From the Genetics Program and § Department
of Biochemistry and Biophysics, Oregon State University, Corvallis,
Oregon 97331
Received for publication, December 20, 2002
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ABSTRACT |
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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
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 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 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 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
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
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.
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
factor-synchronized wild-type yeast and dbf4 yeast at the
permissive and non-permissive temperature, and in
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
factor and temperature sensitivity.
G1-arrested cells were obtained by treating exponentially
growing bar1 cells with 100 ng/ml
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.
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 [-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).
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RESULTS |
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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
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 factor, all cells had a 1C DNA content, confirming that the
pheromone had arrested all cells at START. When released from
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
factor in the presence of HU, cells retained a 1C DNA content throughout the course of the experiment.
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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 factor. Also as
expected, all three MCB gene mRNAs transiently increased when cells
were released from
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
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 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
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, 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
factor. When
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
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
factor release at
25 °C, was strongly inhibited in cells released from
factor at
37 °C, confirming that replication-dependent H2A gene expression was suppressed in
dbf4-arrested cells.
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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 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
factor-treated
cultures. By 30 min after
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
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 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 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 factor.
When
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
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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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 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 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
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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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 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
factor-arrested
dbf4 cells.
When dbf4 cells were released from 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
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
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
swi6 cells (1). Using
factor-synchronized
swi6 cells, we investigated whether MCB gene mRNA
induction at G1/S was required for dNTP accumulation at
G1/S. Flow cytometry showed that
swi6 cells
arrested with a 1C complement of DNA when incubated in
factor and
entered S phase about 40 min after
factor release (Fig.
5A). Asynchronous
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
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
swi6
mutation. When
swi6 cells were released from
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
swi6 cells progressed into S phase. Oddly, dNTP levels generally were higher in asynchronous
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
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
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
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
swi6 cells was consistent with the model
that MCB gene induction at G1/S contributed to dNTP
accumulation.
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DISCUSSION |
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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 factor block and release. We found that
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
factor
release and followed peak MCB gene mRNA levels, which occurred
about 40 min after
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
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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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.
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