ABSTRACT
To study the mechanism by which ultraviolet (UV) light inhibits DNA replication,
we examined the effects of UV 254 nm irradiation on the replication of simian
virus 40 (SV40) DNA and SV40-based plasmid in monkey cells. The study was designed to determine the
relative contributions made by inhibition of replication initiation and chain
elongation to the immediate inhibition of DNA replication following UV
irradiation. We used two-dimensional neutral-alkaline electrophoresis to examine the behaviour of replication
intermediates unambiguously. Kinetic analysis using this technique showed that
initiation of replication started to decline at 15 min post-irradiation. When the pulse label incorporated in SV40 replication
intermediates before irradiation was chased for 1 h, most of the label was
found in mature Form I and II molecules. This indicated that replication
elongation took place on damaged template. We also used a transfection
technique to show that heavily irradiated plasmids replicated efficiently in
unirradiated transfected cells. By the transfection technique, we observed that
UV irradiation of host cells dose-dependently inhibited replication of transfected non-irradiated plasmids, suggesting that the inhibition of DNA
replication is due to a global change in cellular physiology induced by UV.
This change was also apparent from poor staining of the chromatin by
fluorescent-DNA-binding dyes immediately after UV irradiation of intact cells. We
conclude that a significant fraction of chain elongation proceeds on damaged
templates and DNA replication during the acute response of cells irradiated
with UV is mainly controlled by the inhibition of replication initiation.
Organisms must replicate their DNA with high accuracy so as to maintain their
genetic identity. However, DNA is subject to damage by many chemical and
physical agents. Ultraviolet (UV) light is one of the best-studied DNA-damaging agents causing inhibition of DNA replication. Recent
studies have demonstrated three regulatory pathways for controlling DNA
replication after radiation damage. Two are cell cycle regulation pathways
which arrest cells at either the late G
1
phase or the G
2
-M interface, and are mediated by p53 tumor suppressor (
1
,
2
) or Cdc2 protein kinase (
3
) respectively. The third pathway accounts for the immediate inhibition of
replication after radiation damage known as the acute response after
irradiation (
4
,
5
).
Several different mechanisms have been proposed to explain the immediate
inhibition of replication after UV irradiation in the acute response. Early
observations by Kaufmann and Cleaver (
4
) demonstrate that UV induces inhibition of replication specifically at the
level of initiation after low (0.3-2.6 J/m
2
) doses. The effect is detected by velocity sedimentation of pulse-labeled, newly synthesized DNA in alkaline sucrose gradients, and is
reflected by a selective inhibition of synthesis of daughter DNA with the same
length as an average replicon within 30-60 min after irradiation. Recently, inhibition of initiation by X-ray irradiation has been demonstrated in a defined mammalian gene (
5
); initiation in the dihydrofolate reductase (DHFR) gene of an irradiated
Chinese hamster cell (CHO) is completely inhibited within 30 min and does not
resume for 3-4 h. On the other hand, inhibition of chain elongation by UV has also
been observed. For example, DNA synthesis during a 15-min pulse immediately after irradiation of a simple eukaryote,
Dictyostelium discoideum
, is of single-stranded low molecular weight DNA, and the size of nascent strand
generated after UV depends on radiation dose (
6
). Similarly, in the SV40 chromosome in UV-irradiated cells, the daughter strands of SV40 DNA grow only to a size
equal to the interdimer distance along the parental strands, suggesting an
elongation blocking at dimer (
7
). However, explanations for how elongation is affected remain controversial. An
early model proposed that damage in the leading strand blocks chain elongation
and replication fork progression, whereas damage in the lagging strand merely
causes small gaps opposite the damage without blocking the replication (
8
). However, analysis of DNA replication started from an origin near DHFR gene in
UV-irradiated CHO cells indicated that replication can efficiently bypass UV
lesions in a 15 kb region without significant repair or recombination and
pyrimidine dimer containing DNA can be completely replicated in UV irradiated
CHO cells (
9
).
In view of the controversy in the mechanisms of inhibition of DNA replication in
mammalian cells and the recent report that initiation of DNA replication in
mammalian cells is the main step inhibited by ionizing radiation (
5
), we examined the effects of UV light on the replication of SV40 and SV40-based plasmid in monkey cells using experimental protocols specifically to
address the effect of UV irradiation on chain elongation and initiation. SV40
is a small double-stranded DNA virus that replicates in the nucleus, has a chromatin
structure very similar to that of host cells, and uses host enzymes for its
bidirectional replication (
10
). It thus provides an excellent model of a single mammalian replicon. Previous
studies have indicated that, like cellular DNA replication, viral DNA
replication is inhibited by UV irradiation in a dose-dependent fashion as measured by incorporation of [
3
H]TdR (
11
-
13
). In the present study, we used two-dimensional neutral-alkaline electrophoretic analysis to define the replication
intermediates and therefore to differentiate DNA replication from DNA repair
synthesis. Using this technique we show that SV40 DNA replication is rapidly
shut off following UV irradiation, with a concomitant rise of repair synthesis.
However, DNA chains initiated before irradiation can be elongated into complete
SV40 molecules within 1 h. Using a transfection technique, we also found that
SV40-based plasmid irradiated with a high dose of UV can replicate efficiently
in non-irradiated host cells, albeit in an error-prone manner. In contrast, when the host cells were irradiated with
a low dose of UV before transfection of non-irradiated SV40 plasmid, the plasmid's replication was dose-dependently inhibited. The data suggest that the acute response
observed immediately after UV irradiation is mainly controlled by inhibition of
replication initiation. Mechanisms involved in producing a microenvironment in
damaged cells that inhibits the overall DNA replication are also discussed.
CV-1 African Green Monkey kidney cells and Cos-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal calf serum and 50 [mu]g/ml gentamicin (Gibco). SV40 viral stocks were prepared as described (
14
). pSV2CAT and pBKCMV plasmids were purchased from American Type Culture Collection. All restriction enzymes were obtained from New England
Biolabs.
CV-1 cells were infected with SV40 virus at 10 p.f.u./cell as described (
14
). Double-stranded SV40 DNA or SV40-based plasmid was transfected into subconfluent cells by the DEAE-dextran method (
15
).
Cells were irradiated with 254 nm UV light from a low pressure mercury
germicidal lamp at an incident dose rate of 2.5 J/m
2
/s, measured with a model UVX digital radiometer (Ultraviolet Products, San
Gabriel, CA) using a UVX-254 midrange sensor. For irradiation of DNA, SV40-based plasmid was irradiated at a concentration of 1 [mu]g/ml immediately before it was transfected into cells. The UV
source was from a spectrolinker #XL-1000 at an incident dose rate of 25-30 J/m
2
/s, measured as described previously.
For
in vivo
pulse-labeling with [
3
H]TdR, cells were incubated with 5 mCi/ml [
3
H]TdR in DMEM medium supplemented with 2% fetal calf serum for the time periods
indicated in the figure legends. At the end of the labeling period, isotope
solution was quickly removed then cells were lysed with buffer containing 1%
sodium dodecyl sulfate (SDS). For pulse-chase experiments, cells were pulse-labeled for either 10 or 15 min with [
3
H]thymidine before irradiation. Following irradiation, cells were chased for 1 h
with medium containing 10 mM thymidine. For nuclear run-on assay, cells were first lysed with NP-40 lysis buffer (0.2% NP-40, 10 mM Tris-HCl, pH 7.4, 0.2 mM ZnCl
2
) to remove cytoplasm, and subsequently incubated with reaction buffer
containing 10 mM HEPES, 10% glycerol, 20 mM KCl, 7.5 mM MgCl
2
, 0.25 mM dithiothreitol, 5 mM ATP, 1.25 mM GTP, CTP and UTP, 0.6 mM each of
dGTP, dATP, dTTP and 10 [mu]Ci of [
32
P]dCTP. The reaction was performed at 37oC for 20 min in a culture plate where nuclei stayed firmly on the plate
throughout the labeling period. At the end of the reaction nuclei were
extracted by the Hirt method (
16
).
The neutral-alkaline two-dimensional electrophoresis technique was modified from Sundin and
Varshavsky (
17
) and Nawotka and Huberman (
18
). DNA was first electrophoresed in a 1.0% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 7.4). After electrophoresis, the marker lane was
cut off and stained with ethidium bromide (0.5 [mu]g/ml) to determine the position of SV40 DNA in the unstained part of the
gel. The lanes containing the samples were cut with a razor blade and
orientated horizontally in the second dimensional gel tray (20 * 20 cm). DNA was electrophoresed in 1.3% agarose gel in 40 mM NaOH, 1 mM
EDTA. The alkaline gel was run at 4oC with magnetic stirring. Electrophoresis was done at 200 mA for 20 h with
one change of buffer. After electrophoresis, the gel was washed with distilled
water three times, neutralized with 0.1 M Tris-HCl, pH 7.0 and stained with ethidium bromide. After photography, the gel
was processed for autoradiography or fluorography as described previously (
19
).
SV40-based plasmid was harvested from the cells by the Hirt method (
16
). Purified DNA was treated with
Dpn
I or
Mbo
I to remove all non-replicated DNA or newly replicated DNA respectively.
Escherichia coli
MC1061 or HB101 was transformed with enzyme-digested DNA by electroporation using a Gene Pulser apparatus at 24 [mu]F and 2.5 kV with the pulse controller set to 200 ohms. The yield of
plasmid DNA was calculated from the bacterial transformation efficiency using
standard plasmid DNA as a control of transformation efficiency.
Cells were lysed with NP-40 lysis buffer to remove cytoplasm. The nuclei were stained with 1 [mu]g/ml of either ethidium bromide or acridine-ethidium heterodimer (Molecular Probes), or with 300 ng/ml
acridine orange (Sigma), for 5 min. These dyes bind DNA and their fluorescence
intensity is greatly enhanced by binding to DNA. Laser induced-fluorescence imaging was performed using a Meridian ACAS550 cytometer.
We used a two-dimensional neutral-alkaline electrophoresis technique to investigate the pattern of
radioactive label incorporation into SV40 DNA after UV irradiation and to
differentiate between replication and repair synthesis. It has been shown previously that SV40
replication intermediates labeled with [
3
H]TdR can be identified by this method (
17
,
18
). As shown in Figure
1
A by schematic drawing and Figure
1
B by autoradiography, immediately after a 15 min pulse labeling, the nascent DNA
chains of supercoiled SV40 replication intermediates (RI) could be displayed as
a curve that asymptotically approached the position of mature Form I DNA in the
first dimension, whereas replication intermediates containing nicks in the
parental strands were represented by a curve that approached the position of
Form II nicked circles (Fig.
1
C, curves RI and nicked RI respectively). When SV40 DNA was labeled for 15 min
beginning at 30 min after infected cells had been irradiated with 90 J/m
2
of UV light, the intensity of replication nascent DNA curves almost disappeared
and the majority of pulse label was incorporated into nicked circular Form II
DNA and the late replication intermediates (indicated as arrows in Fig.
1
D). When SV40 DNA was pulse-labeled for 15 min at 60 min post-irradiation, the curves of nascent DNA disappeared even after
prolonged exposure and the majority of label was found in the position of Form
II nicked circular DNA and late replication intermediates (indicated as arrows
in Fig.
1
E). The incorporation of radioactive label is poor due to inhibition of [
3
H]thymidine incorporation into DNA 1 h after UV irradiation. The absence of
significant number of labeled replication intermediates recovered from
irradiated cells indicated that initiation of SV40 DNA replication is rapidly
turned off within 30 min after UV irradiation.
The results described above show that the majority of radio-
activity labeled during a short pulse at 1 h or later after UV irradiation is
incorporated into mature SV40 DNA and replication intermediates were not
labeled. The labeled mature DNA could be derived from replication of either
templates not damaged by UV irradiation or from residual DNA replication after
UV irradiation. Alternatively, the mature SV40 pulse-labeled after UV irradiation could be derived from repair synthesis. To
examine whether the labeling of mature DNA due to residual DNA replication or
repair synthesis, we labeled SV40 DNA with [
3
H]TdR beginning at 1 h after irradiation in the presence or absence of the
protein synthesis inhibitor cycloheximide and analyzed SV40 DNA topoisomer
distribution by agarose gel (Fig.
3
). Previous studies have shown that SV40 DNA supercoiling depends on wrapping of
DNA on histone cores and inhibition of protein synthesis would eliminate
nucleosome assembly during replication and therefore reduces superhelical
density of SV40 DNA (
20
). On the other hand, during repair synthesis only a small patch of DNA is
involved and there is no requirement of new nucleosome formation. Therefore,
inhibition of protein synthesis would not affect superhelical density in SV40
DNA undergoing repair synthesis
in vivo
whereas SV40 DNA superhelical density of replication products would be
affected. Figure
3
shows that, in the presence of cycloheximide, labeled SV40 DNA from the control
non-irradiated cells exhibited lower superhelicity than that labeled in the
absence of the drug as a result of reduced number of nucleosomes (compare lane
1 with lane 2 in Fig
3
). On the other hand, topoisomer distribution of SV40 DNA labeled from 1 to 2 h
post-irradiation in the presence of cycloheximide was the same as that labeled
in the absence of the drug (compare lane 3 with lane 4). These results suggest
that the mature SV40 DNA labeled after semiconservative DNA replication is
derived from repair synthesis rather than from residual DNA replication.
Since SV40 replication intermediates could still be clearly observed at 15 min
post-irradiation (Fig.
2
B), this observation suggests that a certain fraction of chain elongation can
proceed immediately after UV irradiation. Therefore, we studied SV40 DNA
replication after irradiation by pulse-chase analysis. If chain elongation can proceed after UV irradiation,
SV40 replication intermediates pulse-labeled just before UV irradiation can mature into Form I or Form II
circular DNA products. On the other hand, if chain elongation is the main step
inhibited after UV irradiation and initiation of replication continues
unabated, then replication intermediates should accumulate in the chased
sample. As shown in Figure
4
A, a significant fraction of the label was incorporated into SV40 replication
intermediates during the 15 min pulse before UV irradiation. Following the 1 h
chase after irradiation, no replication intermediates were observed and the
majority of label was chased into Form I and II DNA molecules (Fig.
4
B). These results suggest that the majority of SV40 DNA replication
intermediates labeled before UV irradiation can continue their chain elongation
to form mature products.
The results obtained by neutral-alkaline two-dimensional gel electrophoretic analysis suggest that although DNA
replication is inhibited after UV irradiation, a significant fraction of chain
elongation proceeds after UV irradiation. To examine whether UV-damaged templates indeed can be elongated, we transfected non-irradiated Cos-1 cells with UV-irradiated SV40-based plasmid. At 48 h after transfection, the
plasmid DNA was extracted and digested with
Dpn
I to remove input parental DNA. The amount of progeny DNA was then examined by
bacterial transformation assay or by Southern blot analysis. As shown in Table
1
, plasmid irradiated with a high dose of UV, which is used to ensure the
presence of UV damages in every plasmid DNA, can still replicate as efficiently
as non-irradiated plasmid. For example, at 900 J/m
2
, which generates ~40-50 dimers per plasmid (
21
), replication of irradiated plasmid was as efficient as that of non-irradiated plasmid when assayed at 48 h post-transfection. Similar results were obtained using Southern blot
analysis (Fig.
5
). These results show that UV irradiated plasmids can replicate efficiently if
transfected cells are not irradiated and contain the initiation protein, i.e.,
T antigen.
To examine whether UV irradiation of transfected cells induces a similar effect
on a non-irradiated SV40-based plasmid, we studied SV40 plasmid replication in UV-irradiated cells. Cos-1 cells were irradiated with low doses of UV before
transfection and the replication of non-irradiated plasmids transfected into these cells was examined. We found
that UV irradiation of cells caused a dose-dependent inhibition of transfected non-irradiated plasmid (Fig.
6
). At 10 J/m
2
, replication of unirradiated plasmid was reduced to 38% of that in the non-irradiated control cells at 24 h post-transfection. This reduction was not due to a lower transfection
efficiency of UV-irradiated cells since similar dose-dependent inhibition of plasmid yield was obtained if UV irradiation
was performed after transfection (data not shown). Since this UV dose is too
low to cause damage in SV40 DNA or the T antigen coding sequence in Cos-1 cells, the inhibition of SV40 plasmid replication suggests that UV
irradiation induces an alteration of cellular physiology that inhibits overall
DNA replication.
Since the results above suggest that inhibition of DNA replication is due to a
global cellular physiological change induced by UV, we examined whether any
gross morphological changes were present in irradiated cells. Irradiation with
90 J/m
2
induced a rapid change in staining of cell nuclei with DNA-binding fluorescent dyes (Fig.
7
). Immediately after UV irradiation, no alteration of chromatin staining by
acridine orange was observed (Fig.
7
, compare A with B). At 30 min post-irradiation, the staining of chromatin of irradiated cell nuclei began to
decline, indicating a change of nuclear structure (Fig.
7
C). At 2 h post-irradiation the average staining intensity and the average staining area
of chromatin of irradiated cell nuclei was reduced to 60 and 25% of the non-irradiated cell nuclei respectively (Fig.
7
, compare A with E). The staining of nuclei recovered at 4 h post-irradiation (Fig.
7
F) indicating that the observed change in dye staining is reversible. The
reduction of chromatin staining after UV irradiation parallels the inhibition
of SV40 DNA replication. Similar reduction of staining was observed using
ethidium bromide as probe and for the cells irradiated at 10-30 J/m
2
(data not shown).
In the present study we used two-dimensional neutral-alkaline gel electrophoresis technique to investigate SV40 DNA
replication after UV irradiation. This technique allows us to examine the
behavior of replication intermediates unambiguously. The results obtained using
this analysis indicate that SV40 DNA replication is rapidly turned off after UV
irradiation. This rapid inhibition of DNA replication is mainly due to the
inhibition of replication initiation because the intensity of replication
intermediate curves unequivocally disappear in the two-dimensional gel (Figs
1
B-E and
2
B-D). If replication initiation was not inhibited, continuing initiation
would cause the stalled replication intermediates to accumulate, which was not
observed in our two-dimensional gel. Our result also suggests that inhibition of SV40 DNA
replication appears to be regulated by an overall inhibition of replication
initiation because irradiation of host cell inhibits the replication of the non-irradiated transfected SV40 plasmid in a dose-dependent manner (Fig.
6
). The present study demonstrates that UV irradiation inhibits mainly the
initiation step of DNA replication
in vivo
and this inhibition is due to a micro-environment induced in the irradiated cells. This conclusion differs from
previous studies of UV effect on DNA replication that elongation is the main
step involved in the shut-down of DNA replication. Our study complements many other more recent
investigations and points to the new concept that radiation causes immediate
inhibition of replication initiation (
5
,
22
-
25
; see Discussion below).
Our data suggest that during the acute phase following UV irradiation, a
significant fraction of chain elongation can proceed on UV-damaged templates. This is shown by the following experiments: (i) SV40
DNA replication intermediates defined by a short pulse with [
3
H]TdR just before UV irradiation can be chased into mature Form I and Form II
molecules (Fig.
4
); (ii) kinetic study by
in vitro
nuclear run-on experiments shows a near normal SV40 DNA replication pattern
immediately or at 15 min after irradiation with 90 J/m
2
of UV (Fig.
2
B) although replication is completely turned off later; (iii) heavily irradiated
SV40 DNA or SV40-based plasmid replicates very efficiently after transfection into
unirradiated cells (Table
1
). The last finding is similar to results obtained by others. For example, UV-irradiated SV40-based plasmid replicates as efficiently as non-irradiated plasmid in repair-proficient human fibroblasts when assayed at 48 h post-transfection (
26
,
27
). In addition, studies measuring survival of transfected SV40 DNA by plaque
assay using transfection with low multiplicity show that survival of SV40 DNA
which contains ~32 pyrimidine dimers is reduced to only 37% of the non-irradiated control (
28
,
29
). Since plaque assay depends on intact early as well as late genes, it is more
sensitive to UV than analysis based on DNA replication assay. Therefore, >32
pyrimidine dimers were needed to reduce DNA replication to 37%. Thus, a
significant fraction of DNA replication must be able to bypass UV damage. The
efficient replication of irradiated plasmid in Cos-1 cells could be due to efficient repair occurring on irradiated plasmids
and/or recombination between transfected plasmid. However, there is no significant increase in the ability of input irradiated
plasmid to transform HB101 during 1 h or more post-UV incubation period in our transfection assay (Table
2
). This result suggests that efficient repair of heavily irradiated plasmid
cannot account for the efficient replication we observed. In addition, a dose
of 900 J/m
2
will generate ~40-50 pyrimidine dimers per plasmid (
21
). Poisson distribution predicts that only a minute insignificant fraction (10
-9
%) of plasmids will contain no UV damage. Thus, we consider the possibility that
replication of this minute fraction or the replication-competent templates generated by recombination is quite unlikely.
Efficient bypass of UV damage during replication of DHFR domain in UV-irradiated CHO cells has been demonstrated (
9
). The bypass is not due to DNA recombination since no pyrimidine dimers were
found in the daughter strands. The same study also cited unpublished results
showing complete replication of pyrimidine dimer containing DNA in a repair-deficient CHO mutant cell line. This last observation is similar to our
transfection data (Table
1
and
2
). Hoy
et al.
also showed that maturation rates of DNA synthesized immediately before or
after UV irradiation of CHO cells is similar (
30
) thus implicating that replication fork can efficiently bypass UV damages. Van
Zeeland and Filon also showed that replication forks paused only briefly at
pyrimidine dimers (
31
). Bypass of UV lesions
in vitro
has also been demonstrated using
E.coli
DNA polymerases I (
32
) and III (
33
-
35
) or in the mammalian system (
22
,
36
,
37
). In our
in vivo
experiment measuring the mutation frequency of SV40 plasmid replicated in Cos-1 cells, putatively mutagenic translesion synthesis was also observed.
Our observations that SV40 DNA replication was inhibited by irradiation of
infected cells with UV light for a prolonged period without accumulation of
stalled replication intermediates (Figs
1
and
2
) and that irradiation of Cos-1 cells inhibited the replication of transfected, unirradiated SV40
plasmid (Fig.
6
) suggest that immediate inhibition of DNA replication during acute phase
response is mainly controlled by replication initiation. This is similar to
results obtained for cells irradiated with ionizing radiation (
23
), in which replication of a small plasmid DNA is inhibited when cells are
irradiated with a dose of X-rays too low to damage the plasmid DNA. That study suggests that immediate
inhibition of DNA replication after radiation damage is regulated at the
initiation levels of DNA replication, and is mediated by a
trans
-acting factor or factors that cause arrest of replication of undamaged
small plasmid DNA in the irradiated cells. A recent study (
24
) of
in vitro
DNA replication reactions carried out by UV-irradiated HeLa cell extracts shows that their replication activity of
unirradiated plasmid DNA decreases following UV irradiation. However,
replication activity can be restored to these extracts by the addition of
purified human single-stranded DNA binding protein, suggesting that UV-induced DNA replication arrest may be mediated through the essential
component of the replication initiation complex thus implicating initiation as
the regulatory step after UV irradiation. Recently, inhibition of DNA
replication specifically at the level of replication initiation after ionizing
irradiation has been demonstrated in the DHFR gene of a CHO cell line by two-dimensional gel replicon-mapping (
5
). In this technique, each class of replication intermediates traces a
characteristic pattern, and fragments that contain initiation sites can be
displayed as a bubble arc. When asynchronously growing cells were irradiated
with X-rays, the initiation bubble arc completely disappeared within 30 min of
radiation treatment and did not resume for 3-4 h. In contrast, the pattern of fragments representing the replication
initiated before irradiation did not appear to change significantly in response
to radiation. Our results as well as this study clearly support the idea that
immediate inhibition of DNA replication after radiation is mainly controlled by
replication initiation.
The mechanism by which overall initiation of DNA replication is rapidly
inhibited after radiation damage is still not understood. Some nuclear-specific signalling mechanisms involving signal transduction pathways have
been invoked to explain the rapid inhibition of DNA replication after UV
irradiation (
38
,
39
). Protein kinases have been implicated in the inhibition of replicon initiation
after ionizing radiation (
40
). To examine the possible involvement of signal transduction pathways in the
rapid inhibition of DNA replication, we studied the effect of tyrosine kinase
inhibitors, genistein and tyrphostin, on SV40 plasmid replication in the UV-irradiated cells. We found no improvement of replication efficiency after
blocking the tyrosine kinase signalling pathway (data not shown). Thus, the
signal to inhibit DNA replication after UV is probably not significantly
mediated by a tyrosine kinase pathway in our system. However, when an
intracellular free radical scavenger,
N
-acetyl-cysteine, was added during the incubation after cells were
irradiated with a UV dose of 10 J/m
2
, the SV40 plasmid replication recovered by 56%, i.e., from 43% (100%) to 67%
(156%) (Table
3
). The free radicals generated by UV irradiation may result in oxidative stress
which triggers an alteration of cellular physiology. Indeed, we found rapid
nuclear and chromatin changes in cells irradiated with UV (Fig.
7
). The nuclear morphological changes may mask the recognition of replication
machinery and produce a rapid mechanism for inhibition of DNA replication. This
idea is also supported by a finding that DNA replication is inhibited by X-rays in chromosomal and plasmid DNA but not in mitochondrial DNA, which is
located in the cytoplasm (
25
).
Table 3
In summary, our observations that replication of SV40 DNA was inhibited by UV
irradiation of host cells and that a rapid alteration of nuclear organization
was induced by UV, suggest that inhibition of DNA replication is the result of
a global change in cell microenvironment after UV irradiation, and is possibly
mediated by the nuclear morphology changes. However, further analysis is needed
to understand the molecular mechanism involved in the regulation of DNA
replication initiation after radiation damage.
This work is supported by a grant from National Science Foundation (NSC) of
Republic of China. Y.-C.W. is the recipient of a postdoctoral fellowship award from NSC.
Relative transformation efficiency (%)
NAC (mM)
+NAC
+UV (10 J/m
2
)
+UV +NAC
a
0
100
0.1
84
43
67
REFERENCES
Return