment of the ADK1 mRNA. In vitro transcription and RNase protection protocols were as described by Schmidt and Merrill (17) except that chicken embryo RNA was used as carrier instead of yeast RNA.

Deoxyribonucleoside Triphosphate Pool Measurements

Approximately 3 × 108 yeast cells were harvested and extracted as described by Muller (18). Each precipitated sample was resuspended in 200 µl of cold deionized H2O and assayed for each of the four dNTPs by the DNA polymerase-based enzymatic method (19), which is based on the incorporation of a limiting dNTP by Klenow DNA polymerase in the presence of an excess of 3H-labeled complementary dNTP with poly(dA-dT) or poly(dI-dC) as template. Raw dNTP pool data (moles per sample) were normalized by dividing by the number of cells per sample (to obtain attomoles/cell) or by the cellular volume per sample (to obtain the molar concentration). Cell number per sample was determined by hemacytometer counting or flow cytometry. Cellular volume per sample was calculated from the A600 of the culture at the time of harvesting (1 OD = 1.0 µl of cellular volume). The relationship between optical density and cellular volume was spectrophotometer-specific and was established experimentally for our instrument using exponentially growing wild-type yeast (42 femtoliters per haploid cell) as a standard (20).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MCB Gene Induction Occurs Independently of Replication Initiation-- MCB gene induction occurs at about the same time as replication initiation in yeast synchronized by a variety of methods (1, 5, 21). To determine whether replication contributes to MCB gene induction, the levels of several MCB gene mRNAs were determined in alpha  factor-synchronized dbf4 mutant cells that were incubated at the permissive temperature in the absence or presence of HU, or were incubated at the non-permissive temperature for Dbf4 protein function.

We first concentrated on the effect of HU on MCB gene induction. HU inhibits ribonucleotide reductase by destroying a tyrosyl radical essential for catalytic activity (22) and has been shown to block replication in a variety of cells. To determine the kinetics with which cells synthesized DNA in the absence of HU and to confirm that cells did not synthesize DNA in the presence of HU, cells were stained with propidium iodide and DNA content was determined by flow cytometry (Fig. 1A). After a 3-h preincubation with alpha  factor, all cells had a 1C DNA content, confirming that the pheromone had arrested all cells at START. When released from alpha  factor in the absence of HU, a shift in DNA content indicative of S phase entry was observed within 40 min. In contrast, when released from alpha  factor in the presence of HU, cells retained a 1C DNA content throughout the course of the experiment.


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Fig. 1.   Effect of HU on replication and MCB gene expression in alpha  factor-synchronized cells. DNA content and MCB gene mRNA levels were determined in yeast that were asynchronously growing (a), arrested in G1 by incubation in alpha  factor (0), or released from alpha  factor for the indicated number of minutes in the absence or presence of HU. A, flow cytometric analysis of DNA content. G1 and G2/M peaks are labeled 1C and 2C, respectively. In the absence of HU, an increase in DNA content, which is indicative of S phase entry, was observed 40 min after alpha  factor release. In the presence of HU, cells retained a 1C complement of DNA throughout the course of the experiment. B, Northern blot analysis of MCB gene mRNA levels. Blots were sequentially hybridized with probes specific for the mRNA product of three MCB-regulated genes: RNR1 (encoding the large subunit of RNR), RNR2 (encoding the small subunit of RNR), and CDC9 (encoding DNA ligase). Blots were co-hybridized with probes for the constitutive ADK1 mRNA and the S phase-specific H2A mRNA, to serve as loading controls and synchrony controls, respectively. C, quantitation of Northern blot mRNA data by densitometry. The signal intensity of the RNR1, RNR2, CDC9, and H2A mRNA band in each lane was normalized to the signal intensity of the ADK1 mRNA band in that lane.

Fig. 1B shows Northern blot analyses of three representative MCB gene mRNAs (RNR1, RNR2, and CDC9). RNR1 and RNR2 encode the large and small subunits of ribonucleotide reductase, respectively. CDC9 encodes DNA ligase. The constitutive ADK1 mRNA, encoding adenylate kinase, was used as a loading control, and the S phase-specific H2A mRNA was used to assess the fraction of replicating cells in the population (15). As expected, the levels of all three MCB gene mRNAs decreased when asynchronous cells were arrested in G1 by treatment with alpha  factor. Also as expected, all three MCB gene mRNAs transiently increased when cells were released from alpha  factor in the absence of HU. MCB gene mRNA levels began to increase by 20 min after release and reached maximal levels by 40 min. Significantly, all three MCB gene mRNAs also increased on schedule when cells were released from alpha  factor in the presence of HU. In contrast, induction of the S-phase-specific H2A gene was almost completely inhibited in the presence of HU, confirming that replication-dependent H2A gene expression was suppressed in HU-treated cells. Fig. 1C shows densitometric quantitation of the Northern blot data. Using the average of both zero time values as the baseline and the 40-min value as the maximally induced level, RNR1 mRNA increased 16-fold in the absence of HU and 18-fold in the presence of HU. Over the same time interval, RNR2 mRNA increased 1.6-fold in the absence of HU and 2.6-fold in the presence of HU, and CDC9 mRNA increased 2.5-fold in the absence of HU and 2.9-fold in the presence of HU. Thus, not only was the timing of MCB gene induction unaffected by the presence of HU, but the magnitude of the induction was unaffected as well. Similar results were obtained when cells were synchronized by release from a cdc15-induced mitotic arrest (data not shown).

Interestingly, although MCB gene mRNA levels increased on schedule in HU-treated cells, they did not decrease on schedule. High MCB mRNA levels persisted in HU-treated cells, long after non-treated control cells showed a decrease in levels (Fig. 1, B and C, and data not shown). In fact, RNR2 mRNA continued to increase in HU-treated cells throughout the duration of the experiment. Persistence of MCB gene messages at late time points in HU-treated cells suggested either that DNA replication was necessary to repress MCB gene expression in late S phase or G2 or that the HU-induced replication arrest was triggering MCB gene expression by a separate mechanism that camouflaged the normal down-regulation of MCB gene expression late in the cell cycle. Experiments below, in which Dbf4 inactivation was used to block replication, favor the latter explanation.

The observation that MCB gene induction in the presence of HU occurred at about the same time and to at least the same extent as in the absence of HU implied that replication per se was not a prerequisite for MCB gene induction. However, as HU blocks only DNA chain elongation and not chain initiation, it remained possible that events closely associated with replication origin firing contributed to MCB gene induction at G1/S.

To address the question of whether replication initiation, as opposed to DNA chain elongation, was required for MCB gene induction, we analyzed MCB gene mRNA levels in alpha  factor-synchronized dbf4 cells incubated at the permissive and non-permissive temperature. Dbf4 protein, together with Cdc7, forms a protein kinase holoenzyme that phosphorylates the origin-associated protein Mcm2 and catalyzes one of the last known steps in the chain of events linking START and origin firing (13). At the non-permissive temperature, dbf4 cells released from alpha  factor are able to bud and accumulate mass but are unable to initiate replication (23).

As shown by the flow cytometry data in Fig. 2A, alpha  factor-synchronized dbf4 cells began to enter S phase about 40 min after release at the permissive temperature (25 °C) but did not enter S phase when released at the non-permissive temperature (37 °C). Fig. 2B shows a Northern blot analysis of RNR1, RNR2, and CDC9 mRNA, as well as SWI4 mRNA. The SWI4 gene contains MCB elements, is induced in late G1, and encodes a transcription factor that may autostimulate MCB gene expression (24). The levels of all four MCB gene mRNAs decreased when asynchronous dbf4 cells were arrested in G1 by treatment with alpha  factor. When alpha  factor was removed and the cells were incubated at the permissive temperature (25 °C), all four MCB gene mRNAs transiently increased, reaching maximal levels in about 20 min. Significantly, when dbf4 cells were released from alpha  factor at the non-permissive temperature (37 °C), RNR1, RNR2, CDC9, and SWI4 mRNA increased to the same extent and with the same kinetics as when cells were released at 25 °C. In contrast, the increase in histone H2A mRNA, which peaked 60 min after alpha  factor release at 25 °C, was strongly inhibited in cells released from alpha  factor at 37 °C, confirming that replication-dependent H2A gene expression was suppressed in dbf4-arrested cells.


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Fig. 2.   MCB gene mRNA levels in alpha  factor-synchronized dbf4 cells incubated at the permissive or non-permissive temperature for replication initiation. DNA content and MCB gene mRNA levels were determined in dbf4 cells (strain MY317) that were asynchronously growing (a), arrested in G1 by incubation in alpha  factor (0), or released from alpha  factor for the indicated number of minutes at the permissive (25 °C) or non-permissive (37 °C) temperature. A, flow cytometric analysis of DNA content. B, Northern blot analysis of MCB gene mRNA levels. Blots were sequentially hybridized with probes specific for the mRNA product of four MCB-regulated genes: RNR1, RNR2, CDC9, and SWI4. Blots were co-hybridized with probes for the constitutive ADK1 mRNA and the S phase-specific H2A mRNA, to serve as loading controls and synchrony controls, respectively. C, quantitation of Northern blot mRNA data by densitometry. The signal intensity of the RNR1, RNR2, CDC9, SWI4, and H2A mRNA band in each lane was normalized to the signal intensity of the ADK1 mRNA band in that lane. D, RNase protection assay of TMP1 (CDC21) mRNA levels. TMP1 encodes thymidylate synthase and is the gene for which MCB element function has been best characterized (5). The constitutive ADK1 mRNA and S phase-specific H2A mRNA were assayed in parallel to correct for unequal loading and to monitor synchrony, respectively. Samples containing only the chick RNA used as carrier were analyzed in lanes labeled "C." A dilution of undigested probe was run in lanes labeled "P." (The undigested ADK1 probe migrated too slowly to fit within the cropped photograph.) To determine absolute mRNA levels in experimental samples, known amounts of synthetic pseudo-mRNA (psi RNA) were assayed in parallel to generate a standard curve. Italicized numbers below each lane show the calculated absolute level of mRNA, in molecules per cell, for each experimental sample.

Densitometric quantitation of the Northern blot results is shown in Fig. 2C. In this experiment, peak RNR1, SWI4, and CDC9 mRNA levels occurred 20 min after pheromone release at both 25 °C and 37 °C. Using the average of both zero-time values as baseline, RNR1 mRNA increased 20-fold at 25 °C and 39-fold at 37 °C, SWI4 mRNA increased 10-fold at 25 °C and 9-fold at 37 °C, and CDC9 mRNA increased 8-fold at 25 °C and 9-fold at 37 °C. Peak RNR2 mRNA occurred 60 min after release at 25 °C and 40 min after release at 37 °C. RNR2 increased 7-fold at both 25 °C and 37 °C. In contrast to MCB gene mRNAs, which increased roughly the same amount at both 25 °C and 37 °C, histone H2A mRNA accumulation was inhibited at 37 °C. H2A mRNA peaked 60 h after pheromone release, by which time it had increased 19-fold at 25 °C and only 2.7-fold at 37 °C. H2A gene transcription is tightly linked to replication (15), and the inhibition of H2A mRNA accumulation observed at 37 °C supports the flow cytometry data showing that very few cells entered S phase in the dbf4 population incubated at 37 °C.

To substantiate the conclusion that MCB gene induction was independent of replication initiation, the levels of a fifth MCB gene mRNA were determined in dbf4 cells released from alpha  factor at the permissive and non-permissive temperature. The TMP1 gene (CDC21), which encodes thymidylate synthase, contains two canonical MCB elements in its upstream region, and is the gene in which the cis-acting function of MCB elements is best characterized (5). Because TMP1 mRNA is difficult to detect by Northern blot assay, an RNase protection assay was used to quantitate this message. In addition, by analyzing known amounts of synthetic TMP1 pseudo-mRNA, the absolute level of TMP1 mRNA in experimental samples was determined. As shown in Fig. 2D, asynchronous cultures had an average of eight TMP1 mRNA molecules per cell, and this number decreased to one molecule per cell in alpha  factor-treated cultures. By 30 min after alpha  factor release at the permissive temperature, TMP1 mRNA had increased to 14 molecules per cell. Significantly, a similar increase in TMP1 mRNA, to 19 molecules per cell, occurred by 30 min after alpha  factor release at the non-permissive temperature.

In summary, the results in Fig. 2 show that MCB gene induction is wholly independent of replication initiation. MCB gene induction occurred at the same time and to an equal extent in dbf4 cells incubated at either the permissive or non-permissive temperature for replication initiation. The results are inconsistent with models in which MCB gene induction is triggered or modulated by signals potentially associated with S phase entry, such as dNTP depletion or RNR-mediated oxidation of thioredoxin to meet the demands of the replication forks for dNTPs (7). It should also be noted that S phase entry was not needed for MCB gene repression at later time points, because MCB gene mRNA levels decreased with similar kinetics at 25 °C and 37 °C at later times after alpha  factor release. Thus, the earlier noted persistence of MCB gene messages in HU-treated cells was probably due to the replication-arrest checkpoint mechanism rather than a requirement for DNA replication for MCB repression in late S phase. In summary, the above results indicated that neither DNA chain initiation nor elongation was necessary for MCB gene induction at the G1/S boundary of the cell cycle.

Deoxyribonucleoside Triphosphates Accumulate at G1/S during the Cell Cycle-- Many MCB genes encode enzymes dedicated to DNA precursor biosynthesis. It was not clear whether induction of these genes at G1/S served to increase dNTP levels or to restore dNTP levels that were depleted by the replication process. To address this question we measured the levels of the four deoxyribonucleoside triphosphates in yeast cells synchronized by alpha  factor block and release.

Fig. 3B shows dNTP pool data represented on a per cell basis. Asynchronous cells contained 1.68 attomoles of dTTP, 1.36 attomoles of dATP, 0.64 attomoles of dCTP, and 0.52 attomoles of dGTP. Thus, asynchronous yeast contained a total of 1.8 × 106 dNTP molecules or about 7.5% of that minimally needed for replication of the genome. The levels of all four dNTPs decreased when asynchronous cells were incubated with alpha  factor. When alpha  factor was removed, the levels of all four dNTPs transiently increased, reaching maximum levels in about 80 min. Parallel flow cytometry measurements of DNA content (Fig. 3A) confirmed that asynchronously growing cells arrested with a 1C complement of DNA when incubated in alpha  factor and that most cells entered S phase about 40 min after pheromone was removed. Similar results were obtained when dNTP pools were measured in cell synchronized by release from a cdc15-induced mitotic block (data not shown).


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Fig. 3.   Deoxyribonucleoside triphosphate levels in alpha  factor-synchronized wild-type yeast. DNA content and dNTP levels were determined in wild-type cells (strain SY2626) that were asynchronously growing (a), arrested in G1 by incubation in alpha  factor (0), or released from alpha  factor for the indicated number of minutes. A, flow cytometric analysis of DNA content. DNA content began to increase at 40 min after release. B, deoxyribonucleotide pool sizes normalized on a per cell basis. C, deoxyribonucleotide pool sizes normalized to cellular volume. Each bar and vertical line represents the mean ± range for duplicate determinations.

In comparing dNTP pool sizes in synchronized cells, it is important to consider cell volume changes during the cell cycle. Volume changes are also an important consideration when comparing dNTP levels in yeast carrying mutations that affect cell size, or when comparing the -fold change in dNTP levels to the -fold change in mRNA levels. For these reasons, the molar concentration of dNTPs is a more useful measure of dNTP pool size changes during the cell cycle. When pools were estimated on a molar basis (Fig. 3C), dTTP increased 8-fold, dATP and dCTP increased 9-fold, and dGTP increased 4-fold by 80 min after release. The average dNTP pool increase was smaller than the increase in RNR1 or TMP1 mRNA (Fig. 2), but this was expected as dNTPs were being consumed by the process of replication. The failure of the small dGTP pool to increase as much as the others could be due to its greater percent consumption by the replication process.

When dNTP pools are represented on a molar basis (Fig. 3C), the decrease in pool sizes at later times in the cell cycle was not as pronounced as when pools were represented on a per cell basis (Fig. 3B). This is because a wave of cell division occurs at about 100 min after alpha  factor release and sharply increases the denominator used in calculating dNTP levels per cell.

The data shown in Fig. 3 are representative of the results obtained in three repeat experiments. Although the absolute dNTP concentration determined in separate experiments varied somewhat, the pattern of low dNTP concentrations in pheromone-arrested cells and multifold increases in dNTP concentrations beginning at about the time of S phase entry was consistently observed. By averaging the results obtained in separate experiments, we estimate the average concentrations of dNTPs in asynchronously growing wild-type cells to be 58 µM for dTTP, 38 µM for dATP, 21 µM for dCTP, and 14 µM for dGTP. These values roughly agree with previously published measurements (18, 25, 26).

The increase in dNTP concentrations upon S phase entry indicates that dNTP synthesis is subject to more than allosteric control. Although ribonucleotide reductase, the principal regulated enzyme in dNTP synthesis, is subject to feedback inhibition by dNTPs, a purely allosteric mechanism for dNTP synthesis control would predict that dNTP levels would either decrease or remain relatively constant when cells started to replicate their DNA. The observation that dNTP levels significantly increased as cells entered S phase implies that non-allosteric mechanisms must exist that allow the cell to more than compensate for the consumption of dNTPs by the replication machinery. A transcriptionally mediated increase in the level of dNTP-synthesizing enzymes is one of several non-allosteric mechanisms that may contribute to increased dNTP synthesis as cells enter S phase. Other mechanisms include phosphorylation control of ribonucleotide reductase or control of the level or activity of proteins that interact with ribonucleotide reductase, such as Sml1 (26).

Deoxyribonucleoside Triphosphate Accumulation Occurs Independently of Replication Initiation-- Deoxyribonucleotide accumulation in wild-type cells began at about the same time as replication initiation. Using the temperature-sensitive dbf4 mutation to block replication, we asked whether replication initiation was required for dNTP accumulation. Cells were released from alpha  factor at the permissive or non-permissive temperature. Flow cytometry established that most of the dbf4 cells arrested with a 1C complement of DNA in alpha  factor and started to replicate their DNA within 40 min of pheromone removal (Fig. 4A). At the non-permissive temperature (37 °C), dbf4 cells retained a 1C DNA content for at least 80 min after pheromone removal and then showed an increase in DNA content that suggested that dbf4 cells were able to bypass the replication initiation block if incubated longer than 80 min at 37 °C. Even when the experiment was repeated at 39 °C, dbf4 cells were still able to bypass the replication initiation block after 80 min. Although DNA replication was evident at later time points, it was clear that replication initiation was inhibited for at least the first 80 min after pheromone release and that the cells could be used to test the relationship between replication initiation and dNTP accumulation.


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Fig. 4.   Deoxyribonucleotide levels in alpha  factor-synchronized dbf4 cells incubated at the permissive or non-permissive temperature for replication initiation. DNA content and dNTP levels were determined in dbf4 cells (strain MY317) and wild-type cells (strain SY2626) that were asynchronously growing (a), arrested in G1 by incubation in alpha  factor (0), or released from alpha  factor for the indicated number of minutes at the indicated temperature. A and B, DNA content and dNTP levels in dbf4 cells. DNA content began to increase 40 min after release at 25 °C but did not increase until 100 min after release at 37 °C. C and D, DNA content and dNTP levels in wild-type cells. DNA content began to increase 40 min after release at both 25 °C and 37 °C. In the dNTP pool histograms, open bars, shaded bars, and black bars represent dNTP levels in asynchronous cells, cells released at 25 °C and cells released at 37 °C, respectively. Indicated values are the mean of duplicate determinations.

Deoxyribonucleotide pool data for synchronized dbf4 cells are shown in Fig. 4B. All four dNTP pools decreased when asynchronous cells were arrested in G1 using alpha  factor. When released at the permissive temperature, the levels of all four dNTPs began to increase by 20 min and reached a maximum by 40 min. A small decrease in dNTPs was observed at 60-80 min, followed by a recovery to maximal levels by 120-140 min. Flow cytometry suggested that the transient decrease and recovery was due to cells completing one replication cycle by 80 min and re-initiating a new cycle by 140 min. The difference between minimal and maximal dNTP levels in dbf4 cells was not as great as in wild-type cells (compare shaded bars in Fig. 4, B and D). This was partly due to higher dNTP levels in alpha  factor-arrested dbf4 cells.

When dbf4 cells were released from alpha  factor at the non-permissive temperature, the levels of all four dNTPs again began to increase by 20 min, but continued to increase throughout the course of the experiment (Fig. 4B). We speculate that dNTPs super-accumulated at 37 °C in dbf4 cells, because they were not being incorporated into DNA. To control for a general effect of elevated temperature on dNTP accumulation, wild-type cells were assayed in parallel with dbf4 cells (Fig. 4, C and D). At both 25 °C and 37 °C, the levels of all four dNTPs in wild-type cells began to increase 20 min after pheromone release and reached a maximum by 60 min. Incubation at the higher temperature increased the maximal level of dATP about 1.7-fold but had little effect on the maximal levels of dTTP, dCTP, or dGTP. Thus, part of the 4.8-fold increase in maximal dATP levels observed in dbf4 cells incubated at 37 °C may be due to a general effect of elevated temperature. If this temperature effect component is subtracted, inactivation of Dbf4 protein led to 3.1-fold increase in maximal dATP levels. Because incubation at 37 °C had no effect on maximal levels of dTTP, dCTP, and dGTP in wild-type cell, we conclude the 2.1- to 2.9-fold increase in maximal dTTP, dCTP, and dGTP levels observed at 37 °C in dbf4 cells was due to inactivation of Dbf4 protein. As dNTP levels increased on schedule in dbf4 cells incubated at the non-permissive temperature, and indeed super-accumulated at later time points, the process triggering dNTP accumulation at the G1/S boundary of the cell cycle was not dependent on Dbf4 function and was therefore not dependent on replication initiation.

Deoxyribonucleoside Triphosphate Accumulation Is Disrupted in Delta swi6 Cells-- The increase in dNTP levels in synchronized cells occurred shortly after the increase in MCB gene mRNA levels. Thus, dNTP accumulation at G1/S may in part be due to a transcriptionally mediated increase in the levels of dNTP-synthesizing enzymes. To test the cause and effect relationship between MCB gene induction and dNTP levels, a yeast strain defective in MCB gene regulation was analyzed. MCB elements bind a complex consisting of Swi6 and either Mbp1 or Swi4 (1-3). In Delta swi6 null cells, MCB genes are expressed but are no longer regulated normally during the cell cycle (1, 2). For example, rather than being expressed at high levels only in G1/S cells, RNR1 and TMP1 mRNA are expressed at an intermediate level throughout the cell cycle in Delta swi6 cells (1). Using alpha  factor-synchronized Delta swi6 cells, we investigated whether MCB gene mRNA induction at G1/S was required for dNTP accumulation at G1/S. Flow cytometry showed that Delta swi6 cells arrested with a 1C complement of DNA when incubated in alpha  factor and entered S phase about 40 min after alpha  factor release (Fig. 5A). Asynchronous Delta swi6 cells (Fig. 5B) had higher levels of all four dNTPs than asynchronous wild-type cells (Fig. 3C). This was consistent with previously published results showing that asynchronous Delta swi6 cells generally have higher levels of MCB gene mRNAs than asynchronous wild-type cells (1, 2). Deoxyribonucleotide measurements in synchronized cells showed that dNTP pool dynamics were significantly affected by the Delta swi6 mutation. When Delta swi6 cells were released from alpha  factor and progressed into S phase, the 1.5-fold increase in dATP and 1.9-fold increase in dCTP levels (Fig. 5B) were significantly less than the 9-fold increase in dATP and dCTP levels observed in wild-type cells (Fig. 3C). Furthermore, dTTP and dGTP did not increase at all, and in fact decreased, as Delta swi6 cells progressed into S phase. Oddly, dNTP levels generally were higher in asynchronous Delta swi6 cells than at any time point in the synchronized population. Although we do not know the basis for this incongruous and yet reproducible result, it is tangential to the main question addressed by the experiment, which was to determine whether the Delta swi6 mutation affected the normal pattern of dNTP accumulation during the cell cycle. The observation that the dATP and dCTP pools continued to show a small residual increase when Delta swi6 cell entered S phase indicated that dNTP pool changes were not entirely mediated by Swi6-dependent MCB gene regulation. Furthermore, we cannot rule out the possibility that pleiotrophic effects of the Delta swi6 mutation affected dNTP levels. Nevertheless, the fact that dATP and dGTP accumulation was partially suppressed and dTTP and dGTP accumulation was completely suppressed in Delta swi6 cells was consistent with the model that MCB gene induction at G1/S contributed to dNTP accumulation.


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Fig. 5.   Deoxyribonucleoside triphosphate levels in alpha  factor-synchronized Delta swi6 yeast cells that express MCB genes throughout the cell cycle. DNA content and dNTP levels were determined in Delta swi6 cells (strain MY409) that were asynchronously growing (a), arrested in G1 by incubation in alpha  factor (0), or released from alpha  factor for the indicated number of minutes. A, flow cytometric analysis of DNA content. DNA content began to increase at 40 min after release. B, dNTP levels. Bars and vertical lines represent the mean ± range for duplicate determinations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MCB gene induction in budding yeast occurs at about the same time after START as bud emergence, spindle pole body duplication, and replication initiation. Mutational analyses had established that bud emergence and spindle pole body duplication are not dependent on replication initiation (8). For example, numerous cdc mutations that prevent replication initiation at the non-permissive temperature do not prevent bud emergence or spindle pole body duplication. Prior to the current study, it was not clear whether MCB gene induction was similarly independent of replication initiation. It was previously shown that three MCB genes were induced at the normal time but at a lower level in cdc4 cells that were incubated at the non-permissive temperature for S phase entry (12). Furthermore, in an earlier report from the Merrill laboratory (7), we speculated that replication initiation may contribute to MCB gene induction via a mechanism involving RNR-mediated oxidation of thioredoxin. In a genetic screen for mutants that expressed MCB-containing reporter genes in the absence of Swi6, all the mutants isolated had undergone recessive mutations in the TRR1 gene encoding thioredoxin reductase (7). The results suggested that reduced thioredoxin either inactivated an activator or activated a repressor of MCB element activity. Because thioredoxin is known to modulate transcription factor activity in other systems (27-32) and to serve as an electron donor in the reaction catalyzed by ribonucleotide reductase, we speculated that increased flux through RNR at the onset of S phase might result in a transient episode of thioredoxin oxidation, which in turn might contribute to MCB gene induction (7). Although such a substrate-regulated mechanism for MCB gene induction was attractive in its simplicity, the results reported herein are inconsistent with the model. MCB gene induction occurred on schedule in the presence of HU or when replication initiation was blocked by Dbf4 inactivation. In addition to negating the specific model that RNR-mediated oxidation of thioredoxin modulated MCB gene activity, the full induction of MCB genes at G1/S in dbf4 cells incubated at the non-permissive temperature established that MCB gene induction, like bud emergence and spindle pole body duplication, is wholly independent of replication initiation.

Although many of the MCB genes induced at G1/S encode enzymes involved in DNA precursor synthesis, it was not known whether the induction of these genes affected DNA precursor levels. To address the question, we monitored dNTP levels in yeast synchronized by alpha  factor block and release. We found that alpha  factor-arrested G1 cells had lower dNTP levels than asynchronous cells and that the levels of all four dNTPs transiently increased when G1-arrested cells were allowed to re-enter the cell cycle (Fig. 3). Peak dNTP levels occurred about 80 min after alpha  factor release and followed peak MCB gene mRNA levels, which occurred about 40 min after alpha  factor release. The relative timing of the increases in MCB mRNA levels and dNTP levels was consistent with the idea that MCB gene induction contributed to dNTP accumulation. Furthermore, our finding that dNTP accumulation at G1/S was partially disrupted in Delta swi6 cells, which have previously been shown to express MCB genes constitutively (1, 2), also was consistent with the model that changes in MCB gene expression contributed to observed changes in dNTP levels. The size and changes in the dNTP pools we observed in synchronized yeast cells are in accord with previous dNTP pool measurements in synchronized mouse embryo cells (33) and Chinese hamster cells (34), except that the dCTP pool was the largest pool in Chinese hamster cells.

Our dNTP pool measurements indicate that asynchronous wild-type yeast contain a total of 1.8 × 106 dNTP molecules per cell, which is less than 8% of the 2.4 × 107 dNTPs minimally required for genome duplication. Thus, to meet the demands of the replication machinery for DNA precursors, increased synthesis of dNTPs must occur during S phase. Allosteric control of dNTP-synthesizing enzymes cannot account fully for increased dNTP synthesis during S phase, because a purely allosteric mechanism cannot explain how the absolute levels of dNTPs can increase as cell enter S phase. Our results support a model in which MCB gene induction serves to increase the levels of enzymes such as ribonucleotide reductase, thymidylate synthase, and thymidylate kinase, which in turn support a higher rate of DNA precursor synthesis. Furthermore, because both MCB gene induction and dNTP accumulation occur in the absence of DNA chain initiation or elongation, our results suggest that cells have evolved a mechanism to pre-emptively rather than reflexively increase their rate of DNA precursor synthesis to ensure that the replication machinery has an adequate supply of deoxyribonucleoside triphosphates.

    ACKNOWLEDGEMENTS

We thank Julie Oughton, Corwin Willard, and the Oregon State University Environmental Health Sciences Center (supported by NIEHS, National Institutes of Health Center Grant ES00210) for expertise and instrumentation required for flow cytometry, Leland Johnston for supplying dbf4 yeast and MCB gene plasmids, and Robert Sclafani for critically reviewing a draft of the manuscript.

    FOOTNOTES

* This work was supported by National Science Foundation (NSF) Grant MCB9728782 and National Institutes of Health (NIH) Grant CA82633 (to G. F. M.) and by NIH Grant GM55134 and NSF Grant MCB0130760 (to C. K. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 541-737-3119; Fax: 541-737-0481; E-mail: merrillg@ucs.orst.edu.

Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M213013200

    ABBREVIATIONS

The abbreviations used are: MCB, Mlu1 cell cycle box; dNTP, deoxyribonucleoside triphosphate; HU, hydroxyurea; RNR, ribonucleotide reductase; YEPD, yeast extract peptone dextrose; START, point at which yeast becomes committed to complete the cell cycle.

    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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