From the Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263
Received for publication, May 1, 2002, and in revised form, October 29, 2002
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ABSTRACT |
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DNA replication is inhibited by DNA damage
through cis effects on replication fork progression and
trans effects associated with checkpoints. In this study,
we employed a combined pulse labeling and neutral-neutral
two-dimensional gel-based approach to compare the effects of a DNA
damaging agent frequently employed to invoke checkpoints, UVC
radiation, on the replication of cellular and simian virus 40 (SV40)
chromosomes in intact cells. UVC radiation induced similar inhibitory
effects on the initiation and elongation phases of cellular and SV40
DNA replication. The initiation-inhibitory effects occurred
independently of p53 and were abrogated by the ATM and ATR kinase
inhibitor caffeine, or the Chk1 kinase inhibitor UCN-01. Inhibition of
cellular origins was also abrogated by the expression of a
dominant-negative Chk1 mutant. These results indicate that UVC induces
a Chk1- and ATR or ATM-dependent checkpoint that targets
both cellular and SV40 viral replication origins. Loss of Chk1 and ATR
or ATM function also stimulated initiation of cellular and viral DNA
replication in the absence of UVC radiation, revealing the existence of
a novel intrinsic checkpoint that targets both cellular and SV40 viral
origins of replication in the absence of DNA damage or stalled DNA
replication forks. This checkpoint inhibits the replication in early S
phase cells of a region of the repetitive rDNA locus that replicates in
late S phase. The ability to detect these checkpoints using the well
characterized SV40 model system should facilitate analysis of the
molecular basis for these effects.
DNA replication is rapidly inhibited when cells are subjected to
DNA damage during the S phase of the cell cycle. In mammalian cells,
this inhibitory effect is generally detected as a decrease in
incorporation of radioactive DNA precursors into newly synthesized DNA.
The dose-dependent magnitude of the inhibitory effect
induced by ionizing radiation and UVC radiation is biphasic in
nature Early studies of these effects suggested that inhibition of initiation
of DNA replication induced by ionizing radiation might occur in
cis as a result of alterations in chromatin structure produced by radiation-induced strand breaks (6). The elongation arrest
observed at high doses of UVC radiation also was thought to occur in
cis when lesions formed a physical block to replication fork
progression (7). However, more recent experiments indicate that the
inhibitory effects of ionizing radiation (8-10) and methylation damage
(11) on episomal DNA replication can occur in trans. Furthermore, the inhibitory effect of ionizing radiation on DNA replication is reduced in cells from ataxia telangectasia and Nijmegen
breakage syndrome patients containing mutations in the ATM
and NBS1 genes, indicating that the products of these genes mediate this effect in trans as part of an intra-S phase
checkpoint response to DNA damage (12, 13). Similarly, in both budding and fission yeast, the DNA replication-inhibitory effect of DNA damage
corresponds in part to a trans-acting intra-S phase DNA damage checkpoint mediated by the products of a number of different genes, including structural and functional homologues of ATM and the
related checkpoint kinase ATR (14, 15). In budding yeast, this and a
similar checkpoint that responds to stalled replication forks repress
late S phase origins of replication in early S phase cells (16-18). In
both fission (19) and budding (17) yeast, this latter checkpoint also
requires homologues of ATR and ATM, in addition to other checkpoint
proteins. A similar checkpoint in mammals requires the checkpoint
kinase Chk1 (20) and is abrogated by the ATM and ATR inhibitor caffeine
(21). Although this latter checkpoint can be induced by the
ribonucleotide reductase inhibitor hydroxyurea in p53-defective cells,
a p53-dependent intra-S phase checkpoint that responds to
hydroxyurea treatment has also been described (22).
Although most of the DNA damage and DNA replication intra-S phase
checkpoints described above have been shown to inhibit initiation of
DNA replication, the molecular targets of these checkpoints remain
unknown. Furthermore, whether these checkpoints can also inhibit
elongation of nascent chains remains unclear. In fact, recent evidence
suggests that UVC radiation also induces a checkpoint that contributes
to the elongation-inhibitory effects of this DNA damaging agent (23).
In addition, the initiation-inhibitory effect of the intra-S phase
checkpoint that responds to stalled forks could be caused by checkpoint
stabilization of protein complexes at stalled replication forks in
early S phase cells (21, 24).
Thus, a detailed understanding of checkpoints and their molecular
targets requires the ability to distinguish initiation from elongation-inhibitory effects, as well as information about the specific conditions under which these effects occur. Most efforts to
distinguish between these effects in cellular replicons have employed
an indirect approach involving velocity sedimentation of pulse-labeled
nascent DNA. Experiments that rely completely on indirect labeling
techniques can be problematical in their interpretation, however,
because of differences in rate of replication fork movement, radiolabel
incorporation at sites of DNA repair, and DNA damage in nascent
radiolabeled DNA molecules. Further characterization of checkpoint
effects also has been hindered by the difficulty with which the
velocity sedimentation technique can be applied to the analysis of DNA
replication in the experimental systems that have proved most useful as
model eukaryotic replicons, those of yeast and small viral DNA
replicons such as simian virus 40 (SV40).
SV40 is a particularly useful model system for studying different
aspects of the regulation of DNA replication because of the small size
of its genome, its replication to high copy numbers, and except for the
viral initiator protein and helicase large T antigen, its dependence on
cellular proteins for DNA synthesis. Cell-free systems that replicate
SV40 DNA have been extensively employed to analyze the effects of DNA
damage on SV40 DNA replication (25). However, most of these studies do
not distinguish between initiation versus
elongation-inhibitory effects. Consequently, the relationship between
the biochemical events detected in these experiments and cellular
responses to DNA damage that inhibit DNA replication in intact cells
remains unclear.
We previously employed SV40 to study the effects of drugs on SV40 DNA
replication in intact cells using an assay that clearly and simply
distinguishes initiation-specific inhibitory effects from even subtle
effects on the elongation phase of SV40 DNA replication (26-28). This
assay employs radiolabeling of replicating SV40 DNA combined with a
neutral-neutral (N/N) two-dimensional gel electrophoresis technique
that separates replicating from nonreplicating molecules of DNA. In
this study, we employed the velocity sedimentation technique and the
neutral-neutral two-dimensional gel-based assay to analyze the
initiation and elongation-inhibitory effects on cellular and SV40 DNA
replication induced by UVC radiation, which is frequently employed to
study DNA damage and checkpoint effects on DNA replication in other
systems. We also asked whether the effects of UVC radiation on cellular
and viral DNA replication require the checkpoint kinase Chk1. Our
results confirm the interpretation of velocity sedimentation
experiments in uninfected cells and show that UVC radiation induces a
Chk1-dependent and caffeine-sensitive checkpoint that does
not require p53 and targets the initiation, but not elongation, phase
of cellular and viral DNA replication. They also reveal the existence
of a novel intrinsic Chk1-dependent checkpoint pathway that
regulates both cellular and viral origins of replication in the absence
of DNA damage or stalled DNA replication forks.
Cell Culture and Materials--
Mouse embryo fibroblasts
(MEF)1 were a gift of Dr.
Tyler Jacks, M.I.T., Boston, MA. MDA041 and TR9-7 cells were a gift of
Dr. George Stark, Lerner Foundation, Cleveland, OH. Cell culture
reagents were purchased from Invitrogen. Aphidicolin was
obtained from Sigma. It was diluted in 100% Me2SO, and
stock solutions were stored at SV40 Virus and Adenovirus Infections--
For SV40 virus
infections, BSC-1 monkey cells were seeded at 5 × 105
cells per 100-mm plate and grown for 24 h until 70-80% confluent in Dulbecco's minimum essential media containing Earle's salts supplemented with 10% fetal bovine calf serum and 1% nonessential amino acids. Cells were infected with SV40 virus (multiplicity of
infection >1) diluted 1:10 in Dulbecco's minimum essential media for
1 h at 37 °C. Virus-containing medium was then removed and
cells were incubated an additional 23 h in fresh medium. MDA041 cells were infected with 5 × 109 infectious units/ml
of recombinant adenovirus expressing either GFP or the dominant
negative Chk1 (Lys-Arg) mutation (Ref. 47; gift of C. Vaziri)
and analysis of UVC and UCN-01 effects on DNA replication was performed
22 h later. Expression of adenovirus-encoded proteins was
confirmed by fluorescence microscopy (GFP) or immunoblotting (dn-Chk1).
UVC Radiation and Treatment with Caffeine or UCN-01--
Culture
medium was briefly removed from infected and uninfected cells just
before irradiation to a sterile bottle in a 37 °C water bath, and
monolayers were irradiated with 254 nm wavelength UVC radiation using a
Stratagene Stratalinker. Medium was then returned to the plates and
cells were cultured for an additional period of time until labeling
and/or harvest of DNA. Caffeine or UCN-01 were added to medium to final
concentrations of 2 mM or 100 nM, respectively,
2 min before UVC irradiation.
Labeling and Velocity Sedimentation Analysis of Cellular
DNA--
Sucrose gradients were prepared from equal volumes of
solutions containing 0.1 M NaOH, 0.9 M NaCl,
and 0.02 M EDTA and either 5 or 20% sucrose. Measurements
of UVC effects on cellular DNA replication were performed by labeling
DNA in UVC-irradiated or sham-irradiated cells with 10 µCi/ml
[3H]thymidine for 15 min beginning at 50 min after
irradiation. Medium was then removed, and monolayers were washed once
with phosphate-buffered saline. They were then trypsinized at room temperature and cells were suspended in 500 µl of 1× SSC. Aliquots of suspensions containing 3 × 105 cells were then
diluted into an equal volume of cell lysis buffer containing 0.2 M NaOH, 0.02 M EDTA, and 0.1% (w/v) Nonidet
P-40 that had been gently layered on top of each sucrose gradient. Cells were allowed to lyse for 3 h at room temperature. The
gradients were then centrifuged in a Beckman SW28 rotor at 20 °C for
2.5 h at 27,000 rpm. They were then distributed into 1-ml
fractions and the DNA in each fraction was precipitated on ice for 15 min after adding 100 µg of herring sperm DNA and perchloric acid to a
final concentration of 4%. The precipitated DNA was collected on GF/C
glass fiber filters prewetted with 1.6 N HCl containing 6%
sodium pyrophosphate. Each filter was washed twice with 20 ml of 4%
perchoric acid, and then twice with 5 ml of 70% ethanol, and finally 5 ml of 100% ethanol. The dried filters were counted in 3 ml of
scintillation mixture.
Labeling and Isolation of SV40 Viral DNA--
SV40 viral DNA
replicating in infected cells was labeled for 20 min with
[3H]thymidine (10 µCi/ml medium) at times after
irradiation as indicated in the figure legends. Viral DNA was harvested
by washing 3 times with phosphate-buffered saline followed by
incubation for 1 h at 37 °C with 3 ml/100-mm culture plate of
100 mM EDTA, 0.6% SDS containing 0.2 mg/ml proteinase K. Cell lysates were scraped into Falcon round-bottomed polypropylene
tubes on ice, and 1 ml of 4 M NaCl was added to each tube
with gentle mixing prior to refrigeration for a minimum of 1 h.
Hirt supernatants were prepared by centrifugation of the precipitates
containing high molecular weight cellular DNA for 30 min at 10,200 × g at 4 °C in a Beckman J-13.1 centrifuge rotor.
Supernatants were extracted twice with 10 mM Tris, 1 mM EDTA (TE)-buffered phenol (pH 7.6) and once with
chloroform:isoamyl alcohol (24:1). DNA was ethanol precipitated,
recovered by centrifugation as above, washed with 70% ethanol,
re-centrifuged, and resuspended in restriction enzyme buffer.
SV40 DNA Replication Analysis--
The effects of UVC radiation
on SV40 DNA replication were analyzed as we described previously (27,
28). The assay employs neutral-neutral two-dimensional gel
electrophoresis using the method of Brewer and Fangman (30) with some
modifications. Purified SV40 DNA was linearized with BamHI
for 4 h at 37 °C, and electrophoresed in a 0.6% agarose gel in
1× TAE buffer containing 0.1 µg/ml ethidium bromide (EtBr) for
25 h at 0.7 V/cm. The lanes were excised from the first dimension
gel, inserted into enlarged preparative wells in second dimension gels
of 1% agarose gel in 0.5× TBE containing 0.5 µg/ml EtBr, and sealed
in place with excess agarose. Second dimension gels were
electrophoresed at 4 °C for 19 h at 4 V/cm in a 1× TBE running
buffer containing 0.5 µg/ml EtBr. Southern blots were hybridized to
[
For fluorographic analyses, gels were dehydrated by gentle agitation
for 1 h in 95% ethanol followed by a change of ethanol and
another 1-h incubation. The dehydrated gels were then impregnated with
5% 2,5-diphenyloxazole in 100% ethanol for 1 h with gentle agitation. Fluor was precipitated during a 45-min incubation in distilled water, and the gels were dried onto Whatman 3MM paper using a
gel dryer for 60 min at 60 °C. Dried gels were exposed to Kodak
XAR-5 film at Two-dimensional Gel Electrophoresis Analysis of Replicating
rDNA--
The expression of p53 was induced ectopically in
exponentially proliferating populations of TR9-7 human fibroblast cells
by the withdrawal of tetracyclin from the medium for 2 days to
synchronize them mostly in G1, as reported previously (29).
They were released from the G1 block by addition of
tetracyclin and allowed to transit to early S phase in the presence of
5 µg/ml aphidicolin (Sigma) for 16 h. Cells were then allowed to
transit into S phase by withdrawal of aphidicolin for 2 (early S phase)
or 6 h (late S phase) before addition of UCN-01 to a concentration
of 100 nM. 50 min later, replication intermediates were
harvested on the nuclear matrix and then separated from nonreplicating
DNA on (N/N) two-dimensional gels as described previously by Brewer and
Fangman (30). Gels were blotted onto Hybond N+ membranes
and probed with radiolabeled sequences specific for a 5.6-kb
EcoRI fragment of DNA in the human rDNA locus. Signals were
detected by PhosphorImager (Amersham Biosciences).
Inhibitory Effects of UVC Radiation on Cellular DNA
Replication--
To compare the effects of UVC radiation on SV40 DNA
replication detected by two-dimensional gel methodology to its effects on cellular DNA replication detected by velocity sedimentation of
pulse-labeled DNA, we first established the precise conditions under
which the cellular effects could be observed in our laboratory using
the velocity sedimentation technique. In this approach, nascent
molecules are pulse-labeled with [3H]thymidine for a
short time in control cells or cells subjected to DNA damaging agents,
and then size-fractionated on alkaline sucrose gradients. A selective
decrease in numbers of small, [3H]thymidine-labeled
origin-proximal nascent molecules compared with larger molecules that
had been extended further from origins before the pulse label indicates
an initiation-inhibitory effect. A decrease in label associated with
nascent molecules of all sizes indicates an elongation-inhibitory effect.
Exponentially proliferating cultures of MEFs were subjected to
various doses of 254-nm wavelength UVC radiation, pulse-labeled with
[3H]thymidine for 15 min beginning 50 min after
irradiation, and then size fractionated on alkaline sucrose gradients.
Nascent DNA molecules pulse-labeled in sham-irradiated control cells
had a broad distribution of sizes from short, origin-proximal molecules near the top of the gradient to longer molecules that had been extended
to regions distal to origins, which migrated closer to the bottom of
the gradient (Fig. 1A, 0 J/m2). Incorporation of label into both
populations was reduced in cells irradiated with 10 J/m2
UVC (Fig. 1A, 10 J/m2),
indicating inhibitory effects on both initiation and elongation, although the greater decrease in numbers of short, origin-proximal nascent molecules compared with large nascent molecules indicated that
the predominant inhibitory effect at this dose of UVC was on initiation
of DNA replication. Similar effects were observed at 25 J/m2 UVC radiation (Fig. 1A, 25 J/m2) except that the labeling of long nascent
DNA molecules was somewhat reduced, suggesting a slightly more
pronounced elongation-inhibitory effect at this higher fluence. These
results are similar to those described earlier except that levels of
UVC radiation reported previously to induce these effects were somewhat
lower compared with our experiments (3). At the highest dose of UVC
radiation (Fig. 1A, 50 J/m2),
the size distribution of nascent molecules was shifted significantly toward shorter molecules, and the pulse label associated with large
molecules was reduced even further. This pattern also has been observed
previously at higher fluences of UVC, and has been attributed to a
pronounced inhibitory effect on elongation coupled to reinitiation of
DNA synthesis beyond a lesion that blocks DNA synthesis on the leading
strand template, or discontinuous synthesis beyond a lesion on the
lagging strand template (2).
Previous studies have reported that caffeine abrogates the
initiation-inhibitory effects of ionizing radiation (31) and UVC
radiation (32), similar to the effects of mutations in the ATM gene (12). We next sought to determine whether caffeine would have a similar effect on DNA synthesis in the UVC-irradiated MEFs
employed in our experiments (Fig. 1B). Irradiation of
caffeine-treated cells with 10 and 25 J/m2 UVC only
slightly reduced the amount of incorporation into short molecules (Fig.
1B). Therefore, caffeine abolishes the initiation-inhibitory effect on cellular DNA replication induced by these fluences of UVC
radiation in MEFs. Caffeine also slightly reduced the inhibitory effect
of 10 J/m2, but not 25 J/m2 UVC radiation on
the labeling of long nascent chains (Fig. 1B, 25 J/m2). These results are consistent with
earlier studies demonstrating an initiation-inhibitory effect of UVC
radiation by a checkpoint that is inhibited by caffeine (32), and
suggest that the elongation-inhibitory effect at higher doses of UVC is
not part of this checkpoint. The initiation-inhibitory checkpoint does
not depend on p53, because UVC radiation had similar effects on DNA
replication in p53
Interestingly, the overall amount of incorporation of
[3H]thymidine in these experiments was somewhat greater
in the sham-irradiated control cells treated with caffeine (Fig.
1B) compared with control cells that were not treated with
this compound (Fig. 1A, note change of scale for the
ordinate, B compared with
A). Furthermore, the size distribution of nascent DNA
molecules in sham-irradiated control cells treated with caffeine was
shifted to somewhat shorter lengths (Fig. 1B, 0 J/m2) compared with the distribution observed
in unirradiated control cells in the absence of caffeine (Fig.
1A, 0 J/m2). The increased
incorporation of [3H]thymidine and shorter lengths of
nascent molecules suggest that caffeine activates origins of
replication independently of its effects on a UVC-induced checkpoint.
Stimulatory effects of caffeine on cellular DNA replication have been
reported previously (32, 33).
Dose- and Time-dependent Effects of UVC Radiation on
SV40 DNA Replication--
Application of the velocity sedimentation
technique for distinguishing between initiation- and
elongation-inhibitory effects on DNA replication is limited to
relatively large nascent molecules. To compare the
dose-dependent effects of UVC radiation observed in
cellular replicons with those induced on the initiation and elongation
phases of DNA replication in the much smaller SV40 genome, we analyzed
replicating SV40 genomes isolated from UVC-irradiated cells using an
assay based on the neutral-neutral two-dimensional gel electrophoresis
technique developed by Brewer and Fangman (30). This method separates
branched replicating molecules from linear, nonreplicating molecules on
the basis of size in the first dimension, and size and shape in the
second dimension. Specific restriction fragments of replicating DNA can
be detected as arcs that arise from nonreplicating DNA fragments by
probing blots of two-dimensional gels with radiolabeled probes.
24 h after viral infection, SV40-infected BSC-1 monkey cells were
subjected to the same fluences of UVC radiation employed in experiments
involving uninfected cells. They were then incubated for 50 min before
replicating SV40 DNA was pulse-labeled with [3H]thymidine
for an additional 15 min. These conditions were identical to those used
in the analysis of UVC effects on cellular DNA replication as described
in the legend to Fig. 1. SV40 DNA was then isolated from cells and
digested with BamHI. Equal aliquots of DNA were fractionated
on two neutral-neutral two-dimensional gels run in parallel. One gel
was blotted to a nitrocellulose membrane and probed with SV40 sequences
to measure the number of SV40 replication intermediates, and the second
gel was fluorographed to detect incorporation of
[3H]thymidine into SV40 RIs as a measure of replication
activity. Signals from the probed blot were measured by PhosphorImager
analysis. [3H]Thymidine incorporation was measured by
densitometric tracing of multiple exposures of the fluor-impregnated
gel to x-ray film within the linear response range of the film. In both
cases, measurements of signals from RIs in the bubble arc were
normalized to signals from fully replicated (1n) DNA to account for any
differences in overall yields of DNA.
PhosphorImager analysis of probed blots indicated a
dose-dependent decrease in signals from SV40 RIs compared
with signals from completely replicated DNA at UVC fluences up to 50 J/m2 (Fig. 2A).
Quantitation of the signals from the bubble arc and 1n DNA revealed
that the number of SV40 RIs decreased in a dose-dependent fashion to ~10% of the number observed in sham-irradiated control samples (Fig. 2B, number RIs). This decrease
suggested that RIs were maturing in the absence of new initiation
events. The ability of RIs to mature implies the absence of a
significant inhibitory effect on elongation of nascent DNA chains. This
expectation was confirmed when replication activity was measured by
quantitating signals from the fluorograph. Fig. 2B shows
that at UVC fluences up to and including 50 J/m2,
incorporation of [3H]thymidine into SV40 RIs decreased
essentially in parallel with decreases in the number of RIs. Thus, the
decrease in incorporation could be completely accounted for by a
decrease in the number of these RIs rather than an inhibitory effect on
elongation.
These results indicate that the predominant inhibitory effect of 10-25
J/m2 UVC radiation on SV40 DNA replication occurred at the
level of initiation, similar to cellular replicons (Fig. 1). Although
elongation was significantly inhibited in cellular replicons at 50 J/m2 (Fig. 1), this effect was absent at this dose in the
SV40 experiments (Fig. 2). This may reflect fewer lesions that block
replication forks in cis within each SV40 replicon because
of their smaller size (see "Discussion"). However, a higher dose of
UVC radiation (500 J/m2) induced elongation-inhibitory
effects in SV40 replicons as well. This was indicated by an increase in
the number of SV40 RIs detected in Southern blots in conjunction with a
continued decrease in their replication activity measured in
fluorographs (Fig. 2, A and B, 500 J/m2). A similar pattern of large numbers of
RIs with reduced replication activity was observed in parallel control
experiments that analyzed SV40 RIs recovered from infected, but
unirradiated cells exposed to the elongation-inhibitor aphidicolin
(Fig. 2B, aphid). Thus, the larger numbers of
SV40 RIs observed at 500 J/m2 UVC radiation compared with
lower doses appeared to reflect the failure of RIs to mature because of
an elongation-inhibitory effect of UVC radiation, similar to the
effects of aphidicolin treatment.
It was also possible, however, that the decrease in number of RIs in
UVC-irradiated cells could reflect their destabilization. To
unambiguously distinguish between these possibilities, we performed an
additional control experiment in which SV40-infected cells were treated
with the elongation-inhibitor aphidicolin during and after UVC
irradiation. If UVC radiation destabilizes RIs, the reduced number of
RIs in UVC-irradiated cells should also be observed in cells exposed to
both UVC and aphidicolin. However, if these decreases are caused by an
initiation-inhibitory effect, aphidicolin treatment of UVC-irradiated
cells should block these decreases by inhibiting the maturation of RIs.
As in Fig. 2A, UVC irradiation of virus-infected cells with
25 J/m2 UVC radiation reduced the number of SV40 RIs to
~20% of the number observed in sham-irradiated controls (Fig. 2,
C and D). In contrast, addition of aphidicolin to
the medium just before irradiation occurred (Fig. 2, C and
D, UVC/aphid) reduced the magnitude of the decrease in SV40 RIs induced by UVC radiation to levels similar to
those observed in cells treated with aphidicolin alone. The decrease in
number of SV40 RIs observed in association with aphidicolin treatment
alone may reflect their partial destabilization by aphidicolin treatment (26). However, the similar numbers of RIs observed in
aphidicolin-treated cells that were also subjected to UVC irradiation indicates that UVC radiation did not cause a further destabilization of
these RIs. Therefore, the decrease in SV40 RIs induced by UVC radiation
is, in fact, related to the maturation of these RIs in the absence of
new initiation events.
The inhibitory effects of UVC radiation on cellular DNA replication are
rapid, occurring maximally over a period of 1 h followed by
recovery of DNA replication a few hours later (3, 7). For comparison,
we analyzed the time-dependent inhibitory effects on SV40
DNA replication induced by 25 J/m2 UVC radiation. Fig.
2E shows that signals from SV40 RIs obtained from blots
probed with SV40 sequences gradually decreased during the first hour
after irradiation. Quantitation of these signals by PhosphorImager
analysis revealed that the number of SV40 RIs observed 1 h after
irradiation amounted to ~20% of those observed in the
sham-irradiated controls (Fig. 2F) similar to the number observed at this fluence after 50 min in the dose-response experiments. Densitometric quantitation of signals from x-ray films exposed to the
fluor-impregnated two-dimensional gel run in parallel demonstrated that
incorporation of [3H]thymidine into SV40 DNA decreased in
parallel with the decrease in numbers of RIs in a similar
time-dependent fashion (Fig. 2F, 3H-TdR incorporation). Thus, the kinetics of the
inhibitory effect of UVC on SV40 DNA replication are rapid, similar to
its effect on cellular DNA replication. As in the dose-response
experiments, the similar number of SV40 RIs compared with their
replication activity (Fig. 2F) indicates the inhibitory
effect is on initiation, and not elongation.
Four hours after irradiation, the number of RIs increased once again
compared with the signals from RIs observed at the 1-h time point (Fig.
2, E and F). Incorporation of
[3H]thymidine also increased at the 4-h time point, and
the magnitude of this increase was similar to the increase in the
number of RIs (Fig. 2, E and F, 240').
Therefore, the increase in number of RIs is related to restoration of
DNA synthesis as SV40 genomes more frequently initiate DNA replication.
The restoration of SV40 DNA synthesis is also similar to that observed
in cellular replicons of uninfected cells several hours after UVC
irradiation (3).
The similarity of the initiation-inhibitory effect of UVC radiation on
the replication of SV40 DNA to its effect on cellular DNA mediated by a
caffeine-sensitive checkpoint suggests that this checkpoint targets
both cellular and SV40 viral origins of replication. To explore this
possibility, we asked whether caffeine could abrogate the inhibitory
effects of UVC radiation on initiation or elongation of SV40 viral DNA
replication. Caffeine treatment of SV40-infected cells irradiated with
25 J/m2 of UVC caused an increase in both the number and
replication activity of SV40 RIs compared with those isolated from
irradiated cells that were not treated with caffeine (Fig.
2G, 25 J/m2). Thus, caffeine
at least partly abrogated the inhibitory effect of UVC radiation on
initiation of SV40 DNA replication, similar to its effect on cellular
DNA replication in UVC-irradiated uninfected cells (Fig.
1B). In addition, treatment of sham-irradiated control SV40-infected cells with caffeine also caused an increase in the numbers and replication activity of SV40 RIs compared with levels observed in the absence of caffeine (Fig. 2G, 0 J/m2). Thus, caffeine also stimulates
initiation of SV40 DNA replication in the absence of DNA damage. These
results confirm the interpretation of the velocity sedimentation
experiments and suggest that both cellular and SV40 DNA replication are
regulated by a caffeine-sensitive checkpoint induced by UVC, and an
intrinsic checkpoint that regulates origins in the absence of DNA
damage or other perturbations.
Chk1 Is Required for UVC-induced and Intrinsic Checkpoints That
Target Cellular and SV40 Viral Origins--
Although caffeine inhibits
both of the checkpoint kinases ATR and ATM (1), UVC radiation activates
ATR, rather than ATM (34). Furthermore, UVC radiation induces the
phosphorylation of Chk1 by ATR (35), and Chk1 functions downstream of
ATR in other checkpoint pathways induced by UVC or replication arrest (34). To determine whether Chk1 plays a role in the caffeine-sensitive pathway that inhibits initiation of DNA replication, we asked whether
the Chk1 kinase inhibitor UCN-01 has effects on cellular DNA
replication similar to those of caffeine. These experiments employed
MDA041 human fibroblasts lacking functional p53. As in MEFS, 10 J/m2 UVC radiation induced an initiation-specific
inhibitory effect on DNA replication indicated by a significant
reduction in the number of shorter nascent DNA molecules (Fig.
3A, 10 J/m2). Treatment of these cells with UCN-01
inhibited the decrease in [3H]thymidine-labeled
origin-proximal nascent molecules induced by UVC radiation (Fig.
3A, 10 J/m2 + UCN-01), similar to the effect of caffeine on UVC-irradiated MEF cells (Fig. 1B). Also similar to caffeine, UCN-01
treatment caused an increase in small nascent DNA strands in
sham-irradiated control cells (Fig. 3A, 0 J/m2 + UCN-01). These results are
consistent with the possibility that Chk1 is required for the
UVC-induced checkpoint that inhibits cellular origin function, as well
as an intrinsic checkpoint that regulates cellular origins of
replication in the absence of DNA damage.
To rule out the possibility these results were related to
Chk1-independent effects of UCN-01, we repeated this analysis in cells
infected with an adenovirus vector expressing a dominant-negative mutant of Chk1, or, as a control, a vector expressing green fluorescent protein (GFP). As expected, DNA replication in MDA041 cells
expressing GFP was affected by UVC and UCN-01 treatments similarly to
uninfected controls (Fig. 3B). However, a number of
differences in these effects were observed in MDA041 cells expressing
dn-Chk1 (Fig. 3C). In the absence of UCN-01 or UVC, these
cells produced slightly more small nascent DNA molecules compared with
parental cells or cells expressing ad-GFP (Fig. 3C, 0 J/m2, compare with 0 J/m2 in panels A and B).
Furthermore, in contrast to its effects in other cell lines, UCN-01
treatment did not change the size distribution of nascent molecules in
dn-Chk1-expressing cells (Fig. 3C). Thus, the intrinsic
checkpoint abrogated by UCN-01 is missing from these cells. Similar to
the combined effects of UCN-01 and UVC in control cells (Fig. 3,
A and B), UVC radiation increased, rather than decreased, the number of small nascent DNA molecules in these cells
compared with the sham-irradiated controls (Fig. 3D,
10 J/m2); the sham-irradiated control
values (0 J/m2) from Fig. 3C
are replotted as a solid line for comparison). Although it
is not clear why UVC caused this increase in the absence of UCN-01,
UCN-01 had little additional stimulatory effect on initiation (Fig.
3D, 10J/m2 + UCN-01). Thus, similar to MEF cells treated with caffeine
(Fig. 1B), both the intrinsic checkpoint and the checkpoint
induced by UVC that inhibit initiation are largely absent from cells
expressing dn-Chk1, indicating these checkpoints require Chk1.
We next asked whether Chk1 is required for the similar,
caffeine-sensitive inhibitory effects on SV40 viral DNA replication. Measurements of number of RIs (Fig. 3E) or of their
replication activity (data not shown) indicated that, like caffeine,
UCN-01 partly abrogated the initiation-inhibitory effect of 10 J/m2 UVC radiation on SV40 DNA replication. Thus, at lower
doses of UVC radiation, initiation of SV40 DNA replication appears to
be regulated at least in part by the same UVC-induced
Chk1-dependent pathway that inhibits cellular origins. The
inhibitory effect of higher doses of UVC radiation was not reversed by
UCN-01 treatment (Fig. 3E), despite the fact that 25 J/m2 UVC radiation exerts predominantly
initiation-inhibitory effects that are partly abrogated by caffeine
(Fig. 2G). The nature of this difference is not clear, but
may be related to more complete abrogation of checkpoints by caffeine
compared with UCN-01 in these experiments. UCN-01 treatment also caused
an increase in the number and activity of SV40 replication
intermediates in sham-irradiated controls (Fig. 3D),
indicating that the intrinsic, Chk1-dependent pathway that
regulates cellular DNA replication also regulates initiation of SV40
DNA replication.
The Chk1-dependent Intrinsic Checkpoint Targets Late S
Cellular Origins of Replication--
Both yeast (16-19, 36) and
mammalian (20, 21) cells harbor checkpoints that block the activation
of late S phase origins of replication when DNA replication is blocked
in early S phase. In mammals, this checkpoint is abrogated by both
caffeine and UCN-01. The increased number of small nascent DNA
molecules detected in UCN-01- and caffeine-treated uninfected cells in
our experiments theoretically could be accounted for by the unscheduled
activation of late S origins in early S phase cells. To test this
possibility, we analyzed the effect of UCN-01 on DNA replication in a
late S replicating fragment of the rDNA locus in the human fibroblast cell line TR9-7 cells. Populations of these cells (which are derived from MDA041 cells) can be easily synchronized in early S phase by the
ectopic induction of p53, which blocks progression through the cell
cycle mostly in G1 (29), and subsequent release of this
block into medium containing aphidicolin. Because the stimulatory effect of caffeine and UCN-01 occurred in the absence of stalled replication forks, our analysis was performed beginning 2 h after release of the aphidicolin block, at which time cells have completely recovered their ability to replicate DNA (data not shown).
Control experiments first established that UCN-01 treatment caused an
increase in incorporation of [3H]thymidine and in the
number of small nascent strands in exponentially proliferating
populations of TR9-7 cells (Fig.
4A), similar to the effect of
UCN-01 on DNA replication in MDA041 cells described above (Fig.
3A). To analyze origin timing in the rDNA locus, we employed
the neutral-neutral two-dimensional gel electrophoresis technique. The
tandemly repeated human rDNA locus contains a polymorphism that
produces a specific EcoRI fragment in some, but not all, repeats. Consistent with an earlier report that this fragment resides
in rDNA repeats that replicate later in later S phase (37), replication
intermediates were not detected in this fragment when replicating DNA
was isolated from cells in early S phase, 2 h after release of the
aphidicolin block (Fig. 4B, early S phase, ( A complete understanding of how DNA damaging agents and intra-S
phase checkpoints inhibit DNA replication requires the ability to
analyze the molecular events involved in two fundamentally different
aspects of replication, those involved in initiation, and those
required for the elongation of nascent DNA chains. We previously
developed an assay that clearly distinguishes between these effects
induced by drugs that inhibit viral DNA replication in the well
characterized SV40 model system (26-28). In this study, we asked
whether this assay can distinguish these effects on SV40 DNA
replication induced by a DNA damaging agent more frequently used to
study checkpoint regulation of cellular DNA replication, UVC radiation.
Our results show that lower fluences of UVC radiation induce
initiation-inhibitory effects on SV40 DNA replication very similar in
magnitude and kinetics to those induced by the same levels of UVC in
cellular replicons of uninfected cells. The initiation-specific effects
of UVC radiation on cellular DNA replication were indicated by a
decrease in small, presumably origin-proximal strands of nascent DNA
(Fig. 1), similar to those reported previously. UVC effects on SV40
viral DNA replication were detected as substantial dose- and
time-dependent decreases in both the number and replication activity of SV40 RIs isolated from SV40 virus-infected cells subjected to the same conditions of irradiation and labeling employed in the
analysis of cellular replicons (Fig. 2). At higher doses, similar to
its effect on cellular replicons, UVC radiation also inhibited the
elongation phase of SV40 DNA replication (Fig. 2, A and
B). These similarities confirm the validity of both
techniques for distinguishing initiation versus
elongation-inhibitory effects and suggest that a common mechanism
underlies these effects in cellular and viral replicons.
Inhibition of initiation of cellular and viral DNA replication by low
doses of UVC radiation was at least partly abrogated by the checkpoint
kinase inhibitors caffeine and UCN-01. The specific inhibition of Chk1
by UCN-01 at the concentration used in these experiments (38, 39)
suggested that this corresponded to abrogation of a
Chk1-dependent checkpoint. This was confirmed by the
observation of reduced inhibitory effects on initiation in cells
expressing a dominant-negative Chk1 mutant in uninfected cells (Fig.
3D). Similar results indicating a role for Chk1 in a
UVC-induced checkpoint were recently obtained by others as well (48).
This checkpoint clearly does not depend on p53, because it is present
in p53-null MEFs (Fig. 1C), and in MDA041 cells (Fig. 3,
A-C), which are functionally p53-null (40), as well as in
SV40-infected cells (Fig. 2), where p53 presumably is inactivated by
SV40 large T antigen. This is consistent with the previously described
roles of Chk1 in p53-independent checkpoints (41). The UVC-induced
checkpoint may be related to S phase checkpoints induced by the
topoisomerase I inhibitor camptothecin (42) or cisplatin (43), which
are also abrogated by UCN-01. It may also be related to a recently
described transient, p53-independent late G1 checkpoint
that blocks entry into S phase in response to UVC radiation, which
depends on ATR and is also abrogated by UCN-01 (44).
Failure of both caffeine and UCN-01 to abrogate inhibitory effects at
higher doses of UVC radiation that block elongation indicates that the
checkpoint detected in our experiments specifically targets the
initiation, and not the elongation phase of DNA replication. Because
elongation effects were observed only at higher doses of UVC radiation,
they are likely to occur in cis as replication forks are
blocked by a larger number of lesions in DNA. This could explain the
elongation inhibitory effects induced by 50 J/m2 UVC
radiation in cellular replicons (Fig. 1), but not in SV40 viral
replicons (Fig. 2). Presumably, the larger cellular replicons contain
more fork-arresting lesions compared with much smaller viral replicons.
The increased frequency of initiation of cellular (Fig. 1) and viral
(Fig. 2) DNA replication observed in unirradiated cells treated with
caffeine or UCN-01 is consistent with earlier reports that caffeine
stimulates initiation of cellular DNA replication in the absence of DNA
damage (32, 33, 45). Our findings indicate that this stimulation is
caused by the abrogation of an intrinsic checkpoint requiring Chk1, and
either ATR or ATM, which targets both cellular and viral origins of
replication. The cellular origins targeted by the intrinsic checkpoint
appear to be normally activated in late S phase (Fig. 4). In fact,
previous studies demonstrated inhibition by a checkpoint of late S
origins in mammalian cells, and this checkpoint was also abrogated by UCN-01 (20) and caffeine (21). However, the activation of late S
origins by caffeine or UCN-01 observed in these earlier studies was
only detected when replication forks were blocked in early S phase, and
did not coincide with the accelerated activation of these origins in S
phase (20, 21). In contrast, the intrinsic checkpoint detected by our
experiments occurs in the absence of stalled forks or other
perturbations and its abrogation results in the accelerated activation
of cellular origins. These seemingly contradictory observations can be
explained if the intrinsic pathway regulates only a subset of late S
origins, the effects on which were not detected in the global analysis
of replication timing performed previously. In fact, mutations in the
budding yeast checkpoint genes RAD53 and ORC2
similarly abrogate both types of checkpoints, one that regulates late S
origins in cells blocked in early S phase that, in checkpoint defective
strains, are activated in these cells with normal kinetics (16, 18,
36), and an intrinsic checkpoint that targets only some late S origins,
which are activated in an accelerated fashion in checkpoint defective strains in the absence of a replication block or other perturbations (16). Furthermore, mutations in Rad3, the fission yeast homologue of
ATR, abrogate both a replication arrest-dependent
checkpoint that targets late S origins (19) and an intrinsic checkpoint that regulates some, but not all of these origins in the absence of a
replication block.2 The
existence of a Chk1 and ATR-dependent intrinsic checkpoint is consistent with the observation in Xenopus of an
increased association of ATR with chromatin during DNA replication
unperturbed by drugs (46).
In summary, our study shows that initiation of cellular and SV40 viral
DNA replication are both regulated by a caffeine-sensitive, p53-independent and Chk1-dependent checkpoint induced by
UVC radiation, and by a novel Chk1-dependent intrinsic
checkpoint that inhibits both cellular and viral origins of replication
in the absence of DNA damage or other perturbations. The intrinsic
checkpoint targets a late S replicating region of the rDNA locus in
uninfected cells. The discovery of an intrinsic checkpoint that
regulates DNA replication in the absence of stalled forks or other
perturbations is important in the context of the frequency with which
checkpoint pathways are defective in cancer cells. For instance,
accumulating evidence suggests that some of the genetic instability in
cancer cells arises at replication forks, which, in cells with defects in the intrinsic checkpoint identified here, are likely to be more
numerous compared with cells with intact checkpoints.
The detection of similar effects of UVC radiation and checkpoint
inhibitors on the initiation and elongation phases of cellular and SV40
DNA replication confirms the interpretation of earlier, less direct
analyses of UVC radiation and caffeine-induced effects on cellular DNA
replication detected by velocity sedimentation of pulse-labeled nascent
DNA. Our results delineate the precise conditions required to observe
these checkpoint effects in the well characterized SV40 experimental
system, and identify proteins that regulate initiation, and not
elongation, as potential targets of these checkpoints. This provides a
framework for further investigating the underlying molecular mechanisms.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
an initial steep component occurs at low levels of damage,
followed by a shallower component at higher levels. Analysis of
cellular DNA pulse-labeled shortly after inducing DNA damage with
ionizing radiation (1), UVC radiation (2, 3), topoisomerase inhibitors (4), and DNA reactive compounds (5) suggest these two components correspond to effects on two fundamentally different processes involved
in DNA replication, initiation of DNA replication at origins of
replication, and subsequent elongation of nascent chains at replication forks.
EXPERIMENTAL PROCEDURES
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
20 °C. Proteinase K, restriction
endonucleases, and Sephadex G-50 spin columns were obtained from Roche
Molecular Biochemicals (Indianapolis, IN). High strength analytical
grade agarose used for two-dimensional gels was obtained from
Bio-Rad. [methyl-3H]Thymidine (1 mCi/ml,
63-67 Ci/mmol) was from Moravek Biochemicals, Inc. (Brea, CA).
[
-32P]dATP (10 mCi/ml, 3000 Ci/mmol) and Hybond
N+ nylon were purchased from Amersham Biosciences.
Prime-It DNA labeling kit was from Stratagene (La Jolla, CA).
Adenovirus vectors expressing GFP or dn-Chk1 were a gift of Cyrus
Vaziri (Boston University School of Medicine). UCN-01 was a gift of the
National Cancer Institute, Bethesda, MD. All other chemicals were
purchased from Sigma and were reagent grade or better.
-32P]dATP-labeled full-length linear SV40 DNA probe
(specific activity ~1 × 109 cpm/µg) prepared by
random priming EcoRI-linearized SV40 DNA. Unincorporated
dNTPs were removed from labeled probe using a Sephadex G-50 spin
column. Blots were hybridized to probe for 16 h at 65 °C,
washed, and exposed to a PhosphorImager screen (Amersham
Biosciences). The mass of replication intermediates was
determined from two-dimensional gel phosphorimages using
ImageQuant software (Molecular Dynamics) to measure the
signals from bubble arcs and 1n DNA. To control for differences in
yield of DNA in different experiments, the relative number of RIs was
calculated for each experiment by normalizing the signal in the bubble
arc to that from the completely replicated 1n DNA, as described
previously (27, 28).
80 °C with exposure times adjusted within the linear
response range of the film. Replication activity was determined from
the [3H]thymidine-labeled DNA signals in x-ray films
scanned by computing laser densitometer (Amersham Biosciences).
Calculations of relative replication activity were performed using
signals from either bubble arcs or completely replicated 1n DNA after
normalizing these signals to the mass of the 1n DNA measured in the
Southern blots run in parallel, as described previously.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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View larger version (20K):
[in a new window]
Fig. 1.
Inhibitory effects of UVC radiation on
initiation and elongation of cellular DNA replication and their
abrogation by caffeine. A, mouse embryo
fibroblasts were irradiated with different fluences of UVC radiation
and then pulse-labeled with [3H]thymidine for 15 min
beginning 50 min postirradiation. The size distribution of
pulse-labeled nascent DNA molecules was determined by velocity
sedimentation on alkaline sucrose gradients as described under
"Experimental Procedures." Gradients were centrifuged from left to
right. B, same as panel A, except that caffeine
was added to the culture medium to a final concentration of 2 mM 2 min prior to irradiation. C, same as
panel A, except that MEFs were derived from embryos
nullizygous for p53.
/
MEFS (Fig. 1C).
View larger version (43K):
[in a new window]
Fig. 2.
Dose- and time-dependent
inhibition of SV40 viral DNA replication by UVC and its abrogation by
caffeine. SV40-infected BSC-1 monkey cells were subjected to
various doses of UVC radiation and incubated for an additional 50 min
before pulse labeling with [3H]thymidine for 15 min
(A and B) or were subjected to 25 J/m2 of 254-nm wavelength UVC radiation and incubated for
the indicated periods of time before pulse labeling with
[3H]thymidine for 15 min (C-F). SV40
replication intermediates recovered from Hirt extracts were separated
on two-dimensional agarose gels and visualized by Southern blotting and
hybridization to 32P-labeled SV40 DNA. The number of SV40
replication intermediates was quantitated by PhosphorImager
analysis. Replication activity was assessed by measuring incorporation
of [3H]thymidine directly into replication intermediates
or into fully replicated SV40 DNA by densitometric analysis of
fluor-impregnated gels run in parallel with the blotted agarose gels
and exposed to x-ray film. C, SV40-infected cells were
sham-irradiated (sham control), or subjected to UVC
radiation alone (UVC 25 J/m2), UVC
radiation after addition of aphidicolin
(aphid/UVC) or with aphidicolin alone
(aphid). G, SV40-infected monkey cells were
treated or not treated with 2 mM caffeine beginning 2 min
before irradiation with 10 J/m2 of 254 nm wavelength UVC
radiation. All values represent the averages of three independent
experiments.
View larger version (30K):
[in a new window]
Fig. 3.
Chk1 is required for UVC-induced and
intrinsic checkpoints that target cellular and viral origins of
replication. A, MDA041 cells were treated as indicated
and pulse-labeled with [3H]thymidine for 15 min beginning
50 min postirradiation. MDA041 cells were similarly analyzed 22 h
postinfection with either a GFP control adenovirus vector
(B) or an adenovirus vector expressing dn-Chk1 (C
and D). Gradients were centrifuged from left to right.
E, SV40-infected cells were treated as indicated and SV40
replication intermediates detected in neutral-neutral two-dimensional
gels were quantitated as described in the text and under
"Experimental Procedures."
)UCN-01). This was the case even after much longer
exposures of blots to PhosphorImager screens (data not shown).
However, replication intermediates were detected in these cells when
they were also treated with UCN-01 (Fig. 4B, early S
phase, (+)UCN-01). In contrast, equivalent numbers of
replication intermediates were detected in DNA isolated from cells
allowed to proceed through S phase for another several hours regardless
of whether or not they were treated with UCN-01 (Fig.
4B, late S phase). Thus, this fragment of rDNA
normally replicates in these cells in late S phase, and UCN-01
treatment accelerates its replication so that it now replicates in
early S phase. These results establish that the
Chk1-dependent intrinsic checkpoint inhibits late S origins of replication in early S phase cells.
View larger version (37K):
[in a new window]
Fig. 4.
UCN-01 accelerates the replication of a
region of the rDNA locus from late to early S phase.
A, nascent DNA pulse-labeled in untreated control cultures
of TR9-7 human fibroblasts or cultures treated with 100 nM
UCN-01 for 50 min were analyzed by velocity sedimentation analysis.
Direction of sedimentation was from left to right. B,
neutral-neutral two-dimensional gel analysis of DNA replication
intermediates from a region of the repetitive rDNA locus isolated from
cells treated or not treated with 100 nM UCN-01 2 h
("early S") or 6 h ("late S") after release from an aphidicolin
arrest in early S phase and 50 min before their isolation.
DISCUSSION
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank George Stark for cell lines, Robert Schultz and the National Cancer Institute, for UCN-01, Bill Kaufmann for communicating results prior to publication, and Cyrus Vaziri for adenovirus vectors and very helpful advice.
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Note Added in Proof |
---|
Recent experiments published after review of our manuscript was complete indicate that Chk1 phosphorylates a downstream checkpoint protein, Cdc25A, in the absence of DNA damage or other perturbations. (Zhao, H., Watkins, J. L., and Piwnica-Worms, H. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 14795-14800). This is consistent with the role for Chk1 in an intrinsic checkpoint revealed by our experiments.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCB 9317011, United States Public Health Service Grant CA 84086, and by shared resources funded by Roswell Park Cancer Center Support Grant P30 CA16056.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.
Current address: Human Genome Sciences, Inc., 9410 Key West Ave.,
Rockville, MD 20850.
§ Both authors contributed equally to the results of this work.
¶ To whom correspondence should be addressed. Tel.: 716-845-7691; Fax: 716-845-1579; E-mail: wburhans@acsu.buffalo.edu.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M204264200
2 J. Huberman, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: MEF, mouse embryo fibroblasts; GFP, green fluorescent protein; RI, replication intermediates.
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