From the
Mimosine has been reported to specifically prevent initiation of
DNA replication in the chromosomes of mammalian nuclei. To test this
hypothesis, the effects of mimosine were examined in several DNA
replication systems and compared with the effects of aphidicolin, a
specific inhibitor of replicative DNA polymerases. Our results
demonstrated that mimosine inhibits DNA synthesis in mitochondrial,
nuclear, and simian virus 40 (SV40) genomes to a similar extent.
Furthermore, mimosine and aphidicolin were indistinguishable in their
ability to arrest SV40 replication forks and mammalian nuclear
chromosomal replication forks. In contrast to aphidicolin, mimosine did
not inhibit DNA replication in lysates of mammalian cells supplied with
exogenous deoxyribonucleotide triphosphate precursors for DNA
synthesis. Mimosine also had no effect on initiation or elongation of
DNA replication in Xenopus eggs or egg extracts containing
high levels of deoxyribonucleotide triphosphates. In parallel with its
inhibitory effect on DNA synthesis in mammalian cells, mimosine altered
deoxyribonucleotide triphosphate pools in a manner similar to that
reported for another DNA replication inhibitor that affects
deoxyribonucleotide metabolism, hydroxyurea. Taken together, these
results show that mimosine inhibits DNA synthesis at the level of
elongation of nascent chains by altering deoxyribonucleotide
metabolism.
Chemical agents that reversibly inhibit DNA synthesis have
proven to be effective tools for investigating DNA replication in
mammalian cells. Until recently, however, all of these agents were
believed to target cellular processes involved in the elongation phase
of DNA replication. In fact, the lack of agents that specifically
inhibit initiation of DNA replication is in part responsible for the
paucity of information that exists on the initiation process.
A
recent candidate for a specific inhibitor of initiation of DNA
replication is the plant amino acid mimosine
(
While mimosine may serve as a useful agent for synchronizing cells,
accurate interpretation of experimental data depends on understanding
the mechanism of mimosine action. The evidence that mimosine inhibits
initiation of DNA replication is indirect, and alternative explanations
exist for the effects described above. For example, mimosine could
inhibit nucleotide metabolism, resulting in delayed arrest of DNA
synthesis at replication forks followed by destabilization of
replication fork structures. Therefore, it was important to determine
whether or not mimosine was a specific inhibitor of initiation of DNA
replication in cellular chromosomes or a general inhibitor of DNA
synthesis at replication forks.
To this end, we compared the effects
of mimosine on nuclear, mitochondrial, and simian virus 40 (SV40) DNA
replication, as well as on DNA replication in Xenopus eggs.
The mechanisms by which DNA replication occurs in these four systems
differ substantially. For example, initiation of mitochondrial DNA
replication occurs throughout the cell cycle, whereas initiation of
nuclear DNA synthesis is limited to S phase. Moreover, mitochondria do
not utilize any of the enzymes required at DNA replication forks in
mammalian nuclei
(17) . Although SV40 and nuclear DNA synthesis
appear to share most of the enzymes required at replication forks,
initiation of SV40 replication differs significantly in that it is
controlled by the viral-encoded origin recognition protein large tumor
antigen (T-ag), and it is not limited to one initiation event per cell
cycle
(18) . The mechanism by which nuclear DNA replication is
initiated is as yet unknown, but it restricts cellular chromosomes to
one initiation event per origin per S phase
(19) . Xenopus eggs provide the most well characterized system that can initiate
replication in non-viral DNA sequences as well as continue DNA
synthesis at replication forks. Like mammalian cells, DNA replication
in Xenopus eggs is restricted to one initiation event per cell
cycle
(20) . However, in contrast to mammalian cells, which
initiate replication at specific chromosomal loci
(21) ,
Xenopus eggs can initiate replication within most (perhaps
all) DNA sequences that are presented to them
(22, 23, 24) . Furthermore, whereas initiation
of replication in mammalian chromosomes requires specific DNA sequences
(25) , initiation of replication in Xenopus eggs does
not
(22, 26) .
The results of our study indicated
that mimosine inhibited DNA synthesis in cellular, mitochondrial, and
SV40 chromosomes to the same extent despite fundamental differences in
the way these types of chromosomes are replicated. Furthermore,
mimosine had the same inhibitory effect on elongation of nascent DNA
chains in both cellular and SV40 replicating chromosomes as did
aphidicolin, a specific inhibitor of replicative DNA polymerases.
Finally, in parallel with its inhibitory effect on DNA synthesis,
prolonged mimosine treatment altered intracellular levels of
deoxyribonucleotide triphosphates (dNTPs) in a manner similar to that
of another DNA replication inhibitor that affects deoxyribonucleotide
metabolism, hydroxyurea.
These results demonstrate that mimosine can
block cell cycle progression within S phase at the level of elongation
of nascent DNA chains by causing the depletion of intracellular pools
of deoxyribonucleotides. This is consistent with our observation that,
in contrast to aphidicolin, mimosine did not inhibit DNA replication in
lysates of mammalian cells supplied with an exogenous source of
deoxyribonucleotides or in extracts of Xenopus eggs or intact
Xenopus eggs, both of which contain high levels of
deoxyribonucleotides. Consequently, studies that have employed mimosine
to block cell cycle progression in late G
DNA was analyzed by neutral-neutral two-dimensional gel
electrophoresis
(32) . Aliquots (10 µl) of purified SV40 DNA
were digested with a 10-fold excess of BamHI for 2 h at 37
°C and then applied to a 0.6% agarose gel in a 40 m
M Tris
acetate and 1 m
M EDTA
(40) containing 3 µg/ml
ethidium bromide and subjected to electrophoresis for 16 h at 1 V/cm
(ambient temperature). DNA was visualized by 360 nm UV irradiation to
excise each lane accurately. Each excised lane was laid across the top
of a 1% agarose gel in 45 m
M Tris borate, 1 m
M EDTA,
and 30 µg/ml ethidium bromide
(28) and embedded with
additional agar. Electrophoresis in the second dimension was carried
out in buffer containing ethidium bromide at 4 °C for 20 h at 6
V/cm. DNA was then transferred to nylon membranes (GeneScreen Plus) and
probed with
We were able to
confirm this observation with mimosine inhibition of nuclear DNA
synthesis. When CHO C400 cells were synchronized with aphidicolin near
the G
If mimosine prevents initiation
of SV40 DNA replication, then incorporation of
[
After incubation of Xenopus sperm nuclei in Xenopus egg extracts supplemented with either mimosine or aphidicolin, the
time course for incorporation of [
Analogous experiments were
carried out with intact Xenopus eggs. Plasmid DNA was injected
into unfertilized Xenopus eggs that were then allowed to
convert the injected DNA into chromatin before activating the eggs in
the presence of Ca
dCTP pools remained unaffected by mimosine
treatment. However, dTTP pools increased, and dATP and dGTP pools
decreased to less than 15% of levels observed in untreated cells (Fig.
8). The decrease in dATP and dGTP pools occurred in parallel with a
gradual decrease in DNA synthesis (Fig. 8). In fact, the relative
amounts of all four dNTPs observed after 3 h of mimosine treatment were
very similar to those previously observed in mouse cells treated with
hydroxyurea
(48) . Since the effect of hydroxyurea on
deoxyribonucleotide metabolism is believed to be responsible for its
inhibitory effect on DNA replication, we concluded that the similar
alterations introduced by mimosine treatment were responsible for the
inhibitory effect of mimosine on DNA replication.
Mimosine has proven useful as an agent for synchronizing
cells near the G
Results presented here show that
mimosine does not act by specifically inhibiting initiation of DNA
replication. Several lines of evidence support this conclusion. First,
the dose response curve for inhibition of DNA synthesis by mimosine and
the kinetics of this inhibition were the same for mitochondrial DNA as
for cellular chromosomes (Figs. 1 and 2). These observations strongly
suggest that mimosine is affecting a metabolic pathway shared by
nuclear and mitochondrial DNA replication and is not targeting a
specific step in the initiation of cellular DNA replication.
Furthermore, mitochondrial DNA replication was efficiently inhibited
throughout the cell cycle. Therefore, the inhibitory effect of mimosine
cannot be confined to late G
The second line of evidence is that the same concentration of
mimosine that inhibited cellular DNA synthesis also inhibited SV40 DNA
synthesis (Fig. 3), a genome whose replication depends on the
same proteins required to replicate cellular chromosomes but whose
initiation process differs sharply from that of the cell in that it
requires a unique viral-encoded protein and undergoes reinitiation
multiple times within a single S phase
(18) . Thus, it is
unlikely that mimosine would specifically block initiation of both
cellular and viral DNA replication. In fact, analysis of SV40
replicating intermediates by two-dimensional gel electrophoresis
(Fig. 4) demonstrated directly that mimosine arrested DNA
synthesis at SV40 replication forks. Moreover, the effects of mimosine
on viral replicating intermediates were indistinguishable from those of
aphidicolin, an established inhibitor of DNA synthesis at replication
forks
(8) . Both drugs produced the same pattern of arrested
SV40 DNA replication intermediates, and both drugs induced a slow
disappearance of replicating intermediates accompanied by conversion of
replication bubble structures containing two replication forks into
structures containing single replication forks. This phenomenon was
strikingly similar to previous reports that aphidicolin induces
destabilization of SV40 DNA replication forks
(43, 44) .
Destabilization of replication forks in cellular chromosomes could also
account for the absence of detectable chromosomal replication forks in
cells treated with mimosine
(6, 7) as well as the
increased fraction of breaks observed at fragile sites in human
chromosomes in the presence of aphidicolin
(51) and the
stimulation of gene amplification in the presence of various DNA
synthesis inhibitors
(52) .
Several characteristics of
the effects of mimosine suggest that it inhibits DNA replication by
altering deoxyribonucleotide metabolism. First, DNA synthesis in
nuclear and viral chromosomes share the same metabolic pathways for
synthesis of deoxyribonucleotides and, although differences in
deoxyribonucleotide metabolism occur in mitochondria compared to nuclei
(54, 55) , mitochondrial DNA synthesis may share some of
these pathways as well. An effect by mimosine on a shared metabolic
pathway involved in dNTP metabolism would provide a simple explanation
for the similarity of the effect of mimosine on all three DNA
replication systems. Second, mimosine did not inhibit DNA synthesis in
lysed cells supplemented with the deoxyribonucleotide substrates
required for DNA synthesis (Fig. 6). Third, similar to the
previous observation that Xenopus embryos are insensitive to
the deoxyribonucleotide synthesis inhibitor hydroxyurea for at least
the first 12 rounds of DNA replication after fertilization
(39) , mimosine did not inhibit replication of DNA templates
injected into Xenopus embryos or incubated in extracts of
Xenopus eggs (Fig. 7 A). The failure of mimosine
and hydroxyurea to inhibit DNA replication in Xenopus embryos
may be related to the presence in these embryos of dNTP stores
sufficient to support complete replication of the DNA in at least 2,500
nuclei
(60) .
Measurement of the relative amounts of dNTPs in
mimosine-treated cells compared with untreated cells demonstrated
directly that mimosine alters intracellular dNTP pools in cultured
cells (Fig. 8). A similar effect on dNTP metabolism by mimosine
was reported recently by Dai et al. (53) . In fact, in
our experiments and in those of Dai et al., mimosine treatment
produced changes in amounts of individual dNTPs relative to levels in
untreated cells that were very similar to those previously reported to
occur in mouse cells treated with hydroxyurea
(48) . In
particular, cells treated with either compound are depleted of dATP and
dGTP to similar levels relative to controls. Since hydroxyurea's
inhibitory effect on DNA replication is believed to be mediated by the
specific depletion of dATP and dGTP pools
(48, 58) ,
mimosine very likely inhibits the elongation phase of DNA replication
via the similar reduction in dATP and dGTP pools observed in our
experiments and by Dai et al. (53) .
How mimosine
effects these changes in dNTP metabolism is not clear. The similarity
of the effects of mimosine and hydroxyurea on dNTP metabolism and DNA
replication in cultured cells and of the lack of effect by either
compound on DNA replication in early Xenopus embryos suggests
that, similar to hydroxyurea
(49) , mimosine inhibits the enzyme
ribonucleotide reductase. Mimosine is a strong iron chelator
(56) , and ribonucleotide reductase has an essential requirement
for iron for proper function
(50) . Thus, mimosine might disrupt
ribonucleotide reductase function through its ability to chelate iron.
In fact, Dai et al. (53) recently demonstrated a
direct inhibitory effect of mimosine on ribonucleotide reductase, and
this effect could be reversed by iron. Similarly, the addition of
excess iron to mimosine-treated cell cultures restores DNA synthesis
and dATP levels to those of untreated controls.
The effect of mimosine on dNTP metabolism could reflect
a direct interaction of mimosine with ribonucleotide reductase, similar
to hydroxyurea. However, mimosine depletion of dGTP and dATP pools
occurs slowly compared to depletion by hydroxyurea, and as reported
here and in Mosca et al. (7) , the kinetics with which
mimosine inhibits DNA replication also are slow compared to those of
hydroxyurea, even when the longer time required to transport mimosine
into the cell is taken into account. These observations and the fact
that the subunit of ribonucleotide reductase which contains iron turns
over fairly rapidly in cells
(57) suggest an alternative
hypothesis: mimosine sequesters iron required for the function of newly
synthesized ribonucleotide reductase as previously synthesized enzyme
is depleted.
The apparent G
Regardless of how mimosine alters dNTP metabolism, these alterations
are expected to block DNA replication at the level of elongation. This
mechanism is consistent with previously published data on mimosine when
two aspects of mimosine inhibition become apparent. First, mimosine,
like other DNA synthesis inhibitors, may induce breaks at replication
forks that eventually cause them to disappear. Second, the action of
mimosine on DNA replication is indirect through alterations in
deoxyribonucleotide pools. The slow kinetics of this effect are very
likely responsible for the ``slow-stop'' phenotype previously
attributed to inhibition of initiation of DNA replication
(7) .
Thus, while mimosine remains a useful agent for efficiently
synchronizing cultured cells, it may not be useful as a tool for
probing late G
We thank Solon Rhode (University of Nebraska Medical
Center) and Robert Kalejto and Joyce Hamlin (University of Virginia)
for sharing results prior to the publication of their articles, Miriam
Miranda for determining the effects of mimosine on development of mouse
preimplantation embryos, Martin Weinberger, Christine Brun, and Joel
Huberman for critical reading of the manuscript, and Joyce Hamlin for
the gift of pneoS13.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
- N(3-hydroxy-4-pyridone)-
-amino propionic acid).
Fluorescence activated cell sorter analyses of exponentially
proliferating mammalian cells arrested with mimosine suggested that
they accumulated in the late G
phase of their cell division
cycle
(1, 2, 3, 4) . More detailed
studies revealed that mimosine inhibited DNA replication throughout S
phase but only after several hours of treatment
(5, 6, 7) . Two observations in these studies
supported the conclusion that mimosine specifically blocked initiation
of new replicons. First, the length of time required for mimosine to
inhibit DNA replication in cells that were synchronized at the
beginning of S phase with aphidicolin was substantially longer than the
time required to inhibit replication in exponentially proliferating
cells. Since aphidicolin allows formation of replication bubbles but
prevents subsequent expansion of these bubbles by inhibiting
specifically DNA polymerases
,
, and
(8) ,
mimosine appeared to inhibit replication only when new initiation
events were required to sustain S phase, analogous to ``slow
stop'' mutations in bacteria or yeast that specifically block
initiation of DNA replication
(9) . Second, replication fork
structures were not detected by two-dimensional gel electrophoresis
after prolonged treatment of cells with mimosine. This observation
suggested that mimosine prevented formation of new replication forks,
while allowing previously existing replication forks (present in
replicons initiated prior to addition of mimosine) to complete
replication. As a result of these studies, mimosine has recently been
employed by a number of laboratories under the assumption that this
drug synchronizes cells just prior to the onset of DNA replication
(1, 3, 4, 10, 11, 12, 13, 14, 15, 16, 38) .
may require
reinterpretation.
Culture and Synchronization of
Cells
CHO(
)
C400 hamster and BSC-1
monkey cells were grown in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum (Life Technologies,
Inc.) and nonessential amino acids. Xenopus Xtc kidney cells
were grown at 23 °C in amphibian medium (Life Technologies, Inc.)
supplemented with 10% fetal bovine serum and 25 m
M HEPES (pH
7.0). For experiments requiring populations of cells synchronized with
aphidicolin at their G
/S boundary, cells were first
collected in G
by isoleucine deprivation
(27) followed by release into complete medium containing 5
µg/ml aphidicolin for 12 h. Mimosine (Aldrich) was stored at 4
°C as a 10 m
M solution in phosphate-buffered saline, and
aphidicolin (Boehringer-Mannheim) was stored as a 2 mg/ml solution in
dimethyl sulfoxide at -20 °C.
Nuclear and Mitochondrial DNA
Replication
Nascent nuclear and mitochondrial DNA (mtDNA)
was radiolabeled for 20 min with 10 µCi/ml
[H]thymidine (62 Ci/mmol) (Amersham Corp.) per
10-cm dish of cells. When mtDNA replication was to be quantified,
culture medium included 5 µg/ml
1-(
-
D-arabinofuranosyl)cytosine to suppress nuclear DNA
replication. Cells were lysed with 3 ml/dish of 50 m
M Tris-HCl
(pH 7.4), 10 m
M EDTA, 400 m
M NaCl, 0.6% sodium
dodecyl sulfate, and 0.2 mg/ml Proteinase K (Boehringer Mannheim) by
incubating them for 16 h at 37 °C. DNA was purified by extracting
the lysate with phenol and chloroform-isoamyl alcohol and was then
precipitated with ethanol and redissolved in 100 µl/dish of 10
m
M Tris-HCl (pH 7.8) and 1 m
M EDTA. Aliquots
containing 8 µg of DNA were digested with various restriction
enzymes according to the supplier's instructions and then
fractionated by electrophoresis in 0.8% agarose gels in a Tris
borate-EDTA buffer
(28) .
H-DNA was visualized by
exposing dried gels to preflashed x-ray film for various lengths of
time at -70 °C with an intensifying screen
(29) after
they had been processed for fluorography
(30) . Total DNA
replication was quantified by precipitation with trichloroacetic acid
(28) . Mitochondrial DNA replication was quantified by scanning
densitometry of autoradiograms that were exposed within the linear
response range of the film.
SV40 DNA Replication
BSC-1 monkey cells
(50% confluence) in 10-cm dishes were infected with SV40 virus (m.o.i.
= 10). At 24 h post-infection, either 0.4 m
M mimosine
or 5 µg/ml aphidicolin was added to the medium, and 3 h later,
nascent viral DNA was labeled with 50 µCi/ml
[H]thymidine (80 Ci/mmol) (Amersham) for 15 min.
The cells were then lysed with 3 ml/plate of 10 m
M EDTA, 0.6%
SDS
(31) . After 5 min, 1 ml of 5
M NaCl was added
dropwise to each plate, and lysates were transferred gently to Falcon
2059 tubes, chilled on ice overnight to precipitate cellular DNA, and
then centrifuged in a Beckman JS13.1 rotor at 10,000 rpm for 30 min (4
°C) to remove cellular DNA. DNA in the supernatant was extracted
with phenol and chloroform-isoamyl alcohol, precipitated with ethanol,
dissolved in distilled water (100 µl/dish of cells), and then
adjusted to 150 m
M NaCl with a 10
buffer stock (New
England BioLabs) appropriate for digesting DNA with BamHI.
P-labeled SV40 DNA, and replication
intermediates were visualized by exposure to Phosphorimager screens
(Molecular Dynamics). Alternatively, gels were prepared for
fluorography to visualize nascent
[
H]thymidine-labeled DNA as described above.
Early Labeled DNA Fragment Assay
CHO C400
cells were synchronized in Gby isoleucine deprivation and
then subjected to one of three protocols. G
phase cells
were prepared by releasing them into Dulbecco's modified
Eagle's medium supplemented with nonessential amino acids and 10%
fetal bovine serum (complete medium) for 2 h. Mimosine-arrested cells
were prepared by releasing them into complete medium containing 0.4
m
M mimosine for 12 h. Aphidicolin-arrested cells were prepared
by releasing them into complete medium containing 5 µg/ml
aphidicolin for 12 h and then replacing the aphidicolin-containing
medium with prewarmed complete medium and incubating them for an
additional 5 min. Cells were harvested by trypsinization, counted, and
transferred to 1.5-ml microcentrifuge tubes (5 million cells/tube) on
ice, washed with cold hypotonic buffer
(33) , and finally
swollen for 10 min on ice in hypotonic buffer. Cells were pelleted by
centrifugation, supernatant completely removed, and then lysed in the
same microcentrifuge tube by homogenization with 20 strokes of a Kontes
pellet pestle. Nuclei were then isolated by centrifugation at 14,000
rpm for 3 min (4 °C) in an Eppendorf 5415C microcentrifuge and
resuspended in 100 µl of S phase extract prepared from CHO K1 cells
(33) . To this mixture, 25 µl of 5
replication
mixture
(33) was added; the mixture was then transferred to a
1.5-ml microcentrifuge tube containing 20 µl of lyophilized
[
-
P]dATP (6000 Ci/mmol) (Amersham). Tubes
were incubated at 37 °C, and 25-µl aliquots were taken at 10,
20, 30, 60, and 120 min. These samples were added to 300 µl of 0.5%
SDS, 50 m
M Tris-HCl (pH 8.0), and 10 m
M EDTA to stop
the reaction. Acid-precipitable radioactivity was measured from
50-µl aliquots of this cell lysate by addition of trichloroacetic
acid
(28) . DNA was isolated from the remaining sample by
digesting it with 100 µg/ml Proteinase K for 2 h at 56 °C and
then extracting once with phenol:chloroform and once with chloroform
alone before adding 100 µl of 7.5
M ammonium acetate and
adjusting the solution to 80% ethanol. DNA was precipitated,
resuspended in EcoRI buffer (New England BioLabs), digested
with 15 units EcoRI, and fractionated by electrophoresis in an
0.65% agarose gel containing 45 m
M Tris borate and 1
m
M EDTA
(28) for 14 h at 2.3 V/cm and 21 °C. Gels
were dried and exposed to Kodak XAR-5 film at room temperature.
DNA Replication in Xenopus Eggs and Egg
Extracts
Xenopus egg extracts and Xenopus sperm chromatin were prepared as described by Blow and Laskey
(20) . Sperm chromatin was added to 20 µl of extract
supplemented with 0.5 µl of [-
P]dATP
(6000 Ci/mmol) (Amersham) to a final concentration of 3 ng of
DNA/µl extract, and the indicated concentrations of mimosine or
aphidicolin were added. Extracts were incubated at 21 °C, and at
the indicated times, 2.5-µl aliquots were removed, and DNA was
precipitated with trichloroacetic acid
(28) . For analysis of
plasmid replication, 1.6 ng of pneoS13
(34) was injected into
unactivated Xenopus eggs that were activated 2 h later by
addition of Ca
and Ca
ionophore
A24187
(35) . Ten eggs were collected at each of the indicated
times and pooled, and plasmid DNA was isolated from each pool of eggs.
One-tenth of the plasmid DNA was supplemented with 300 ng of
bacteriophage
DNA to provide an internal standard to monitor
subsequent digestions and to ensure complete digestion with
DpnI
(36) . DNA was digested for 1 h with 5 units of
DpnI (New England BioLabs) per ng of isolated plasmid DNA in
buffer 4 (New England BioLabs) supplemented with 200 m
M NaCl
to prevent cleavage of hemimethylated DNA
(36) . All forms of
DpnI-resistant DNA as well as identical aliquots that were not
digested with DpnI were subsequently converted to linear
molecules with PvuI, and the products fractionated by
electrophoresis in 0.65% agarose gels in 45 m
M Tris borate and
1 m
M EDTA for 15 h at 2.4 V/cm
(28) . DNA was then
transferred to a nylon membrane (Hybond-N+, Amersham) by capillary
blotting using 0.4
M NaOH and 0.6
M NaCl. DNA on the
membrane was hybridized with pUC19
P-DNA
(37) , and
the amount of linear monomeric plasmid DNA was determined by scanning
the filters in a Betagen Betascope 603. Identical aliquots of plasmid
DNA were digested with 5 units of MboI (New England BioLabs)
in buffer 4 (New England Biolabs). DNA was hybridized with
P-labeled neomycin gene (0.75-kb PvuII fragment
of pSV2neo). The amount of the largest MboI DNA digestion
product, a 0.56-kb
P-DNA band, was determined by scanning
filters in the Betascope.
Measurement of dNTP Pools
dNTPs were
extracted according to the method of Skoog and Nordenskjold
(48) . Briefly, 10-cm plates containing 10
exponentially proliferating CHO C400 cells were washed once with
phosphate-buffered saline and then scraped into ice-cold 60% methanol
(1 ml/plate). Lysates were stored at -20 °C overnight and
were then centrifuged to remove cell debris. The supernatant was
evaporated to dryness, and dNTPs were redissolved in 100 µl/plate
water. Amounts of dNTP in the extracts were determined by measuring
incorporation by Klenow enzyme of complementary
[
H]dNTPs in DNA synthesis reactions using 10
µl of extract and poly(dA
dT) or poly(dI
dC) as a template
(39) .
Mimosine Inhibits Both Nuclear and Mitochondrial
DNA Synthesis
To determine whether the effects of mimosine
were specific to nuclear DNA synthesis, the ability of mimosine to
inhibit [H]thymidine incorporation into both
mitochondrial and nuclear DNA was tested in CHO cells. One group of
cells was treated with various concentrations of mimosine for 3 h and
then, in the continued presence of mimosine, incubated with
[
H]thymidine for 20 min. Total cellular DNA was
isolated, digested with EcoRI restriction endonuclease, and
then fractionated by gel electrophoresis. A second group of cells was
treated in the same manner except that
1-(
-
D-arabinofuranosyl)cytosine was included to
specifically inhibit nuclear DNA synthesis and thus reveal the presence
of mitochondrial DNA restriction fragments
(30) . These data
revealed that mimosine inhibited both mitochondrial and nuclear DNA
synthesis (Fig. 1). Moreover, the sensitivities of mtDNA and nuclear
DNA synthesis to mimosine were indistinguishable. Both mitochondrial
and nuclear DNA synthesis were inhibited to the same extent at the same
concentrations of mimosine (Fig. 2 A), and the rates of
recovery for both mitochondrial and nuclear DNA synthesis upon removal
of mimosine were the same (Fig. 2 B). These results
suggested that mimosine acts on some metabolic pathway that is common
to both nuclear and mitochondrial DNA replication. Furthermore, since
mtDNA replication occurs throughout the cell cycle and the length of
the mimosine treatment of exponentially proliferating cells in these
experiments was a small fraction of the total time required for cell
division (
20 h), mimosine must inhibit mtDNA replication
throughout the entire cell cycle, not just during S phase.
Figure 2:
Characteristics of mimosine inhibition of
mitochondrial and nuclear DNA replication were the same. A,
exponentially proliferating cultures of CHO C400 cells were treated for
3 h with mimosine at the indicated concentration.
[H]Thymidine was added for 20 min. Nuclear DNA
synthesis (
) was quantified in cells not treated with araC by
acid precipitation of total cellular DNA, while mtDNA synthesis (
)
was quantified in cells treated with araC by densitometry of
fluorographs as described in Fig. 1. AraC suppressed nuclear DNA
synthesis and thus revealed mtDNA synthesis (Fig. 1). Relative DNA
synthesis was expressed as a percentage of the amount detected in
untreated cultures or just prior to addition of mimosine (time =
0). B, exponentially proliferating cultures of CHO C400 cells
were treated with 0.4 m
M mimosine for 3 h to completely arrest
DNA synthesis and then released from mimosine arrest by washing them
twice with fresh medium without mimosine and culturing them in the
absence of mimosine for the times indicated.
[
H]Thymidine was added for 20 min, and both
nuclear and mitochondrial DNA synthesis was measured as in panel A. C, cells were synchronized at their
G
/S boundary with aphidicolin (see ``Materials and
Methods'') and then released from the aphidicolin block in the
presence of 0.4 m
M mimosine ( solid symbols).
Replicating mtDNA was monitored as in previous experiments except that
nuclear DNA replication was suppressed by not withdrawing aphidicolin
from the culture medium (rather than the addition of araC).
Exponentially proliferating cells were also treated with 0.4
m
M mimosine and analyzed in parallel as in panel A.
In
previous studies, a crucial observation was that mimosine took
substantially longer to arrest DNA synthesis in cells that had been
synchronized at the beginning of their S phase with aphidicolin than in
unsynchronized cells that were proliferating exponentially
(7) .
Aphidicolin does not prevent synthesis of short RNA-p-DNA chains that
precede formation of Okazaki fragments
(40) . Thus, aphidicolin
allows both SV40
(41) and cellular
(5) chromosomes to
initiate DNA replication by forming replication forks but limits
subsequent DNA synthesis according to the dose and time of exposure.
Therefore, the delayed response to mimosine suggested that mimosine
inhibition did not occur until new initiation events took place to
continue S phase, analogous to the ``slow-stop'' phenotype
that characterizes mutations in genes required specifically to initiate
replication in bacteria and yeast
(9) .
/S boundary (shortly after entry into S phase) and
released from the aphidicolin block into medium containing mimosine,
inhibition of DNA replication did not begin until 2-3 h and was
complete after a 4-5-h incubation (Fig. 2 C). In
contrast, inhibition of DNA synthesis in exponentially proliferating
cells began 30-60 min after mimosine addition and was completed
after 2-3 h (Fig. 2 C). These data on mimosine
inhibition of nuclear DNA synthesis are in excellent agreement with
those of Mosca et al. (7) . However, we observed the
same phenomenon with mtDNA synthesis. Since mtDNA replication responded
in an identical manner even though mtDNA replication is not affected by
aphidicolin, the delayed response phenotype in G
/S cells
cannot be related to synchronization of chromosomal replicons by
aphidicolin. The fact that mimosine inhibition of nuclear and mtDNA
synthesis were indistinguishable under all conditions strongly suggests
that mimosine does not specifically inhibit some step in the initiation
of cellular chromosome replication.
Mimosine Inhibits DNA Synthesis at SV40 Replication
Forks
To examine directly the effects of mimosine on DNA
replication forks, replication of SV40 DNA was analyzed in
mimosine-treated virus-infected monkey cells. At 24 h post-infection,
BSC-1 cells were cultured for 3 h with different amounts of mimosine.
Uninfected cultures were treated in parallel. Cells were then incubated
with [H]thymidine for 20 min before DNA was
extracted, digested with EcoRI, and fractionated by gel
electrophoresis. Ethidium bromide staining of the gels indicated that,
prior to addition of mimosine, SV40 DNA had replicated to the same
extent in both treated and untreated cells (data not shown).
Fluorographic detection of
H-DNA in the same gels revealed
that both nuclear and SV40 DNA synthesis were inhibited strongly by 200
µ
M mimosine (Fig. 3).
H]thymidine into SV40 replicating intermediates
will cease only when all of the viral replicating intermediates present
prior to mimosine treatment complete their replication cycle
(18, 42) . At that time, no SV40 replicating
intermediates will be present. However, if mimosine blocked DNA
synthesis at replication forks, then replicating intermediates will be
present but inactive. To distinguish between these two possibilities,
virus-infected BSC-1 cells were treated with 0.4 m
M mimosine
for either 3 or 6 h and then incubated with
[
H]thymidine for 20 min to radiolabel replicating
intermediates. Viral DNA was isolated and cut at the single
BamHI site located in its termination region for replication
to produce a collection of replication bubble structures
(``
-structures'') of various sizes in the absence of
replication fork structures (``Y-structures'') (Fig.
4 A). The products of this digestion were then fractionated by
the neutral-neutral two-dimensional gel electrophoresis technique of
Brewer and Fangman
(32) to identify bubble and fork structures
(Fig. 4 A). Newly synthesized SV40
H-DNA was
detected by fluorography (Fig. 4 B). Total SV40 DNA was
detected by transferring the DNA from the gel to a nylon filter and
then hybridizing it with SV40
P-DNA (Fig. 4, C and D).
Figure 4:
Mimosine arrests SV40 replication forks.
Replicating SV40 DNA was radiolabeled in three parallel groups of
virus-infected BSC-1 cells. At 24 h post-infection, one group of cells
was treated with 0.4 m
M mimosine, one group with 5 µg/ml
aphidicolin, and one group left untreated for either a 3- or 6-h
period. SV40-replicating DNA was then radiolabeled for 20 min with
[H]thymidine, and SV40-replicating intermediates
were purified, digested at the single BamHI site located in
the termination region to produce a population of replication bubbles,
and then analyzed by neutral-neutral two-dimensional gel
electrophoresis ( A). The migration pattern of unreplicated
SV40 DNA, unreplicated cellular DNA remaining in the Hirt supernatant,
replication bubbles ( solid line), and replication
forks ( dotted line) are indicated. B,
H-DNA was visualized by fluorography. C and
D, DNA in duplicate gels was transferred to a membrane,
hybridized with SV40
P-DNA, and detected by phosphorimage
analysis.
The results showed clearly that mimosine
arrested DNA synthesis at replication forks. Cells treated with
mimosine for 3 h no longer synthesized SV40 DNA, as determined by
incorporation of [H]thymidine
(Fig. 4 B), but they still contained at least 60% as many
SV40 replicating intermediates as untreated cells
(Fig. 4 C), as determined by Phosphorimager analysis of
the two-dimensional gels. The slow disappearance of SV40 replicating
intermediates observed at 3 and 6 h after addition of mimosine
(Fig. 4 C) resulted from inhibition of DNA synthesis at
replication forks because experiments carried out in parallel with
aphidicolin instead of mimosine gave identical results (Fig.
4 D). Furthermore, ``Y-arcs''
(Fig. 4 A) produced by molecules containing a single fork
also were observed in DNA recovered from mimosine or aphidicolin
treated cells but not from untreated cells (Fig. 4, C and D). These molecules result from a break at one of the
two forks in a single replicating intermediate; breaks at both forks
would produce simple linear molecules. These results are consistent
with previous studies showing that prolonged exposure to aphidicolin
produces breaks at SV40 DNA replication forks
(43, 44) .
Thus, the effects of mimosine and aphidicolin on SV40 DNA replication
were indistinguishable; inhibition of viral DNA synthesis at
replication forks followed by destabilization of replication forks and
slow disappearance of replicating intermediates.
Mimosine Does Not Block Entry of Mammalian Cells into
S Phase
To determine whether mimosine blocks cells before
or after they have initiated DNA replication ( i.e. entered S
phase), nuclei from mimosine-arrested mammalian cells were tested for
their ability to carry out DNA replication in an extract prepared from
S phase CHO cells. These extracts, when supplemented with the four
deoxyribonucleotide substrates for DNA synthesis, the four
ribonucleotide substrates for RNA primer synthesis, and an ATP
regenerating system, can support DNA synthesis in nuclei from S phase
cells but cannot initiate replication in nuclei from Gphase cells
(22, 33) . CHO C400 cells were
synchronized in their G
phase by first arresting their
proliferation through isoleucine deprivation and then releasing them
into complete medium for 2 h. Other cells were synchronized at the
beginning of S phase by first arresting them through isoleucine
deprivation and then releasing them into complete medium containing 5
µg/ml aphidicolin for 12 h. A third group of cells was released
into complete medium containing 0.4 m
M mimosine for 12 h.
Nuclei were then isolated from each population and incubated in CHO
cell extract supplemented with a reaction mixture that included
[
-
P]dATP. As previously reported
(22, 33) , nuclei from cells that had entered S phase
(arrested with aphidicolin) continued to synthesize DNA when placed in
these cell extracts, while nuclei from cells in G
phase did
not (Fig. 5 A). Nuclei from cells arrested in mimosine behaved
like nuclei from cells arrested in S phase (Fig. 5 A),
revealing that mimosine did not arrest cells prior to initiation of DNA
replication.
Figure 5:
Mimosine arrests nuclear DNA synthesis
after formation of replication forks. CHO C400 cells were synchronized
in their Gphase, arrested with aphidicolin ( Aph),
or arrested with mimosine ( Mim) and subjected to early labeled
fragment analysis as described under ``Materials and
Methods.'' Isolated nuclei were allowed to synthesize DNA in the
presence of [
-
P]dATP, and the amount of
acid-precipitable
P-DNA was measured at the indicated
times ( A). The number of nuclei per microliter of extract was
counted, and the DNA synthesis was expressed as a percentage of input
DNA (6.6 pg of DNA/nucleus). B, alternatively, DNA was
extracted from nuclei radiolabeled for 20 min as in A,
digested with EcoRI, and then fractionated by gel
electrophoresis. Nuclei were also isolated from exponentially
proliferating CHO C400 cells and radiolabeled as in panel A. The gel was dried and exposed to film. Indicated are
the positions of mtDNA fragments ( mt), DNA fragments from the
amplified dihydrofolate reductase locus, and the DNA fragment
containing the origin of bidirectional replication ( OBR-1) 17
kb downstream from the dihydrofolate reductase gene. The schematic
representation at the bottom represents the 273-kb amplified
dihydrofolate reductase gene region that includes another structural
gene, 2BE2121. Horizontal arrows indicate directions
of transcription.
The pattern of early labeled DNA restriction fragments
from the dihydrofolate reductase gene locus was examined to determine
whether mimosine-arrested cells could be distinguished from
aphidicolin-arrested cells. The dihydrofolate reductase locus in CHO
C400 cells has been amplified about 500-fold by stepwise selection in
methotrexate and comprises approximately 3% of the total DNA
(45) . As a result, restriction fragments from this locus can be
visualized above a background smear of genomic DNA. An origin of
bidirectional DNA replication (OBR-1) has been mapped by a number of
different methods to a site about 17 kb downstream of the dihydrofolate
reductase gene
(21) . The restriction fragment containing this
origin of bidirectional DNA replication constitutes the earliest
labeled DNA in this region of the genome and can be identified as such
by synchronizing CHO C400 cells at the beginning of their S phase in
aphidicolin, isolating nuclei from these cells, and then briefly
radiolabeling these nuclei in CHO cell extracts as described above. The
restriction fragment containing OBR-1 appears as a prominent
P-DNA band above a smear of
P-DNA fragments
generated from throughout the CHO C400 genome
(22, 33) .
The same pattern of early labeled
P-DNA restriction
fragments was observed with nuclei isolated from either
aphidicolin-arrested cells (Fig. 5 B, APH) or
from mimosine-arrested cells (Fig. 5 B, MIM). In
contrast, nuclei isolated from G
phase cells revealed only
P-DNA bands from the mitochondrial genome (Fig.
5 B, G
), and nuclei from
non-synchronized, exponentially proliferating cells (Fig. 5 B,
EXP) incorporated radiolabel equally well throughout their
genome. Thus, nuclei from cells synchronized with mimosine continue DNA
synthesis in vitro in a manner indistinguishable from cells
synchronized with aphidicolin, demonstrating that mimosine, like
aphidicolin, arrests proliferating mammalian cells after they have
initiated DNA replication.
Mimosine Does Not Inhibit DNA Synthesis in Cell
Extracts
To determine whether or not mimosine inhibited
directly any of the proteins required for DNA synthesis at replication
forks, exponentially proliferating CHO C400 cells were treated with
mimosine for 3 h to arrest DNA synthesis, and then the cells were lysed
and incubated further in the presence of unlabeled dGTP, dCTP, dTTP,
and [-
P]dATP under conditions that support
DNA synthesis in S phase nuclei
(46) . Whereas incorporation of
radiolabeled DNA precursors in vivo was strongly inhibited by
this treatment (Figs. 1 and 2), incorporation of
[
-
P]dATP in vitro was not
inhibited either by pretreatment of cells with mimosine or by
pretreatment of cells with mimosine and addition of mimosine to the
cell lysate (Fig. 6). In contrast, aphidicolin effectively inhibited
DNA synthesis in vivo or in vitro (Fig. 6).
These results demonstrated that mimosine does not act directly on some
protein at replication forks but indirectly through cellular
metabolism. In fact, the observation that DNA synthesis was
reproducibly greater in lysates from cells pretreated with mimosine
relative to untreated cells suggested that mimosine had reduced the
intracellular dATP pool size, thereby increasing the specific
radioactivity of [
-
P]dATP in the lysate.
Figure 6:
Mimosine does not inhibit DNA synthesis in
cell lysates. Exponentially proliferating CHO C400 cells were cultured
either in the presence (+) or absence (-) of 0.4 m
M
mimosine for 3 h ( Mimosine Cells). Cells were
then lysed in the presence of 0.4% Nonidet P-40, supplemented with the
four deoxyribonucleotide substrates for DNA synthesis, the four
ribonucleotide substrates for RNA primer synthesis, and
[
-
P]dATP (6000 Ci/mmol, Amersham) (46).
Lysates were then incubated for 1 h at 32 °C in the presence
(+) or absence (-) of either 0.4 m
M mimosine
( Mimosine
Lysate) or 5 µg/ml aphidicolin
( Aphidicolin
Lysate). The amount of
acid-precipitable
P-DNA present at the end of the reaction
was expressed relative to untreated lysates from cells that had not
been treated with mimosine.
Mimosine Does Not Inhibit DNA Synthesis in Xenopus
Eggs
In contrast to mammalian cells, Xenopus eggs
and egg lysates can efficiently initiate de novo replication
of exogenously introduced nonviral DNA templates. Replication is
independent of DNA sequence but dependent on the presence of a
chromatin substrate that is organized into a nuclear structure
(22, 47) . Thus, Xenopus provided an
opportunity to test the effects of mimosine on a process of initiation
of DNA replication that is directed entirely by cellular proteins.
-
P]dATP
into Xenopus sperm DNA was monitored by acid precipitation of
radioactively labeled DNA. As was observed with mammalian cell lysates
supplemented with deoxyribonucleotide precursors of DNA synthesis,
mimosine had no effect on DNA synthesis in Xenopus egg
extracts, although aphidicolin completely arrested DNA synthesis under
the same conditions (Fig. 7 A).
and a Ca
ionophore
(35) . This procedure increased the efficiency
of plasmid DNA replication at least 10-fold during the first S phase
(35, 36) . At various times after activation, plasmid
DNA was purified, linearized, and then digested with either
DpnI (Fig. 7 B) or MboI
(Fig. 7 C). Plasmid DNA that was resistant to
DpnI had undergone at least one round of replication, whereas
plasmid DNA that was sensitive to MboI had undergone at least
two rounds of DNA replication
(36) . DNA products were
fractionated by gel electrophoresis and identified by
blotting-hybridization using appropriate
P-labeled DNA
probes.
Figure 7:
Mimosine does not inhibit DNA replication
in Xenopus eggs. A, replication of Xenopus sperm chromatin was measured at the indicated times after addition
of sperm chromatin to Xenopus egg extracts in the presence
() or absence (
) of 2 m
M mimosine or to
Xenopus egg extracts supplemented with 8 µg/ml aphidicolin
(
). Incorporation of [
-
P]dATP into
acid-precipitable DNA was expressed as a percentage of the total sperm
DNA added to the extract. B, Xenopus eggs were
injected with an 18-kb plasmid DNA (pneoS13) and incubated in the
presence of either 2 m
M mimosine (
) or 40 µg/ml
aphidicolin (
) or in the absence of either inhibitor (
).
The fraction of plasmid DNA that had replicated at least once was
determined by measuring the fraction of DpnI resistant,
full-length plasmid DNA monomers ( DpnI resistant full-length
plasmid)/(total full-length plasmid). C, samples of plasmid
DNA isolated from the same injected eggs shown in panel B also were digested PvuI and MboI to determine
the fraction of plasmid DNA that had replicated at least twice. The
amount of the largest MboI DNA digestion product was measured.
D, the sensitivities of exponentially proliferating CHO C400
cells (
) and Xenopus Xtc kidney cells (
) to 0.4
m
M mimosine were compared. The amount of
[
H]thymidine incorporated into cellular
H-DNA was expressed as a percentage of the incorporation
observed in untreated cultures (time = 0). Since Xtc cells
divide once every 40 h while CHO C400 cells divide once every 20 h, the
time scale for Xtc cells was divided by 2 and plotted on the same time
scale as CHO C400 cells to compensate for the difference in their cell
cycle times.
Incubation of Xenopus eggs with mimosine had no
effect on either the first or subsequent rounds of replication of
plasmid DNA, whereas incubation with aphidicolin completely inhibited
replication under all conditions. In fact, newly fertilized embryos
continued to divide and develop to the tadpole stage when cultured in
the presence of high levels of mimosine or when mimosine was injected
into the eggs. This result was remarkably similar to the previous
observation that the deoxyribonucleotide synthesis inhibitor
hydroxyurea also has no effect on DNA replication in Xenopus embryos, presumably because they contain large stores of
deoxyribonucleotide precursors of DNA synthesis
(39) . The
inability of mimosine to inhibit DNA replication was not species
specific because Xenopus cells in culture were as sensitive to
mimosine as CHO C400 cells (Fig. 7 D). Therefore, both
the initiation and elongation phases of DNA replication in Xenopus eggs or egg lysates were resistant to mimosine at concentrations
that were 5-10-fold greater than required to inhibit DNA
synthesis in either mammalian or Xenopus somatic cells in
culture. Similar results were obtained with mouse preimplantation
embryos. Mouse 2-cell embryos cultured in the presence of 0.4
m
M mimosine developed up to the blastula stage at the same
rate and with the same efficiency as did 2-cell embryos that were
cultured in the absence of mimosine. In contrast, aphidicolin rapidly
arrested embyronic development as expected.
Mimosine Alters Deoxyribonucleotide
Metabolism
Many of the results described above suggested
that mimosine inhibits DNA replication by altering a metabolic process
related to deoxyribonucleotide metabolism. We directly tested this
possibility by measuring the size of intracellular pools of dNTPs in
control and mimosine-treated CHO C400 hamster cells. dNTPs were
extracted from cells and assayed for their ability to support DNA
synthesis in reactions containing Klenow enzyme, complementary
[H]dNTPs, and poly(dA
dT) or poly(dI
dC)
templates
(48) . Inhibition of cellular DNA synthesis by
mimosine was assayed in parallel cell cultures by measuring
incorporation of [
H]thymidine into
acid-precipitable DNA.
Figure 8:
Mimosine alters intracellular levels of
deoxyribonucleotide triphosphates. dNTPs were extracted from control
cells and cells treated with 0.4 m
M mimosine for various
lengths of time, and the relative amounts of all four dNTP pools were
determined for each extract (--, dNTP pools relative to
controls). DNA synthesis was measured in parallel cultures by pulse
labeling DNA with 5 µCi/ml [H]thymidine for
an additional 20 min after extracts were made (- - -,
% DNA replication). All values are normalized to the values obtained in
control experiments using untreated cultures (100%). Values represent
the averages of at least two independent
experiments.
/S border. However, whether or not this
drug targets some specific step required for initiation of DNA
replication in cellular chromosomes or generally inhibits DNA synthesis
at replication forks after they have formed remained an open question
(see Introduction). In the first case, mimosine would arrest cells in
their G
period prior to the onset of DNA synthesis, while
in the latter case, mimosine would arrest cells at the beginning of
their S phase after the onset of DNA synthesis. If mimosine prevented
initiation of DNA replication, it would provide a useful tool for
dissecting the initiation process.
or the S phase of the cell
cycle.
Figure 3:
Mimosine inhibits SV40 as well as nuclear
DNA replication. SV40-infected BSC-1 monkey cells were treated with
various concentrations of mimosine for 3 h beginning at 24 h
post-infection. Replicating DNA was then labeled with
[H]thymidine for 30 min. Viral DNA was recovered
by Hirt extraction and restricted with EcoRI; equivalent
amounts of sample were fractionated by agarose gel electrophoresis, and
H-DNA was detected by fluorography. Uninfected cells were
treated similarly.
The third line of evidence
supporting the hypothesis that mimosine arrests DNA synthesis at
replication forks stems from the ability of cell extracts to support
DNA synthesis in nuclei isolated from S phase cells but not in nuclei
isolated from Gphase cells
(22, 33) . The
fact that nuclei isolated from either mimosine- or aphidicolin-arrested
cells carried out DNA synthesis in these extracts
(Fig. 5 A) demonstrated that these nuclei had already
entered S phase. Furthermore, the pattern of DNA fragments that were
rapidly labeled in nuclei from mimosine-arrested cells was
indistinguishable from the pattern produced in nuclei from
aphidicolin-arrested cells (Fig. 5 B). Therefore, both
mimosine- and aphidicolin-arrested chromosomal DNA replication in S
phase just after DNA synthesis had been initiated in vivo at
specific sites within these fragments.
(
)
arrest observed in some
mimosine-treated cells is correlated in some cases with altered
post-translational modifications of proteins implicated in cell cycle
regulation
(38, 59) . However, the apparent lack of an S
phase arrest in these cells may simply reflect levels of functional
ribonucleotide reductase that are sufficient to support completion of
one S phase after mimosine treatment begins but not sufficient to
support significant amounts of DNA synthesis when a second S phase
begins many hours later. Consequently, cells would accumulate with a
G
content of DNA, which could be misinterpreted as a block
in late G
(2, 3) . This potential
misinterpretation could affect the conclusions reached by a number of
studies that employed mimosine to block cell cycle progression in late
G
(1, 3, 4, 10, 11, 12, 13, 14, 15, 16, 38, 59) .
cell cycle regulatory events related to the
onset of the S phase of the cell cycle.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.