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INTRODUCTION |
DNA replication is at the center of the eukaryotic cell cycle, and
its regulation under conditions of stress, or after exposure to
DNA-damaging agents, is under the control of intricate pathways that
are only now beginning to be characterized. Although the mechanisms
regulating DNA replication after DNA damage are studied intensively and
a considerable amount of information is now available (reviewed in Ref.
1), the mechanisms that regulate DNA replication in response to other
types of stress, such as heat stress, are much less studied and
understood. However, heat stress severely compromises cellular function
and survival, constitutes a likely event in the life cycle of an
organism, and is a form of stress organisms had to deal with during evolution.
It is well documented that exposure of cells to heat strongly inhibits
DNA replication, and it is thought that this inhibition underlies the
increased heat sensitivity of S-phase cells, as compared with cells in
G1 or G2 (2-5). The formation of chromosome aberrations in heated S-phase cells is often considered a consequence of adverse effects on steps of DNA replication (2), as heat is
inefficient in inducing DNA damage directly (6-9). Indeed, heat
inhibits several nuclear processes associated with
semiconservative replication of DNA including the incorporation of
radiolabeled precursors into acid-insoluble material (10, 11), the
initiation of new replicons (3, 4, 12, 13), the elongation of the DNA
fiber at the replication fork (5), the synthesis and deposition of new
histones into chromatin, and the reorganization of nascent DNA into
mature chromatin (14, 15).
Although the above studies identify heat-sensitive steps of DNA
replication, they provide only limited information as to the molecular
mechanism(s) involved. It is possible that the observed inhibition is a
passive consequence of heat-induced inactivation of DNA replication
factors, as well as of damage induced in chromatin by the precipitation
of denatured protein on the nuclear matrix (14-18). Alternatively,
however, some of the inhibitory effects could be the consequence of a
process, equivalent to a checkpoint, which actively stalls DNA
replication to prevent grave errors and facilitate repair, much the way
ionizing radiation inhibits replicon initiation by activating the
ataxia telangiectasia mutated pathway (1, 19-21).
In an effort to characterize putative regulatory components of DNA
replication in cells exposed to heat, we introduced the simian virus 40 (SV40)1-based in
vitro DNA replication assay. In this assay, the replication of
plasmids carrying the SV40 origin of DNA replication (ori) is accomplished in vitro using either crude cytoplasmic
extracts from human cells, or proteins purified from such extracts
(22-26). Only one protein of viral origin is required, the SV40 large
tumor antigen. The assay provides a well controlled and molecularly defined system to study regulatory aspects of DNA replication, and
allows the separation of regulatory effects targeting DNA replication
factors from effects of heat on the substrate DNA.
Using this assay we reported (27) that cytoplasmic extracts prepared
from heated cells had a reduced DNA replication activity, as compared
with extracts prepared from non-heated cells, and that this reduction
in DNA replication activity was specifically caused by an inactivation
of replication protein A (RPA) (28-30). Addition of recombinant RPA
(rRPA) in these reactions completely reversed the inhibition. Because
RPA levels were similar in extracts of heated and non-heated cells and
because RPA was relatively resistant to direct heat inactivation, we
proposed that the observed response reflected the activation of a
checkpoint that targeted and inactivated RPA. A regulatory role for RPA
in DNA replication has also been suggested in cells exposed to ionizing
radiation (31-35).
RPA is an essential component of eukaryotic DNA metabolism and has
activity as the primary single-stranded DNA-binding protein (for
reviews, see Refs. 36 and 37). It is a heterotrimer composed in humans
of 70-kDa (hRPA1), 29-kDa (hRPA2), and 14-kDa (hRPA3) subunits. Genetic
and biochemical studies suggest a role for RPA in the initiation and
elongation stages of DNA replication (36, 37). RPA is also required for
nucleotide excision repair and homologous DNA recombination (38-41).
RPA2 is phosphorylated in a cell cycle-dependent manner and
in response to DNA damage, but the regulatory significance of these
phosphorylations has not been established (36, 37). RPA also interacts
with a variety of proteins involved in DNA metabolism including Rad51
(42), Rad52 (43, 44), XPA (45-47), and p53 (48-50). Finally, RPA is phosphorylated by the DNA-dependent protein kinase and by
ataxia telangiectasia mutated (51-55). Several of these
interactions and modifications are compatible with a regulatory
function for RPA in DNA replication, and with a role in the cellular
responses to DNA damage, although direct evidence is still lacking.
Because significant phosphorylation of RPA2 was not
observed after exposure to heat, it was speculated that if RPA is a
target of processes regulating DNA replication, it should be modified by mechanisms other than phosphorylation (27). Recently, evidence has
been presented that interaction with nucleolin, a major nucleolar protein involved in ribosome biogenesis and diverse other biological processes (for reviews, see Refs. 56 and 57), can regulate the
replication functions of RPA (58).
Here, we extend our studies on the regulation of DNA replication in
cells exposed to heat shock and inquire whether the previously reported
effects of heat on in vitro DNA replication and the
associated inactivation of RPA (27) derive from an interaction with
nucleolin. The results demonstrate RPA-nucleolin interactions perfectly
correlating with in vitro DNA replication activity and
suggest that this interaction reflects an early step of the cellular
response to heat stress.
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MATERIALS AND METHODS |
Cell Culture--
A human malignant melanoma cell line
(SK-MEL-28) was used for all experiments. Cells were obtained from ATCC
(HTB-72) and were grown in Dulbecco's modified minimum essential
medium supplemented with 25 mM glucose, 10%
heat-inactivated calf serum, and antibiotics at 37 °C in an
atmosphere of 5% CO2. For experiments 5 × 105 cells/ml were seeded in either 75-cm2 (15 ml) or 150-cm2 (30 ml) tissue culture flasks and allowed to
grow for 3 days. At this time, the growth medium was changed and cells
were incubated for 24 h in fresh growth medium before use in
experiments. Cells were exposed to heat by submerging sealed flasks in
a water bath set at the desired temperature with an accuracy of
0.05 °C. Temperature was monitored with a calibrated mercury
thermometer. After treatment cells were trypsinized, collected, counted
in a cell counter, and used for extract preparation.
Measurement of DNA Replication in Vivo--
To evaluate DNA
replication either by incorporation of radioactively labeled precursors
into total DNA, or by alkaline sucrose gradient centrifugation (see
below), 5 × 105 SK-MEL-28 cells from a growing
culture were seeded into 75-cm2 tissue culture flasks (10 ml) and were allowed to grow for 3 days. This protocol yields results
equivalent to those obtained using the growth protocol described above,
which was selected when large numbers of cells were needed for extract
preparation. Cells were exposed to heat for the indicated periods of
time and promptly returned to 37 °C. At various times thereafter,
depending on the experimental protocol, 3 µCi/ml
[3H]thymidine (PerkinElmer Life Sciences) was
added and cells were incubated at 37 °C for 15 min. Total DNA
synthesis was measured as described previously (59). Briefly, cells
were trypsinized, loaded on glass microfiber filters (Whatman GF/A),
washed with cold trichloroacetic acid, rinsed with deionized water, and
counted for 3H activity. DNA synthesis was calculated as
the percentage of incorporation in sham-treated controls.
The size distribution of nascent DNA was measured in heated and
non-heated cells in 5-20% linear alkaline sucrose gradients (59).
They were prepared in a buffer containing 0.1 M NaOH, 0.9 M NaCl, 0.01 M EDTA, pH 12.5, in 20-ml
polyallomer tubes for a total volume of 17 ml. Cells (2.5 × 105 in 50 µl) were lysed for 3 h by gently layering
on 0.3 ml of lytic solution (0.5 M NaOH, 0.02 M
EDTA, pH 12.5, and 0.1% Nonidet P-40), in a cut 1-ml syringe.
Subsequently, the cell lysate was gently layered on the top of the
gradient and centrifuged for 90 min in a Sorval RC60 centrifuge
equipped with an AH 627 rotor, at 26,000 rpm, 20 °C. After
centrifugation, gradients were fractionated (1 ml) and incorporated
activity measured as described above. Results are presented as
percentage of total [3H]thymidine activity per fraction
after correction for the relative inhibition of total DNA synthesis
resulting from heat exposure.
Cytoplasmic Extract (S100) Preparation--
The method used to
prepare cytoplasmic cell extracts for in vitro SV40 DNA
replication has been described previously (27, 60). Briefly, cells
(1-2 × 108) were washed in PBS, resuspended in
hypotonic buffer (10 mM HEPES, pH 7.5, 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM
phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol), and
disrupted either by three freeze/thaw cycles, or in a Dounce
homogenizer (20 strokes, B pestle). The cell lysate was adjusted to 140 mM KCl by adding 10% volume of 10× cytoplasmic buffer
(0.3 M HEPES, pH 7.5, 1.4 M KCl, 0.03 M MgCl2) and was incubated in ice for 15 min.
Subsequently, the cell lysate was centrifuged first at 3,300 × g for 15 min and then at 100,000 × g for
1 h (S100), and the cleared supernatant was dialyzed against 100 volumes of dialysis buffer (20 mM HEPES, pH 7.5, 20%
glycerol, 50 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol) at 4 °C. The dialysate was centrifuged at
15,000 × g for 20 min and protein concentration
determined by the Bradford assay (Bio-Rad). The extract was either used
immediately or snap-frozen and stored at
80 °C for later use. In
some experiments the final KCl concentration was adjusted to 200 mM by appropriately increasing salt concentration in the
10× cytoplasmic buffer.
In Vitro DNA Replication Assay--
The SV40-based in
vitro DNA replication assay was described previously (60).
Briefly, reaction mixture (25 µl) contained 40 mM HEPES,
pH 7.5; 8 mM MgCl2; 0.5 mM
dithiothreitol; 3 mM ATP; 200 µM each CTP,
GTP, and UTP; 100 µM each dATP, dGTP, and dTTP; 40 µM [
-32P]dCTP (0.001 µCi/ml, 3000 cpm/pmol; PerkinElmer Life Sciences); 40 mM creatine
phosphate; 1.25 µg of creatine phosphokinase; 0.15 µg of
superhelical plasmid DNA; 100-200 µg of cytoplasmic extract; and 0.5 µg of SV40 large T antigen. The reaction mixture was incubated at
37 °C for 1 h. Reactions were terminated by addition of 20 mM EDTA. Subsequently, 25 µg of denatured salmon sperm
carrier DNA and 1 ml of 10% cold trichloroacetic acid were added to
each sample. The resulting precipitate was collected onto Whatman GF-C glass fiber filters, and 32P incorporation into DNA was
determined by liquid scintillation counting. To analyze replication
products, gel electrophoresis was performed. Samples were treated with
0.1% sodium dodecyl sulfate, followed by 15-min digestion with RNase A
(Sigma, 20 µg/ml) at 37 °C. Proteinase K was then added (Sigma,
200 µg/ml), and samples were incubated at 37 °C for 30 min. DNA in
the sample was purified in a QIAquick column (Qiagen) and
electrophoresed in 1.2% agarose gel at 6.5 V/cm.
Detection of RPA-Nucleolin Complex by
Immunoprecipitation--
Interactions between RPA and nucleolin were
investigated by immunoprecipitation carried out using the IMMUNOcatcher
kit (CytoSignal, Irvine, CA) according to the manufacturer's
instructions. Briefly, SK-MEL-28 cells (2 × 106) were
treated and collected for whole cell extract preparation by incubation
(4 °C for 1 h) in 200 µl of extraction buffer (supplied with
the kit). The supernatants obtained by centrifugation were incubated
with protein A/G beads pre-absorbed with pre-immune serum for 30 min at
room temperature and centrifuged. Clarified supernatants were then
incubated with anti-RPA2 antibody (PAb34A, a gift from Dr. J. Hurwitz)
for 1 h at room temperature (or overnight at 4 °C). Thirty µl
of protein A/G beads was added to the supernatants and incubated for
1 h at room temperature. Beads were collected, washed three times
in extraction buffer, and washed once in PBS, and the
immunoprecipitates subjected to Western blotting.
Western Blot Analysis--
Proteins were separated using 10%
SDS-polyacrylamide gel electrophoresis and transferred to PVDF-Plus
membrane (Osmonics Inc.). The membrane was blocked for 1.5 h with
5% powdered milk in PBS, washed twice with PBS, and incubated
overnight at 4 °C with primary antibody. Subsequently, the membrane
was washed three times in PBS containing 0.1% Tween 20, incubated with
a horseradish peroxidase-conjugated goat anti-mouse, or goat
anti-rabbit, secondary antibody for 1.5 h and washed five times in
PBS containing 0.1% Tween 20. Proteins were detected with
ECLplus (Amersham Pharmacia Biotech) and visualized in a
Storm 860 Phosphor- Imager (Molecular Dynamics). RPA1 and RPA2 were
detected using monoclonal antibodies PAb34A and PAb70A (obtained from
Dr. J. Hurwitz). RPA3 was detected using polyclonal antibody Ab11
(obtained from Dr. S.-H. Lee). Nucleolin was detected either with a
polyclonal antibody 134 (obtained from Dr. P. Bouvet) or with a
commercially available monoclonal antibody C23 (Santa Cruz
Biotechnology Inc., Santa Cruz, CA).
Immunofluorescence Microscopy--
SK-MEL-28 cells grown on
coverslips were washed once with cold PBS and cold cytoskeleton buffer
(10 mM Hepes, pH 7.4, 300 mM sucrose, 100 mM NaCl, and 3 mM MgCl2), as
described previously (61). After extraction for 2 min on ice with 0.5%
Triton X-100 in cytoskeleton buffer containing proteinase inhibitors
(61), cells were fixed for 20 min at room temperature with 4%
formaldehyde in PBS, rinsed with PBS, and incubated with 0.5% Nonidet
P-40 in PBS for 5 min. Primary antibody (anti-nucleolin, C23) was then added and incubated for 1 h at room temperature. After rinsing three times with 0.5% Tween 20 in PBS, secondary antibody (donkey anti-mouse IgG conjugated with Texas Red; Molecular Probes, Eugene, OR)
was applied and incubated for 1 h at room temperature. Coverslips were then rinsed three times with 0.5% Tween 20 in PBS and mounted onto glass slides. Conventional fluorescence or confocal microscopy was
used to detect antigens.
 |
RESULTS |
Transient Inhibition of DNA Replication after Exposure of Cells to
Heat--
As a first step in our investigations, we characterized the
effect of heat on DNA replication in SK-MEL-28 cells. Cultures were
exposed to 44 °C for various periods of time and the effect on DNA
replication determined. Fig.
1A shows the results obtained as a function of time. In line with earlier observations (see Introduction), exposure to heat strongly inhibited ongoing DNA replication. The inhibition was biphasic, with a steep component that
dominated in the first 20 min and reduced DNA synthesis to 20% of
control, followed by a shallow component evident between 20 and 90 min
that further reduced, to nearly zero, DNA synthesis. The effect of heat
shock on DNA replication was transient and recovered to control levels
8 h after a 15-min exposure to 44 °C (Fig. 1B).

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Fig. 1.
Effects of heat shock on replicative DNA
synthesis in SK-MEL-28 cells as assayed in vivo by the
incorporation of [3H]thymidine. Panel A,
total DNA synthesis after exposure of cells to 44 °C for different
periods of time as indicated. DNA replication activity was measured by
a 15-min pulse with [3H]thymidine at 37 °C, given
immediately after heat shock. The mean and standard error from three
experiments are shown. Panel B, kinetics of recovery of
heat-induced inhibition of DNA replication. SK-MEL-28 cells were
exposed to 44 °C for 15 min and returned to 37 °C. At the
indicated times, a 15-min pulse with [3H]thymidine was
given and incorporation into acid-insoluble material measured. The mean
and standard error from two experiments are shown. Panel C,
alkaline sucrose density gradient profiles of SK-MEL-28 cells exposed
to 44 °C for 0 min (circles), 5 min
(triangles), 10 min (inverted
triangles), 20 min (squares), and 30 min
(diamonds). Cells were exposed to a 15-min pulse with
[3H]thymidine immediately after heat shock. Plotted is
the percentage of total activity in each fraction as a function of the
fraction number. For samples exposed to heat, the incorporation values
for each fraction have been multiplied by the observed inhibition of
DNA replication. Sedimentation is from left to right. Early fractions
contain small nascent DNA produced in the early stages of DNA
replication. Shown for calibration purposes are also the locations of
sedimentation maxima for T7 (37 kilobase pairs) and T2 (167 kilobase
pairs) phage DNA.
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The effect of heat shock on the replicon initiation and chain
elongation stages of DNA replication was evaluated with alkaline sucrose density-gradient centrifugation. Fig. 1C shows the
results obtained. Exposure to heat inhibited replicon initiation
manifested by a reduction in the activity of early fractions, which
represent the top of the gradient and therefore low molecular weight
material. However, heat also inhibited, to a similar degree, chain
elongation manifested by a reduction in material incorporated in the
remaining portion of the gradient. Although only results obtained after exposure of cells to 44 °C are presented here, the results in Fig.
2A indicate that an effect on
DNA replication was clearly observed at temperatures above
40 °C.

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Fig. 2.
Panel A, SV40 DNA replication activity
of SK-MEL-28 cell extracts (S100) prepared from cultures exposed for 30 min to the indicated temperatures. Shown for comparison is also
cellular DNA replication measured by a 15-min
[3H]thymidine incorporation in intact cells at 37 °C.
Panel B, SV40 DNA replication in extracts of SK-MEL-28 cells
exposed to 44 °C for the indicated periods of time and processed for
extract preparation immediately thereafter (closed
circles). The open circles trace
results of practically identical reactions assembled with 1 µg for
rRPA. Shown are the mean and standard error from three experiments.
Panel C, agarose gel of SV40 DNA replication products for
reactions similar to those in panel B.
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Inhibition of SV40 DNA Replication in Extracts of Heated
Cells--
Several processes may contribute to the above inhibition of
DNA replication in cells exposed to heat shock. First, the coordinated function of the components of DNA replication machinery may be impaired
at temperatures above 40 °C, without associated irreversible changes
in either the chromatin substrate, or the DNA replication factors.
Second, one or more key DNA replication factors may be heat-sensitive
and may be rapidly inactivated in cells exposed to heat. Third,
heat-induced alterations in chromatin structure may modify the
substrate DNA and may inhibit its efficient replication. Fourth, cells
may sense heat damage in chromatin and other cellular structures and
activate signaling pathways, equivalent to a checkpoint, that actively
inhibit DNA replication.
To discriminate between these possibilities, we tested DNA replication
activity in extracts of heated cells using the SV40 in vitro
DNA replication assay. By measuring DNA replication with untreated
plasmid DNA and tumor antigen, the assay removes the effects of heat on
DNA replication substrate and allows the study of effects on key DNA
replication factors. These effects can include direct heat
inactivation, as well as the targeted inactivation by regulatory processes.
Fig. 2A shows SV40 DNA replication activity in extracts
(S100 fraction) prepared from SK-MEL-28 cells exposed for 30 min to temperatures between 38 °C and 44 °C, normalized to the activity of extracts from non-treated cells. Heat shock suppressed in
vitro DNA replication activity in a
temperature-dependent manner only above 40 °C. The
levels of DNA replication inhibition in vitro paralleled
those in vivo, suggesting that the assay reproduces essential components of the cellular processes underlying DNA replication inhibition.
The effect of different exposures to 44 °C on SV40 DNA replication
is shown in Fig. 2B. A rapid reduction in activity was observed in cells exposed to 44 °C for 5-30 min. Surprisingly, however, the replication activity increased and reached near control levels after exposures for 60 and 90 min. A similar response was also
observed when the products of DNA replication were analyzed by agarose
gel electrophoresis (Fig. 2C). There was no difference in
the distribution of DNA replication products in the different samples,
suggesting that mainly initiation events were reduced, or that all
steps of DNA replication were equally affected. A specific effect on
chain elongation would have increased the abundance of slowly migrating
replication intermediates and can therefore be excluded. Results
obtained after addition of rRPA to the reactions are discussed below.
The high levels of DNA replication activity in extracts of cells
exposed to 44 °C for 60-90 min suggests that the transient inhibition between 5 and 30 min reflects a regulatory process targeting
an essential replication factor(s) rather than the irreversible denaturation of heat-sensitive DNA replication factors; the latter would have persisted as exposure times were increased beyond 30 min.
Inactivation of Replication Factors and Inhibition of DNA
Replication after Heat Shock in Vitro--
We inquired whether the
high levels of DNA replication activity after heat shock for 60-90 min
reflected an inherent heat resistance of the DNA replication factors.
Extracts of non-heated cells were exposed to heat and tested for
in vitro SV40 DNA replication activity. Fig.
3A shows that, contrary to the
exquisite resistance observed when heating was administered in intact
cells, heating in vitro for only 10 min at 41 °C led to a
>80% loss of DNA replication activity. The inhibition was not
directly reversible, as subsequent incubation at 37 °C for 30 min
did not recover DNA replication activity (Fig. 3A).
Additionally, DNA replication per se was sensitive to heat
as incubation of reactions assembled with non-heated cell extract to
temperatures as low as 39 °C reduced DNA replication activity by
over 50%. Incubation at 41 °C completely halted DNA replication
(Fig. 3B).

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Fig. 3.
Panel A, effect on SV40 DNA
replication of a 10-min exposure to 41 °C of an extract (S100)
prepared from non-heated SK-MEL-28 cells. Shown are results with
reactions assembled immediately after treatment or following a 30-min
incubation at 37 °C. Panel B, effect of temperature on
SV40 in vitro DNA replication activity. Reactions were
assembled with extract (S100) prepared from non-heated SK-MEL-28 cells
and incubated at 37 °C (closed circles),
39 °C (open circles), and 41 °C
(inverted triangles). Incorporated activity was evaluated at
the indicated times. The mean and standard error from three experiments
are shown.
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RPA as a Target of a Pathway That Regulates DNA Replication in
Cells Exposed to Heat--
Recently, we provided evidence that RPA is
the target of a pathway that regulates DNA replication in HeLa cells
after heat shock (27). We inquired whether a similar mechanism was
active in extracts of heated SK-MEL-28 cells. The results in Fig.
2B indicate that 1 µg of rRPA restored DNA replication to
near control levels and abolished heat-induced fluctuations in SV40 DNA
replication activity. The effect of RPA in rescuing DNA replication was
specific as bovine serum albumin was ineffective in modifying DNA
replication activity (data not shown). RPA inactivation can be ruled
out as the cause of the reduced DNA replication activity at 5-30-min exposures as RPA retained significant activity in extracts of cells
exposed to 44 °C for 60-90 min. These results point to a regulatory
pathway involving RPA, which is heat-sensitive.
RPA-Nucleolin Interactions after Heat Shock--
The above results
point to RPA as the target of a pathway that regulates DNA replication
in heated cells and raise the question as to the mechanism involved. We
explored the possibility that RPA activity is regulated by an
interaction with nucleolin as described recently (58). Whole cell,
rather than S100, extracts were assayed to examine interactions in a
large component of the cellular protein pool. Fig.
4A shows the results obtained
with extracts of heated cells following immunoprecipitation with an anti-RPA2 antibody. A substantial increase was observed in the amount
of interacting nucleolin after heat shock despite the decrease in the
amount of immunoprecipitated RPA (Fig. 4A). An effect was detectable even after 5 min, but a dramatic increase was observed in
cells exposed for 20-30 min. After longer exposure times, the interaction became attenuated and returned to near control levels after
90 min. Thus, the interaction between RPA and nucleolin mirrors
perfectly SV40 DNA replication activity in extracts from heated cells
(Fig. 2B). There were no significant changes in nucleolin, or in the three RPA subunits, in extracts of heated cells (Fig. 4B), so the observed interaction was not resulting from
altered availability of the interacting proteins. Heating extracts
in vitro could not reproduce the heat-induced interaction
between RPA and nucleolin (Fig. 4C), suggesting the
operation of processes that are perturbed or cannot be activated in the
extract.

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Fig. 4.
Heat shock induced an interaction between
nucleolin and RPA in intact cells but not in whole cell extracts.
Panel A, cells heated at 44 °C for the indicated periods
of time were lysed for whole cell extract preparation and
immunoprecipitated with an anti-RPA-2 antibody. Immunocomplexes were
analyzed by Western blotting using either an anti-nucleolin or an
anti-RPA-2 antibody. Panel B, Western blot analysis for the
different RPA subunits of the whole cell extracts used in the
immunoprecipitation experiment shown in panel A. Panel C, whole cell extracts prepared from non-heated
SK-MEL-28 cells were exposed in vitro to 44 °C for the
indicated periods of time, immunoprecipitated, and analyzed as outlined
in panel A.
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Contrary to results obtained with HeLa cells (27), an evaluation of RPA
levels in S100 extracts of SK-MEL-28 cells showed reductions after heat
shock that could explain the observed inhibition of SV40 DNA
replication, without the need to invoke an interaction with nucleolin
(Fig. 5A). The absence of this
reduction in HeLa cells and its transient occurrence in SK-MEL cells
raised the possibility that it is caused by the interaction with
nucleolin, i.e. a lower extractability of the RPA-nucleolin
complex than of RPA alone from the nucleus during S100 preparation. To
address this possibility, S100 extracts were prepared from heated and non-heated SK-MEL-28 cells using either the standard concentration of
140 mM KCl for extraction during preparation (see
"Materials and Methods"), or 200 mM KCl to enhance
protein extraction from the nucleus. We also increased from 10 to 30 min the incubation time at 4 °C for extraction after homogenization.
Increasing extraction time reduced the difference in RPA levels between
heated and control cells without compromising the inhibition of SV40
DNA replication (Fig. 5, compare A with B).
Increased extraction by high salt further limited the observed
reduction in RPA levels in S100 extracts of heated cells. At the same
time, however, high salt eliminated the inhibition of DNA replication
in S100 extracts and the RPA-nucleolin interaction in whole cell
extracts (Fig. 5C). These results confirm that the
RPA-nucleolin interaction after heat shock is rather common and occurs
in different cell lines such as HeLa (58) or SK-MEL-28 cells, while the
trapping of the complex in the nucleus is cell
line-dependent and more pronounced in SK-MEL-28 cells.

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Fig. 5.
Heat shock reduced the extractability of RPA
into S100 fraction in SK-MEL-28 cells. Panel A,
SK-MEL-28 cells were exposed to 44 °C for the indicated periods of
time and S100 cell extracts prepared. Thirty µg of these extracts
were examined by Western blotting for the levels of RPA subunits.
Panel B, effect of 200 mM KCl extraction during
S100 preparation on SV40 in vitro DNA replication in
extracts of heated cells. SK-MEL-28 cells were exposed to 44 °C for
30 min and S100 extracts prepared using either the standard extraction
protocol (140 mM KCl), or 200 mM KCl.
Replication activity was compared with that of extracts of non-heated
cells. Panel C, whole cell extracts prepared in the presence
of KCl at concentrations similar to those used in panel
B were immunoprecipitated and analyzed by Western blotting
as described in Fig. 4A.
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Heat-induced Translocation of Nucleolin into the
Nucleoplasm--
The near exclusive localization of nucleolin into the
nucleolus and of RPA in the nucleoplasm in non-stressed cells is
expected to prevent an interaction between the two proteins and raises the question as to how this interaction is facilitated after heat shock. Recent studies demonstrated a translocation of nucleolin from
the nucleolus into the nucleoplasm after exposures to 44 °C longer
than 60 min (58). We investigated whether a translocation of nucleolin
can be observed in heated SK-MEL-28 cells in a time frame correlating
with the observed inhibition of in vitro DNA replication.
Cells were exposed to 44 °C for periods ranging between 0 and 90 min
and nucleolin localization determined. Fig.
6 shows representative images of the
results obtained. In control cells (0-min exposure), nucleolin was
localized, as expected, practically exclusively in the nucleolus. The
nucleolar staining was specific for nucleolin, as pre-immune serum did
not produce any staining (data not shown). There was evidence for
translocation of nucleolin into the nucleoplasm even in cells exposed
to 44 °C for 5 min, indicated by a diffuse staining in the nuclear
region. However, nucleoli remained clearly visible in these samples.
The pattern of staining changed significantly after 10 min at 44 °C
and remained qualitatively similar for exposure times up to 30 min. The
images obtained after these exposure times showed a diffuse nuclear
staining with little or no definition of the nucleolus. Notably, after
exposure for 60 or 90 min at 44 °C, a preferential localization of
nucleolin in the nucleolus was again evident. These results exactly
parallel the in vitro inhibition of DNA replication (Fig.
2B) as well as the interaction between nucleolin and RPA
(Fig. 4A) in cells exposed to heat stress.

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Fig. 6.
Heat shock induced a relocalization of
nucleolin from the nucleolus into the nucleoplasm. SK-MEL-28 cells
grown on coverslips were exposed to 44 °C for the indicated periods
of time and were subsequently fixed and stained with an antibody
against nucleolin and a Texas Red-conjugated secondary antibody as
described under "Materials and Methods." Cells were analyzed using
a confocal microscope. Shown are representative images of cells
exposed for different times to 44 °C.
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The observation that a translocation of nucleolin from the nucleolus
into the nucleoplasm could be observed immediately after exposure to
heat indicated that the underlying mechanism is fast. As a next step we
investigated, therefore, the persistence of the response in an effort
to correlate it with the kinetics of DNA replication recovery observed
in intact cells (Fig. 1B). Cells were exposed to 44 °C
for 15 min and returned to 37 °C. At various times thereafter, cells
were fixed and stained for nucleolin localization. The results obtained
are shown in Fig. 7. Nucleolin
translocation is at maximum immediately after heat and returns to the
nucleolus ~1 h after heating, long before cellular DNA replication
has recovered completely (Fig. 1B).

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Fig. 7.
Heat-induced translocation of nucleolin from
the nucleolus into the nucleoplasm is transient. SK-MEL-28 cells
grown on coverslips were exposed to 44 °C for 15 min and returned to
37 °C. At various times thereafter, cells were stained and analyzed
as outlined in the legend of Fig. 6. Shown are representative images of
cells allowed to recover from heat shock for the indicated times.
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DISCUSSION |
RPA-Nucleolin Interactions Inhibit SV40 DNA Replication in Extracts
of Heated Cells--
The results presented above confirm and extend
previous observations on the effect of heat on DNA replication
activity, as measured by the SV40 in vitro DNA replication
assay (27). We show that even modest exposures of SK-MEL-28 cells to
heat (5-10 min) cause measurable reduction in DNA replication activity
(Fig. 2B). Evidently, inhibitory effects on DNA replication
can precede the development of lethal events, as short exposures to
heat only have a small effect on colony-forming ability (10-20%
reduction). As in the case of HeLa cells (27), inhibition of DNA
replication in SK-MEL-28 cells was the result of a specific
inactivation of RPA (Fig. 2B), rather than the result of
nonspecific and widespread heat-induced inactivation of one or more DNA
replication factors.
Inactivation of RPA in heated cells was not caused by obvious
post-translational modifications, as the electrophoretic mobility in
SDS-polyacrylamide gels remained unchanged for all subunits (Fig.
4B). Furthermore, addition of rRPA effectively rescued DNA replication in extracts of heated cells, a result that is compatible with the absence of RPA-modifying activities. If RPA-modifying activities with inhibitory effects were present in the extract, they
would have also modified added rRPA and would have reduced the
efficiency of the rescuing process.
Inhibition of SV40 DNA replication in extracts of heated cells
correlates well with an RPA-nucleolin interaction (Fig. 4A). Nucleolin is involved in ribosome biogenesis and is known to be required for the first step of pre-rRNA processing in which the 5'
external transcribed spacer undergoes endonucleolytic cleavage (57, 62,
63). However, nucleolin is a multifunctional protein, has been found in
a replication complex (64), and has been recently characterized as an
RPA-interacting protein (58). The latter report showed that nucleolin
inhibits SV40 DNA replication and that this inhibition can be rescued
by the addition of stoichiometric amounts of rRPA. Evidence was also
presented that an RPA-nucleolin interaction inhibits the initiation
steps of DNA replication, while leaving unaffected the single-stranded
DNA binding activity of the protein. Although not examined directly, it
is likely that an RPA-nucleolin interaction also affects RPA-requiring
events in the elongation stages of DNA replication. These observations in aggregate provide a mechanistic explanation for the reported effects
of heat on SV40 DNA replication (27) and point to nucleolin as a
potential regulator.
Effects of Heat on Nucleolin Localization in Vivo--
The
possible relevance of the above biochemical observations to the
regulation of DNA replication in heated cells is indicated by the
observed intracellular relocalization of nucleolin (Fig. 6). Although
nucleolin exclusively localizes in the nucleolus of unstressed cells
(56, 57), exposure to heat (58) or other types of stresses (65) leads
to its relocalization into the nucleoplasm. This relocalization
potentially enables an interaction with RPA, which according to the
results presented here, and those previously published (58), is
expected to inhibit DNA replication. In line with this expectation,
nucleoplasmic regions with increased accumulation of nucleolin show
reduced incorporation of DNA synthesis precursors (58). A potentially
relevant observation that hints to intriguing regulatory mechanisms is
that RPA3, the RPA subunit that directly interacts with nucleolin (58),
localizes in the nucleolus of non-heated cells without being associated
with RPA1 and RPA2 (66).
Although earlier observations suggested that nucleolin translocation
and RPA-nucleolin interactions require exposures to 44 °C longer
than 60 min (58), our results demonstrate effects at the earliest heat
exposure times measured (5 min). This places the sensitivity of
nucleolin translocation and of RPA-nucleolin interaction in the same
exposure range as the inhibition of DNA replication (Fig.
1A) and is in line with a regulatory role for nucleolin in
DNA replication under conditions of stress. Further evidence for a
regulatory role of nucleolin in DNA replication is provided by the
similarities between the kinetics of recovery of in vitro
DNA replication (27) and the kinetics of intracellular nucleolin
relocalization after exposure to heat (Fig. 7); both process are nearly
completed within 1-2 h.
On the basis of these observations, a model can be developed for the
regulation of DNA replication in heated cells whereby translocation of
nucleolin from the nucleolus into the nucleoplasm inhibits DNA
replication by a direct interaction with RPA. Although the ultimate
target of this nucleolin-mediated inhibitory process, which may be
equated to a checkpoint response, is RPA, the signal that initiates the
pathway and the up-stream events that lead to nucleolin relocalization
remain unknown. We previously speculated (27) that heat-induced
chromatin damage (67) might be generating the initiating event.
However, other mechanisms of activation cannot be ruled out at present.
One can only speculate on the upstream events that lead to nucleolin
translocation. Release of nucleolin from the nucleolus can be mediated
by a loss of its pre-rRNA binding substrate, which occurs in heated
cells as heat exposure decreases rRNA synthesis (68, 69). However,
because nucleolin is a phosphoprotein that can be modified by several
kinases including Cdc2, protein kinase C
, and casein kinase II (56,
57), and can also be methylated and ADP-ribosylated, it is possible
that its translocation into the nucleoplasm is the result of
post-translational modifications. Indeed, phosphorylation of nucleolin
regulates some of its functions during cell growth and in the cell
cycle, and may also modulate its activities in response to heat stress.
Heat shock may alter the phosphorylation state of nucleolin by
activating an unidentified kinase, or a phosphatase, and may mediate in
this way its translocation into the nucleoplasm. The signal leading to
this modification must be transient as nucleolin returns to the
nucleolus 1-2 h after heat exposure (Fig. 7).
It is particularly interesting that the nucleolin-dependent
pathway of DNA replication regulation is itself heat-sensitive and can
be inactivated after prolonged (60-90 min) exposures to heat (Fig.
2B). This was demonstrated by the reduced interaction of
nucleolin with RPA (Fig. 4A) and by the reduced
intracellular relocalization of nucleolin (Fig. 6) in cells exposed to
heat for 60 min or longer. Thus, an excessive amount of stress can cause a collapse of the protective responses mounted by the cell, suggesting that experiments to characterize them should be carried out
under carefully selected conditions.
The results in Fig. 4C indicate that RPA may not
constitutively interact with nucleolin and that this interaction may
not be induced by heating extracts in vitro. It is possible
that for a productive interaction between nucleolin and RPA a
particular set of posttranslational modifications is required and/or
the presence of nuclear organizing structures such as the nuclear matrix, to facilitate these interactions. It may be relevant in this
regard that nucleolin is found to bind matrix attachment region
elements on chromosomal DNA (70) and that the DNA replication machinery
is thought to be attached to the nuclear matrix. An auxiliary role of
DNA in RPA-nucleolin interactions is indicated by the observation that
RPA-nucleolin interactions can be disrupted at relatively low salt
concentrations (200 mM KCl; Fig. 5C) in cellular
extracts, whereas they require nearly 500 mM salt in an RPA
column made with single-stranded DNA-cellulose (58). Elucidation of the
specific requirements for RPA-nucleolin interactions will enhance our
understanding of the underlying regulatory mechanism(s).
Other Mechanisms in the Regulation of DNA Replication in Heated
Cells--
Although the translocation of nucleolin into the
nucleoplasm explains the initial inhibition of DNA replication, a
comparison between the kinetics of nucleolin relocalization into the
nucleolus (Fig. 7) and of the recovery of DNA synthesis in
vivo (Fig. 1B) suggest that other processes must be
involved in maintaining the initial inhibition. Nucleolin, by virtue of
its fast translocation, may be a first line of defense activated
immediately after exposure of cells to heat and causing an immediate
reduction in DNA replication activity. This fast response may prevent
or reduce DNA replication on a heat-modified nuclear matrix (67) that
may cause complications undermining genomic stability, or even cell
survival. However, this translocation, or the resulting inhibition of
DNA replication, may serve as a signal for the activation of
longer-lived regulatory processes that maintain the inhibition of DNA
replication for up to 8 h (Fig. 1B). According to this
model, the nucleolus may serve as a sensor of heat damage (71) and
nucleolin as the signaling molecule.
The release of nucleolin into the nucleoplasm helps to explain earlier
observations that the dissolution of the nucleolus in response to
mitosis-promoting factor during premature chromosome condensation was
deficient in heated cells (72). It has been reported that nucleolin is
involved in chromatin decondensation by virtue of its ability to
interact with histone H1 (73) and might also be involved in the
dissolution of the nucleolus during mitosis. Its release from the
nucleolus after heat may thus affect this process.
The Heat Resistance of DNA Replication Factors--
A surprising
but potentially very significant observation of the work presented here
is that supralethal exposures to heat do not measurably alter the DNA
replication activity of several factors required for in
vitro SV40 DNA replication. Obviously, this statement holds only
for factors required for SV40 DNA replication and excludes key
components of the eukaryotic DNA replication machinery such as the
components of the origin recognition complex and the mini chromosome
maintenance proteins that are not required for in
vitro SV40 DNA replication. The presence of active SV40 DNA
replication factors at nearly normal concentrations and activities in
extracts of cells exposed to 44 °C for 60 or 90 min suggests that
these factors are neither irreversibly inactivated nor somehow precipitated on cellular structures (67). As these factors completely lose their activities for SV40 DNA replication when heated in vitro, even at a temperature as low as 41 °C (Fig.
3A), it becomes evident that the cellular environment must
provide a high level of protection against heat-induced denaturation
and inactivation. It will be particularly important to characterize the
determinants of this heat resistance.
Finally, the possibility that the interaction between nucleolin and RPA
increases the amount of protein on the nuclear matrix, where DNA
replication occurs and therefore RPA is expected to accumulate, is
intriguing. It suggests that, in addition to the passive process of
protein precipitation on the nuclear matrix as a result of heat-induced
denaturation (67), active processes involved in either the signaling of
a stress response or the repair of the induced damage can contribute to
this effect.
The above observations in aggregate suggest the operation of an active
mechanism in the regulation of DNA replication in cells exposed to heat
shock. The mechanism operates by the inactivation of a key DNA
replication factor, RPA, mediated via direct interaction and
association with nucleolin. Although nucleolin is normally found
exclusively in the nucleolus in non-stressed cells, it translocates into the nucleoplasm after heat exposure, thus enabling the interaction with RPA. RPA targeting by nucleolin takes place in an environment preserving the activity of several essential DNA replication factors. It is likely, therefore, that the contribution of regulatory processes, such as the one relying on RPA-nucleolin interactions, to DNA replication inhibition after heat shock is more significant than presently thought. Characterization of the upstream components in this
pathway will not only enhance our understanding of DNA replication and
its regulation under conditions of stress but will also help to
identify targets for therapeutic intervention, or parameters predicting
the response of tumor cells to heat treatment. Hyperthermia is used as
an adjuvant to radiation therapy (74), and this information should be
helpful in the development of improved therapeutic strategies.