Enhanced S phase delay and inhibition of replication of an undamaged shuttle vector in UVC-irradiated xeroderma pigmentosum variant
Sharon K. Bullock1,,
William K. Kaufmann and
Marila Cordeiro-Stone2,
Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, 517 Brinkhous-Bullitt Building, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525, USA
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Abstract
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Xeroderma pigmentosum variant (XP-V) cells are defective in bypass replication of UVC-induced thymine dimers in DNA because they lack a novel DNA polymerase (polymerase
). In this study the effects of UVC on S phase cells were compared in fibroblasts derived from normal donors (IDH4) and XP-V patients (CTag) and immortalized by expression of the SV40 large T antigen. These transformed fibroblasts did not activate the G1 checkpoint or inhibit replicon initiation when damaged by UVC or
-rays. The transformed XP-V cells (CTag) retained the increased sensitivity to UVC-induced inhibition of DNA strand growth previously observed with their diploid counterpart. Cell cycle progression analyses showed that CTag cells displayed a stronger S phase delay than transformed fibroblasts from normal individuals (IDH4) after treatment with only 2 J/m2 UVC. Low doses of UVC also caused a lag in CTag cell proliferation. The extent of replication of an episomal DNA (pSV011), not previously exposed to radiation, was measured after the host cells were irradiated with 13 J/m2 UVC. Replication of pSV011 was barely affected in irradiated IDH4 cells. Plasmid replication was inhibited by 50% in irradiated CTag cells and this inhibition could not be accounted for by increased killing of host cells by UVC. These results suggest that even in transformed cells UVC induces DNA damage responses that are reflected in transient cell cycle arrest, delay in proliferation and inhibition of episomal DNA replication. These responses are enhanced in CTag cells, presumably because of their bypass replication defect. The accumulation of replication complexes blocked at thymine dimers and extended single-stranded regions in chromosomal DNA might sequester replication factors that are needed for plasmid and chromosomal replication. Alternatively, aberrant replication structures might activate a signal transduction pathway that down-regulates DNA synthesis.
Abbreviations: ATM, gene mutated in ataxia telangectasia; BrdU, bromodeoxyuridine; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; MEM, minimum essential medium; PI, PhosphorImager units; pol
, DNA polymerase
; PRR, post-replication repair; RPA, replication protein A (single-stranded DNA-binding protein); XP-V, xeroderma pigmentosum variant.
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Introduction
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The mechanisms that underlie the inhibition of DNA replication by carcinogen-induced DNA damage are not thoroughly understood. Both passive and active mechanisms are thought to contribute to the overall inhibition. Direct blockage of the replication apparatus by a bulky template lesion represents a passive mechanism of inhibition of DNA replication. In contrast, an active process requires the generation of some type of stress signal, its transduction to sites away from the primary lesion and inhibition of DNA synthesis even in replication units that did not incur any direct damage.
Velocity sedimentation analyses of radiolabeled nascent DNA revealed that UVC-induced inhibition of DNA replication results from inhibition of both replicon initiation and DNA chain elongation (1). Inhibition of DNA chain elongation reflects physical blockage of the replication machinery by template lesions. This passive, cis-acting effect of UVC-induced lesions on DNA replication is overcome by post-replication repair (PRR), which includes pathways leading to bypass replication of blocking lesions and elimination of daughter strand gaps (2). Thus, PRR promotes damage tolerance and completion of replication of the damaged genome. Although UVC induces daughter strand gaps in normal cells, the frequency and half-life of these single-stranded DNA regions are increased in PRR-defective cells, such as those derived from xeroderma pigmentosum variant (XP-V) patients. XP-V cells have a normal capacity for nucleotide excision repair (36), but are deficient in bypass replication of UVC-induced thymine dimers (1,4,712). This bypass defect is caused by frameshift mutations in the gene encoding DNA polymerase
(pol
) (13,14). This novel DNA polymerase has been shown to efficiently bypass cis,syn-cyclobutane thymine dimers in vitro by incorporating adenines opposite this photoproduct (12,15). The enzymes responsible for mutagenic bypass of UVC-induced photoproducts in human cells are polymerase
(16) and hRev1 (17). These proteins, or another error-prone DNA polymerase (18,19), must be the ones that eventually catalyze the bypass of cyclobutane thymine dimers in XP-V cells, hence their hypermutability to UVC (20,21).
The biochemical mechanisms that underlie radiation-induced inhibition of replicon initiation are less clear. It occurs at sites away from the primary DNA lesion (a trans effect) in response to a signal transduction pathway. Therefore, it is the final result of an active process in which the DNA damage is recognized by molecular sensors and the information transmitted to the sites of action by effector molecules. The inhibition triggered by ionizing radiation is dependent on the ATM (gene mutated in ataxia telangectasia) protein kinase (2224) and reflects the activity of an intra-S phase checkpoint (22,24). UVC-induced DNA lesions also inhibit initiation of new replicons in S phase cells (1,25). The primary kinase initiating this response, however, seems to be the ATM- and Rad3-related kinase ATR (26,27). Little is known about the effector molecules involved in the S phase checkpoint responses, although cyclin E/Cdk2 and cyclin A/Cdk2 are thought to be involved (28,29). Proteins that participate in assembling pre-replication complexes at the origins and/or control replicon firing are likely substrates for regulation by the S phase checkpoint. Among the potential candidates are Cdc6 (3032) and Dbf4/Cdc7 (3335), in addition to the replication factors themselves. Whatever its mechanism, inhibition of replicon initiation contributes to the UVC-induced inhibition of S phase progression in human cells.
In this study we have analyzed the effects of UVC on SV40-transformed cells derived from normal (IDH4) and XP-V (CTag) donors. Their bypass defect and the strong inhibition of maturation of DNA replication intermediates after low doses of UVC suggested that XP-V cells might be a useful model system for detecting trans-acting pathways of inhibition of DNA replication, other than inhibition of replicon initiation. Replication of an undamaged shuttle vector was inhibited more strongly in CTag than in IDH4 cells when the host cells were irradiated with low doses of UVC (13 J/m2). Flow cytometric analyses showed that UVC-induced inhibition of progression through S phase was much more pronounced in CTag than in IDH4 cells. These results suggest that UVC-induced blockage of chain elongation in active replicons also results in inhibition of replication in undamaged DNA.
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Materials and methods
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Cell culture
IDH4 and CTag are cell lines derived from human fibroblasts by transformation with the gene for SV40 large T antigen. The IDH4 line originated from diploid fibroblasts of an apparently normal fetus (36) and was received from Dr Jerry Shay (University of Texas Southwestern Medical Center). The CTag line was generated in our laboratory (37) from secondary cultures of XP-V fibroblasts XP4BE (CRL1162; American Type Tissue Collection, Rockville, MD). These cells were maintained in monolayer cultures on polystyrene plates (Falcon, Lincoln Park, NJ) in Eagle's minimum essential medium (MEM) with Earl's salts, supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), 50 µg/ml gentamicin (Elkins-Sinn, Cherry, NJ) and 2 mM L-glutamine. The medium for IDH4 also contained 1 µM dexamethasone (Sigma Chemical Co., St Louis, MO). Secondary cultures of diploid dermal skin fibroblasts, derived from a normal individual (GM3348) and an XP-V patient (CRL1162; XP4BE), were used as positive controls in analyses of checkpoint activity. GM3348 and CRL1162 cells were maintained in Eagle's MEM supplemented with 2 mM L-glutamine, 2x MEM vitamins, 2x MEM amino acids (both essential and non-essential), and 15% FBS (Hyclone Laboratories). Culture medium and supplements were obtained from Gibco BRL Life Technologies (Rockville, MD).
Episomal DNA
Two SV40-based plasmid molecules were used for the in vivo DNA replication studies. We followed the replication of one plasmid (pSV011), while using the other (M13mp2SV) as an internal control for losses that occurred during DNA purification. pSV011 is a 2.9 kb plasmid constructed by inserting a 200 bp fragment containing the core of the SV40 origin of replication (HindIIISphI fragment) into pUC18 (38). M13mp2SV is a 7.4 kb plasmid containing the same 200 bp SV40 origin fragment inserted into the unique AvaII site of M13mp2 (39).
UVC and
-irradiation
Cells to be irradiated with UVC were first rinsed with Hank's balanced salt solution (Gibco BRL) warmed to 37°C and then placed under a UV lamp emitting mostly at 254 nm. The incident fluence rate was calibrated using a UV radiometer (UVP, Upland, CA). UVC irradiation of the plasmid DNA was done by placing 50100 µl of DNA in 10 mM Tris, 1 mM EDTA, pH 7.5, under the UV lamp. The incident fluence rate was adjusted to 10 J/m2/s. Exposure of cells to
-rays was via a 137Cs source (Gammacell of Canada). Control (sham-treated) cells were taken out of the incubator and handled in the same manner as described above, with the exception of exposure to radiation.
Inhibition of DNA synthesis by
-irradiation
Cells were seeded at 150 000200 000/60 mm plate (3 plates/condition) and incubated for 2 days. Cultures were irradiated with 0 or 4 Gy, incubated for 30 min at 37°C and pulse labeled with [3H]thymidine (15 µCi/ml) between 30 and 60 min after irradiation. Cultures were washed with ice-cold Hank's balanced salt solution (Gibco BRL) and the cells lysed in 1ml of 0.3M NaOH at 37°C for 1 h. The absorbance of cell lysates at 260 nm was recorded and a fixed volume used to collect acid-insoluble macromolecules onto GF/C glass filters. 3H counts were quantified by scintillation counting and normalized for differences in cell number by dividing the radioactivity by the absorbance at 260 nm. This normalized value was taken to represent the rate of DNA synthesis in each sample.
UVC-induced alterations in nascent DNA
Cells were incubated with [14C]thymidine (20 nCi/ml) for 2 days in order to label DNA uniformly. The cultures were irradiated with increasing fluences of UVC and incubated at 37°C for 30 min. Then, cells were pulsed with [3H]thymidine (50 µCi/ml) for 15 min, harvested and lysed on top of 36 ml of 520% alkaline sucrose gradients (8). These gradients were centrifuged at 25 000 r.p.m. for 5 h in a SW28 rotor (Beckman Instruments, Palo Alto, CA) and fractionated from the bottom. The amount of 3H and 14C radioactivity in each fraction was measured by scintillation counting. We normalized for differences in the number of cells per sample by dividing the 3H radioactivity in each fraction by the total 14C radioactivity recovered in the gradient (1,8,25,40).
Radiation-induced alterations in cell cycle progression
Cells were seeded at 350 000/100 mm plate (4 plates/condition) and incubated for 2 days. Cultures were exposed to
-rays, incubated at 37°C for 6 h and pulsed for 2 h with 10 µM bromodeoxyuridine (BrdU). In other experiments cells were irradiated with UVC (0 or 2 J/m2) then immediately pulsed for 1 h with 10 µM BrdU. The cultures were fed with fresh medium and incubated for 0, 7.5 or 15 h. Alternatively, the cultures were pulsed for 1 h with 10 µM BrdU at 0, 7.5 or 15 h after irradiation with 0 or 2 J/m2 UVC. Cells were harvested, then stained with propidium iodide and fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody (Becton Dickinson, Franklin Lakes, NJ), as described (41). Flow cytometric analyses were done on a FACScan station with Cyclops software (Becton Dickinson).
Plasmid replication inside UVC-irradiated cells
CTag and IDH4 cells were seeded at 400 000450 000 cells/100 mm plate and incubated for 1 day. Serum-free medium (4.5 ml of MEM) containing 7.5 µg pSV011 and 37.5 µg Transfectam (Promega) was added to each plate (25 plates). After 5 h at 37°C, 4.5 ml of MEM containing 20% FBS was added to each plate without removing the DNA/Transfectam mixture. The cells were incubated for an additional 15 h (starting from the end of the 5 h incubation in serum-free medium). At that point cells were irradiated with increasing fluences of UVC (03 J/m2) and incubated for another 15 h. Plasmid DNA was isolated from the cells by the Hirt extraction procedure (42). After the neutralization step, 250 ng M13mp2SV was added to each sample to control for losses incurred during DNA purification. The plasmid DNA samples were ethanol precipitated, then treated with RNase (Promega) at a concentration of 2 µg/ml. DNA was purified by extraction with equal volumes of a 1:1 (v/v) mixture of phenol and chloroform/isoamyl alcohol (24:1 v/v), ethanol precipitated, then dissolved in 10 mM Tris, 1 mM EDTA, pH 7.5. DNA was restricted with DpnI (Boehringer Mannheim, Indianapolis, IN) to digest the unreplicated DNA. The DNA samples were then digested with EcoRI (Boehringer Mannheim) and analyzed by quantitative Southern hybridization. Full-length pSV011 genomes (resistant to DpnI) were considered to be from molecules that replicated inside the human cells.
Quantitative Southern hybridization
Following DpnI and EcoRI treatments, the plasmid DNA samples were fractionated in 1% agarose gels containing 0.2 µg/ml ethidium bromide. Gels were run in 0.04 M Trisacetate, 1 mM EDTA at 1 V/cm for ~18 h. DNA was transferred to a nylon filter by capillary action. Approximately 25 ng of pSV011 DNA was labeled with [
-32P]dCTP by random priming. The filter was probed with the radiolabeled pSV011 DNA and exposed to a phosphor screen that was later scanned with a Storm 840 PhosphorImager (Molecular Dynamics, Sunnydale, CA). Sequence homology between the two plasmids enabled radiolabeled pSV011 to also hybridize with M13mp2SV, therefore, only radiolabeled pSV011 was used as probe. The radioactivity associated with the full-length (2.9 kb) pSV011 molecules (resistant to DpnI) and the 4.2 kb DpnI restriction fragment of M13mp2SV was quantified using ImageQuant software (Molecular Dynamics). These values are referred to as PhosphorImager (PI) units. The PI units determined for the pSV011 2.9 kb band divided by that of the M13mp2SV 4.2 kb fragment were taken to represent the extent of pSV011 replication.
UVC-dependent cell loss
The same protocol described above for the analysis of plasmid replication was used to determine the UVC-dependent loss of transfected human cells. In subsets of identically prepared cultures cells were trypsinized and the total numbers of cells per plate were determined at times corresponding to transfection, UVC irradiation and harvesting of replicated plasmid DNA. Transfected cultures were either sham-treated or irradiated with 2 J/m2 UVC. In parallel, the average numbers of cells per plate were also measured in cultures that were not transfected or irradiated.
UVC-induced alterations in cell proliferation
IDH4 and CTag cells were seeded at 50 000 cells/60 mm plate (3 plates/condition) and incubated overnight. The following day (day 1) the total numbers of cells per plate were determined using a Coulter counter (Coulter Corp., Miami, FL). On day 2 cell cultures were irradiated with 0, 1 or 2 J/m2 UVC and the total numbers of cells per plate were determined each day thereafter for 7 days.
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Results
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Radiation-induced inhibition of DNA synthesis in transformed cell lines
Expression of SV40 large T antigen oncoprotein by the transformed cell lines used in this study led to the prediction that damage-induced pathways that are p53 dependent would be inactivated. Accordingly,
-rays and UVC did not activate the G1 checkpoint in CTag or IDH4 cells and did not interfere with the entry of cells into S phase (results not shown). Because our interest focused on the response of S phase cells, we next measured inhibition of DNA synthesis by
-rays and UVC. Human cells with intact S phase checkpoint responses inhibit by ~50% incorporation of [3H]thymidine into acid-insoluble macromolecules 3060 min after treatment with low doses of ionizing radiation (2224). This response has been correlated with inhibition of replicon initiation. Using this approach we determined that exposure to 4 Gy
-rays elicited this S phase response from normal diploid (GM3348) and XP-V fibroblasts (CRL1162). IDH4 and CTag cells, however, were completely refractory to inhibition of DNA synthesis in this assay (Figure 1
).
The effect of UVC on replication of chromosomal DNA in IHD4 and CTag cells was assayed by alkaline sucrose gradient analysis. This assay measures changes in the steady-state size distribution of nascent DNA molecules labeled with [3H]thymidine 3045 min after irradiation (1,4,8,25). Dose-dependent decreases in [3H]thymidine incorporation (normalized to the number of cells added to the gradient) were associated with intermediates of DNA replication of 5x1072x108 Da. Labeling of nascent DNA in this size range was inhibited most noticeably in CTag cells. The sum of the normalized 3H radioactivity incorporated into these large DNA intermediates is expressed as a percentage of the same quantity determined in the unirradiated controls and plotted against the fluence of UVC (Figure 2
). Slopes of the linear regression lines shown in Figure 2
defined D0 values of 5.8 J/m2 for IDH4 and 2.5 J/m2 for CTag cells. This 2.3-fold increased sensitivity to inhibition of DNA replication by UVC in the XP-V cell line was determined to be statistically significant (P < 0.05) by ANOVA.

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Fig. 2. UVC-induced inhibition of DNA replication. Logarithmic cultures of IDH4 and CTag cells were incubated for 2 days in medium containing [14C]thymidine. Cells were irradiated with increasing fluences of UVC, incubated at 37°C for 30 min and pulsed with [3H]thymidine for 15 min. Cells were lysed on top of alkaline sucrose gradients and subjected to velocity sedimentation. The 3H radioactivity in each gradient fraction was normalized to cell number (total 14C radioactivity in the gradient). The sum of normalized 3H c.p.m. incorporated into large intermediates of DNA replication (Mr 5x1072x108) was expressed as a percentage of the same sum measured in cells that were not irradiated and plotted against the UVC dose. The D0 values for UVC-induced inhibition of DNA replication, calculated from the linear regression slopes, were 5.8 J/m2 for IDH4 ( ) and 2.5 J/m2 for CTag ().
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For both CTag and IDH4 cells, however, treatment with low doses of UVC did not inhibit [3H]thymidine incorporation into replication intermediates of <5x107 Da (not shown). A reduction in labeling of this size class of nascent DNA is indicative of radiation-induced inhibition of replicon initiation (1,25). These results and those illustrated in Figure 1
indicate that both CTag and IDH4 cells lost the capacity to respond to radiation damage (either UVC or
-rays) by inhibiting initiation of new replicons. The transformed XP-V cells (CTag), however, did retain the enhanced UVC sensitivity to inhibition of DNA elongation (Figure 2
) previously reported for their parental cells (CRL1162) (8).
Inhibition of pSV011 replication in UVC-irradiated human fibroblasts
Purified pSV011 DNA (not exposed to UVC) was transfected into CTag and IDH4 fibroblasts. The host cells were irradiated 15 h later with low doses of UVC. After another incubation of 15 h, the extent of pSV011 replication was quantified. These time points were chosen on the basis of experiments that measured the time course of replication of pSV011 in CTag and IDH4 cells (not shown). Both cell lines showed very similar kinetics and levels of plasmid replication. After a lag period of ~1215 h, the yield of replicated pSV011 increased steadily between 18 and 30 h. The probability that a significant fraction of the intracellular pSV011 (2.9 kb, a few molecules per cell in ~5% of the population) would sustain any direct damage was considered negligible at the low UVC doses used to irradiate the host cells (13 J/m2). Accordingly, pSV011 recovered from the irradiated cells did not contain sites sensitive to nicking by T4 endonuclease V, which recognizes cyclobutane pyrimidine dimers in DNA (not shown).
The recovery of replicated pSV011 from cells exposed to 0, 1 or 2 J/m2 UVC is illustrated in Figure 3
. PI units were determined for the pSV011 replication product (2.9 kb, DpnI-resistant) in different experiments, normalized to the DNA recovery control (4.2 kb band of M13mp2SV) and expressed as a percentage of the corresponding values for unirradiated cells (Figure 4
). Different symbols represent data from three or four independent experiments with IDH4 (Figure 4A
) and CTag (Figure 4B
) cells and the horizontal dashes indicate average values. Irradiation of CTag cells with 1 J/m2 decreased pSV011 replication to 53% of that in sham-treated controls. As the UVC fluence was increased to 2 and 3 J/m2, however, this value decreased only gradually (if at all). In contrast, IDH4 cells irradiated with 1 and 2 J/m2 showed average values for pSV011 replication near 100%. Irradiation of IDH4 cells with 3 J/m2 reduced average pSV011 replication to ~71% of the control. The Wilcoxon two-sample rank test was used to determine that the results with CTag and IDH4 cells were significantly different (P < 0.05) at every dose of UVC tested.

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Fig. 3. pSV011 replication inside UVC-irradiated cells. IDH4 and CTag cells were transfected with undamaged pSV011, as described in Materials and methods. Following an incubation of 15 h, cells were irradiated with the indicated fluences of UVC, then incubated for an additional 15 h. Hirt-extracted plasmid DNA was mixed with a fixed amount of an internal standard (M13mp2SV) prior to purification. DNA was restricted with DpnI, linearized with EcoRI and fractionated on 1% agarose gels containing 0.2 µg/ml ethidium bromide. M represents the lane containing size markers (HindIII-digested DNA). The arrows point to the 4.2 kb fragment of DpnI-digested M13mp2SV (4.2 M13) used as the internal standard and to the replicated, linearized full-length pSV011 DNA (2.9 pSV).
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Fig. 4. Inhibition of pSV011 replication inside UVC-irradiated human cells. The extent of pSV011 replication in the in vivo replication assays was determined by quantitative Southern hybridization, as illustrated in Figure 3 . Normalized PI units were expressed as a percentage of the same values determined for the unirradiated controls and plotted against UVC fluence. Different symbols represent the results of independent experiments (three or four) with IDH4 (A) and CTag (B) cells. The same symbols are used in (A) and (B) to denote experiments carried out in parallel. Average values were determined by: (i) taking the logarithm of individual ratios between normalized PI values for irradiated cells and the corresponding sham-irradiated control for each experiment; (ii) calculating averages of the log values for each UVC dose; (iii) transforming them back to average ratios that were plotted as the percentages illustrated by the horizontal dashes.
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The magnitude of cell loss induced by UVC was determined under the same experimental conditions used to measure inhibition of pSV011 replication. The average cell numbers per plate at transfection (A), irradiation (B) and plasmid harvest (C) for treated and untreated IDH4 and CTag cells are shown in Figure 5
. The open bars indicate that untreated IDH4 and CTag cells displayed similar proliferation patterns. The average numbers of cells per plate dropped to 46 (CTag) and 57% (IDH4) in cultures transfected with plasmid DNA, compared with untreated cultures harvested at the same time after seeding (Figure 5
, bar B, 15 h after transfection, compare open and hatched bars). These results reflect the toxicity of Transfectam used as the lipofection reagent. During the following 15 h there was almost no change in cell number in the transfected and sham-irradiated IDH4 and CTag cells (Figure 5
, compare hatched bar B with the hatched bar C). In the UVC-treated cultures the average numbers of cells per plate 15 h after irradiation decreased to 80 (CTag) and 86% (IDH4) of the number determined for sham-irradiated cells (Figure 5
, compare hatched and filled bars C). We divided the PI units determined for pSV011 replication in sham-treated and irradiated cells by the cell numbers obtained 15 h post-UVC for the sham-treated and irradiated CTag and IDH4 cultures (Figure 5
), respectively, in order to normalize the data shown in Figure 4
(2 J/m2 only). We found that normalization for cell number reduced the apparent UVC-induced inhibition of pSV011 replication illustrated in Figure 4
for both CTag and IDH4 cells. Nonetheless, the degree of pSV011 replication inside irradiated (2 J/m2) CTag cells (average of 55% of control) remained statistically different (P < 0.05) from the value obtained for IDH4 cells (average of 109% of control).

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Fig. 5. UVC-dependent loss of cells transfected with pSV011. IDH4 (top) and CTag (bottom) cultures were transfected with pSV011, as described in Materials and methods, and incubated for 15 h. Selected plates were irradiated with 0 or 2 J/m2 UVC and incubated for an additional 15 h. The average cell number per plate (five plates each) was quantified for transfected cells at the time of irradiation (hatched bar B), as well as 15 h later for sham-treated (hatched bar C) and UVC-irradiated cultures (filled bar C). In parallel, the average cell number per plate (three plates each) for cultures that were not transfected or irradiated was determined at the same time points (open bars A, B and C).
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Flow cytometric analyses of cell cycle progression
In the context of this study it was important to evaluate the effects of a low dose of UVC (2 J/m2) on the progression of CTag and IDH4 cells through S phase. In one set of experiments IDH4 and CTag cells were irradiated with 0 or 2 J/m2 UVC and pulsed for 1 h with BrdU at 0, 7.5 or 15 h after irradiation. The cell cycle distributions for IDH4 (Figure 6A
) and CTag cells (Figure 6B
) were very similar in the absence of UVC (top), as expected for cell cultures in logarithmic growth. When IDH4 cells were irradiated with 2 J/m2, the distribution of BrdU-labeled cells changed only slightly (Figure 6A
, bottom). In contrast, two distinct populations of cells in S phase were detected at 7.5 and 15 h after irradiation of CTag cells (Figure 6B
, bottom). One of these represented S phase cells labeled with BrdU (see arrows in Figure 6B
, bottom). The other population was comprised of CTag cells in S phase that did not incorporate BrdU (see arrowheads in Figure 6B
, bottom). At 15 h post-UVC cells that were in S phase and actively synthesizing DNA corresponded to a sizeable fraction of the CTag population (47%). However, the same CTag population also contained another 18% of cells that were in S phase by DNA content (based on propidium iodide staining) but did not show any BrdUFITC fluorescence (see arrowhead between the G1 and G2 populations). The BrdU-labeled CTag cells that were cycling through S phase between 7.5 and 15 h were presumably those cells that were not in S phase at the time of irradiation. These cells were able to repair UVC-induced DNA damage and, subsequently, incorporate BrdU when in S phase. The CTag cells that were in S phase at the time of irradiation (40%) displayed a prolonged delay and a fraction of them (~45%) arrested as cells that did not incorporate BrdU. In contrast, IDH4 cells that were in S phase at the time of irradiation, as well as those cells that were not, were able to progress through the cell cycle with only a slight delay.

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Fig. 6. UVC-induced alterations in cell cycle progression: pulsing at increasing times post-UVC. Logarithmic cultures of IDH4 (A) and CTag (B) cells were irradiated with 0 or 2 J/m2, then pulsed with 10 µM BrdU for 1 h at 0, 7.5 or 15 h post-UVC. Ethanol-fixed cells were stained with propidium iodide and anti-BrdU FITC-conjugated antibody and analyzed by flow cytometry. Arrows in (B) point to S phase cells that incorporated BrdU during the pulse but were in G1 during the UVC exposure. The arrowheads indicate cells in S phase by DNA content that did not incorporate BrdU. These cells were presumably arrested in S phase by the UVC treatment.
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We also irradiated IDH4 and CTag cells with 0 or 2 J/m2 UVC, pulsed them immediately with BrdU for 1 h and then incubated the cultures in fresh medium for 0, 7.5 or 15 h (Figure 7
). The goal of this experiment was to label cells that were in S phase (or entered S phase) in the first hour after UVC treatment and to follow their fate during the next 15 h. The progression of non-irradiated cells through the cell cycle is shown in the top three panels of Figure 7A and B
. The data revealed that the IDH4 and CTag populations contained cells throughout S phase at the time of the BrdU pulse (Figure 7A and B
, upper left). By 7.5 h the BrdU-labeled cells moved to later phases of the cell cycle and were found primarily in the late S and G2 phases. Then the majority of BrdU-labeled cells divided and were found in G1 by 15 h. When the cells were irradiated with 2 J/m2 UVC the smooth transition through the cell cycle observed in the absence of UVC was significantly altered (Figure 7A and B
, bottom panels). In IDH4 cells movement from S phase towards G2 occurred at a slower rate than observed in the absence of UVC (Figure 7A
). In comparison, there was almost no movement of S phase cells (BrdU-labeled) in the CTag population at 7.5 h post-UVC (Figure 7B
). By 15 h after irradiation a fraction of BrdU-labeled CTag cells were still in S phase. This fraction was 11-fold higher than that detected in the sham-irradiated CTag population (the corresponding ratio in IDH4 was only 1.3). The results in Figure 7
(arrowheads) also show that IDH4 and CTag cells that were in G1 at the time of irradiation (i.e. were not labeled with BrdU) were capable of moving into S phase with similar kinetics.

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Fig. 7. UVC-induced alterations in cell cycle progression: pulsechase protocol. IDH4 (A) and CTag (B) cultures in logarithmic growth were irradiated with 0 or 2 J/m2, then pulsed immediately with BrdU for 1 h. Cells were fed with fresh medium and incubated for the indicated times following the end of the pulse. Cells were analyzed by flow cytometry, as specified in the legend to Figure 6 . The arrowheads point to unlabeled cells in S phase by DNA content. These cells were presumably in G1 at the time of irradiation and BrdU pulse and later entered S phase at about the same rate in both IDH4 and CTag cultures. In this protocol cells that were in S phase in the first hour after UVC irradiation incorporated BrdU but did not move away from S phase at the same rate as the sham-irradiated cells.
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Effect of UVC on cell proliferation
The differential effect of low doses of UVC on IDH4 and CTag cells was also demonstrated by following the increase in cell number in populations exposed to 0, 1 or 2 J/m2 UVC (Figure 8
). Irradiation on day 2 barely affected proliferation of IDH4 cells (Figure 8A
). The number of CTag cells per plate, however, remained approximately constant for 2 days before resuming logarithmic growth. After the UVC-induced delay, proliferation in the irradiated cell populations occurred at rates that were similar to that observed in the sham-treated control.
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Discussion
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The S phase checkpoint is presumed to safeguard eukaryotic cells against replication of damaged DNA. How it signals to the DNA replication machinery to produce DNA damage-dependent delays in S phase is still under investigation. One component of the S phase checkpoint response is inhibition of replicon initiation (1,22,25,40). This transient inhibition maximizes the probability that DNA repair will remove template lesions prior to replicon firing. Earlier studies have suggested that inhibition of replication of episomal DNA by exposure of host cells to low doses of radiation occurs at the level of initiation (43). It is conceivable that the S phase checkpoint also modulates DNA elongation and maturation, but experimental evidence for such responses is lacking.
PRR does not eliminate the primary DNA damage, but promotes damage tolerance by preventing or repairing daughter strand gaps opposite template lesions. We are interested in the relationship between activation of the S phase checkpoint and the capacity of human cells for PRR. Delays in S phase progression should also protect cells by extending the time available for replication fork bypass and daughter strand gap repair. In this study we examined the responses of S phase cells to UVC in populations of human fibroblasts that are proficient in PRR (IDH4) and in XP-V fibroblasts (CTag) that lack the pol
-dependent pathway of translesion synthesis across cyclobutane thymine dimers. Although these cells are from different individuals, it is implied that other genetic differences between them do not impact on their responses to UVC or
-rays.
Another goal of these studies was to determine whether S phase delay and inhibition of episomal DNA replication could be induced by UVC in cells in which DNA damage-dependent inhibition of initiation of chromosomal replicons could not be detected. The SV40-transformed cell lines CTag (XP-V) and IDH4 did not respond to
-rays (Figure 1
) or UVC (not shown) by inhibiting replicon initiation, but differed in their D0 values for the UVC-induced inhibition of DNA elongation (Figure 2
). CTag cells also responded to low doses of UVC more strongly than IDH4 cells by inhibiting episomal DNA replication (Figures 3 and 4
), arresting in S phase (Figures 6 and 7
) and delaying cell proliferation (Figure 8
). We suggest that these responses were triggered primarily by UVC inhibition of DNA elongation and were not the result of inhibition of replicon initiation.
CTag and IDH4 fibroblasts expressing the SV40 large T antigen displayed radioresistant DNA synthesis (Figure 1
) indicative of reduced inhibition of replicon initiation. Transformation of human skin fibroblasts with large T antigen is known to inactivate G1 checkpoint function via binding of p53 and pRB to the oncoprotein (41,44) and to reduce the efficiency of global genomic repair of cyclobutane pyrimidine dimers (45). Expression of large T antigen also attenuates the G2 checkpoint response to DNA damage induced by ionizing radiation, possibly through enhanced expression of cyclin B1 and the kinase activity of mitosis-promoting factor (41,46,47). Large T antigen may attenuate the S checkpoint response of inhibition of replicon initiation through effects on the S phase cyclin-dependent kinase complexes cyclin E/Cdk2 and cyclin A/Cdk2. Both cyclin E and cyclin A are transactivated by E2F1 (48). Inactivation of pRB by large T antigen would enhance expression of the Cdk2 regulatory subunits. ATM function can be restored in SV40-transformed AT cells (49), demonstrating that the SV40 large T antigen does not inactivate ATM directly. The enhanced inhibition of plasmid DNA replication in UV-irradiated XP-V cells suggests that there is active signaling from sites of DNA synthesis arrested at cyclobutane pyrimidine dimers to the DNA synthetic machinery in other replicons. An alternative possibility is that inhibition of DNA replication at sites of pyrimidine dimers sequesters replication factors that are needed to replicate undamaged DNA. These two hypotheses cannot be distinguished by the data presented here. We do not favor the latter explanation, however, because many replication factors are regulated by E2F1 and should be overexpressed in SV40-transformed cells.
UVC fluences of 12 J/m2 deposit the same low density of lesions in genomic DNA in both CTag and IDH4 cells (12 cyclobutane dimers/75 kb, reduced further by nucleotide excision repair). Because of the bypass replication defect in XP-V cells, however, arrest of replication forks at unrepaired lesions and the formation of daughter strand gaps are enhanced in CTag cells. These cis effects of DNA photoproducts are likely to generate molecular signals that, when transmitted to other replication sites, inhibit the replication machinery in undamaged DNA sequences (trans effect). The observation that UVC-irradiation of host CTag cells carrying undamaged episomal DNA led to a reduction in recovery of episomal DNA replication products (Figures 3 and 4
) seems to reflect the operation of a trans-acting mechanism of inhibition of DNA replication (43,50,51).
Delays in S phase progression, reductions in rate of cell proliferation and inhibition of episomal DNA replication are viewed as related outcomes of a signal transduction pathway initiated by accumulation of aberrant intermediates of DNA replication, such as blocked replication forks and daughter strand gaps. These abnormal structures accumulate in bypass-defective XP-V cells, even after low fluences of UVC. Since the putative initiating signal is higher in XP-V cells and the S phase checkpoint responses are enhanced, it is expected that intermediate events in the pathway will be easier to detect in this cell type. Recent results from Cleaver and collaborators (52) appear to support this prediction. Using SV40-transformed human fibroblasts these investigators observed an increased frequency of UVC-induced hMre11 foci in XP-V cells (~15-fold higher than in SV40-transformed fibroblasts from normal donors). All nuclear foci containing hMre11 were also positive for PCNA-specific staining and about half of the PCNA-positive cells (those in S phase) contained hMre11 foci. The authors' interpretation was that UVC induced association of hMre11 (and by inference the complex containing Rad50 and Nbs1) with replication forks arrested at photoproducts (52). The hMre11/Rad50/Nbs1 complex is involved with double-strand break repair and DNA recombination pathways (53,54). Accordingly, nuclear foci staining for Mre11 were induced at higher frequency by ionizing radiation (3354% of irradiated cells, regardless of the XP phenotype), but they did not co-localize with PCNA foci (52). It remains to be determined whether hMre11 recombination complexes are recruited by stalled replication forks or by double-stranded DNA breaks occurring at forks that encounter DNA lesions (i.e. restricted to S phase cells). It is intriguing that sister chromatid exchanges (requiring homologous recombination) are also enhanced in SV40-transformed XP-V cells exposed to UVC (55). Even though hMre11 foci are induced at much lower frequencies in primary human fibroblasts, these foci are more likely to be detected in XP-V than in normal fibroblasts (52). The enhanced S phase delays observed with CTag cells were also observed with XP-V cells that were not transformed with SV40 large T antigen (unpublished observations).
Replication protein A (RPA, single-stranded DNA-binding protein) participates in most DNA metabolic processes and is hyperphosphorylated in cells exposed to ionizing radiation (56), UV (57) and other DNA-damaging agents (58). Thus, its modification in response to DNA damage has received considerable attention (5663). Whether RPA is phosphorylated in response to DNA damage by ATM kinase, DNA-PK, ATR or cyclin-dependent kinases continues to be a matter of intense debate. Conceivably, the type of DNA damage and how it interacts with the DNA replication machinery might dictate the specific mechanism used to activate the S phase checkpoint. For instance, rapid phosphorylation of RPA was observed in human cells treated with topoisomerase inhibitors (58). This response was dependent on DNA-PK and ongoing replication, but independent of p53 and ATM. Shao and collaborators (58) suggested that the encounter of a replication fork with a topoisomeraseDNA cleavage complex might lead to the juxtaposition of replication-associated RPA and DNA-PK associated with double-stranded DNA ends. Phosphorylation of RPA under these conditions may be a signal to the S phase checkpoint machinery and/or contribute to replicative arrest. UVC does not cause double-strand breaks directly, although some are likely to be generated from replication forks breaking at single-stranded DNA regions that are created by blockage of leading strand synthesis at a photoproduct and uncoupling of lagging strand synthesis (64). These single-stranded DNA regions should be coated by RPA. In comparison with transient binding of RPA to single-stranded DNA during normal DNA replication, the RPA molecules coating the extended single-stranded DNA formed during replication of UVC-damaged DNA could be particularly vulnerable to hyperphosphorylation, perhaps by a chromatin-bound kinase (62). Hyperphosphorylated RPA might recruit factors necessary to enforce the S phase checkpoint and/or to facilitate recovery of the stalled replication forks. The latter could be mediated by DNA polymerase switching and bypass replication or a recombination pathway, such as that proposed for the repair of stalled replication forks in Escherichia coli (65).
In summary, inhibition of DNA replication and S phase delay provide more time to repair damaged DNA before and after it is replicated. This is an important protective mechanism for the prevention of carcinogenesis (66). The data in this paper suggest that human cells might be endowed with two pathways (or two branches originating from a single pathway) for induction of S phase delay following UVC irradiation. These pathways lead to inhibition of replicon initiation and DNA elongation. The former is clearly the end point of signal transduction mechanisms, while the latter appears to result from the passive effects of DNA lesions on DNA replication followed by activation of an inhibitory trans-acting process.
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Notes
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1 Present address: Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0058, USA 
2 To whom correspondence should be addressed Email: uncmcs{at}med.unc.edu 
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Acknowledgments
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We are grateful to Cheryl Cistulli and Cynthia Behe for help with treatment of human cells with
-rays and flow cytometric analyses. This work was supported by PHS grant CA55065.
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Received August 7, 2000;
revised October 10, 2000;
accepted October 16, 2000.