Carcinogenesis vol.22 no.2 pp.233–241, 2001
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
1Present
address: Department of Radiation Oncology, Medical College of
Virginia, Virginia Commonwealth University, Richmond, VA 23298-0058,
USA
2To
whom correspondence should be addressed
Email: [email protected]
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 η). 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
γ-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 1–3 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 η, DNA
polymerase η; PRR, post-replication repair; RPA, replication protein A (singlestranded DNA-binding protein); XP-V, xeroderma pigmentosum variant.
© Oxford University Press
Introduction
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 UVCinduced lesions on DNA replication is overcome by postreplication 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 (3–6), but are deficient in bypass replication of UVCinduced thymine dimers (1,4,7–12). This bypass defect is
caused by frameshift mutations in the gene encoding DNA
polymerase η (pol η) (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 ζ (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
(22–24) 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
233
S.K.Bullock et al.
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 (30–32) and Dbf4/Cdc7 (33–35), 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 SV40transformed 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 (1–3 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.
Materials and methods
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, 2⫻ MEM vitamins, 2⫻ 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 (HindIII–SphI
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).
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 1 ml of 0.3 M
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 5–20% 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 γ-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 400 000–450 000 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
(2–5 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 (0–3 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.
UVC and γ-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 50–100 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 γ-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.
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 Tris–acetate, 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 [α-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.
Inhibition of DNA synthesis by γ-irradiation
Cells were seeded at 150 000–200 000/60 mm plate (3 plates/condition) and
incubated for 2 days. Cultures were irradiated with 0 or 4 Gy, incubated for
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.
234
UVC-induced S phase responses in XP-V
Fig. 1. Inhibition of DNA synthesis by γ-rays in diploid and transformed
human fibroblasts. Logarithmically growing cultures of diploid (GM3348
and CRL1162) and transformed (IDH4 and CTag) fibroblasts, derived from
normal (GM3348 and IDH4) or XP-V (CRL1162 and CTag) donors, were
irradiated with 0 or 4 Gy γ-rays. After a 30 min incubation at 37°C, cell
cultures were pulse labeled with [3H]thymidine for 30 min, then harvested
and lysed. Incorporation of [3H]thymidine into acid-insoluble
macromolecules was measured by scintillation counting. DNA synthesis
rates in the cultures exposed to γ-rays were expressed relative to the values
obtained with sham-irradiated cultures. The ~50% inhibition of DNA
synthesis observed with the diploid fibroblasts is thought to reflect radiationdependent inhibition of replicon initiation. The transformed cells did not
display any inhibition of DNA synthesis after exposure to γ-rays. Hence,
they have lost the S phase checkpoint response of inhibition of replicon
initiation.
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.
Results
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, γ-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 γ-rays and
UVC. Human cells with intact S phase checkpoint responses
inhibit by ~50% incorporation of [3H]thymidine into acidinsoluble macromolecules 30–60 min after treatment with low
doses of ionizing radiation (22–24). This response has been
correlated with inhibition of replicon initiation. Using this
approach we determined that exposure to 4 Gy γ-rays elicited
this S phase response from normal diploid (GM3348) and
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 5⫻107–2⫻108) 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 (n)
and 2.5 J/m2 for CTag (d).
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 30–45 min after irradiation (1,4,8,25). Dosedependent decreases in [3H]thymidine incorporation (normalized to the number of cells added to the gradient) were
associated with intermediates of DNA replication of 5⫻107–
2⫻108 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
235
S.K.Bullock et al.
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. Hirtextracted 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 λ 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).
XP-V cell line was determined to be statistically significant
(P ⬍ 0.05) by ANOVA.
For both CTag and IDH4 cells, however, treatment with
low doses of UVC did not inhibit [3H]thymidine incorporation
into replication intermediates of ⬍5⫻107 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 γ-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 ~12–15 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 (1–3 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
236
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 shamirradiated 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.
determined for the pSV011 replication product (2.9 kb, DpnIresistant) 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.
The magnitude of cell loss induced by UVC was determined
UVC-induced S phase responses in XP-V
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).
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).
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
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.
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 BrdU–FITC 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 UVCinduced DNA damage and, subsequently, incorporate BrdU
237
S.K.Bullock et al.
Fig. 7. UVC-induced alterations in cell cycle progression: pulse–chase
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.
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.
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
238
Fig. 8. UVC-induced alterations in cells proliferation. IDH4 (A) and CTag
(B) cells were seeded at ~50 000 cells/60 mm plate. The average cell
number per plate was determined every day (average of 3 plates/condition)
for the period of time indicated. On day 2 (arrow) the cultures were
irradiated with 0 (s), 1 (j) or 2 (m) J/m2 UVC.
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 BrdUlabeled CTag cells were still in S phase. This fraction was 11fold 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.
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
UVC-induced S phase responses in XP-V
at rates that were similar to that observed in the shamtreated control.
Discussion
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 damagedependent 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 η-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 γ-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 γ-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 1–2 J/m2 deposit the same low density of
lesions in genomic DNA in both CTag and IDH4 cells (1–2
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 bypassdefective 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 (~15fold 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 PCNApositive 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 doublestrand break repair and DNA recombination pathways (53,54).
Accordingly, nuclear foci staining for Mre11 were induced at
higher frequency by ionizing radiation (33–54% of irradiated
cells, regardless of the XP phenotype), but they did not colocalize 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
239
S.K.Bullock et al.
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 (56–63). 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
topoisomerase–DNA 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 singlestranded 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.
Acknowledgements
We are grateful to Cheryl Cistulli and Cynthia Behe for help with treatment
of human cells with γ-rays and flow cytometric analyses. This work was
supported by PHS grant CA55065.
240
References
1. Kaufmann,W.K. and Cleaver,J.E. (1981) Mechanisms of inhibition of DNA
replication by ultraviolet radiation in normal human and xeroderma
pigmentosum fibroblasts. J. Mol. Biol., 149, 171–187.
2. Kaufmann,W.K. (1989) Pathways of human cell postreplication repair.
Carcinogenesis, 10, 1–11.
3. Cleaver,J. (1972) Xeroderma pigmentosum: variants with normal DNA
repair and normal sensitivity to ultraviolet light. J. Invest. Dermatol., 58,
124–128.
4. Lehmann,A.R., Kirk-Bell,S., Arlet,C.F., Patterson,M.C., Lohman,P.H.M.,
de Weerd-Kastelein,E.A. and Bootsma,D. (1975) Xeroderma pigmentosum
cells with normal levels of excision repair have defect in DNA synthesis
after UV-irradiation. Proc. Natl Acad. Sci. USA, 72, 219–223.
5. Wang,Y.C., Maher,V.M., Mitchell,D.L. and McCormick,J.J. (1993)
Evidence from mutation spectra that UV hypermutability of xeroderma
pigmentosum variant cells reflects abnormal, error-prone replication on a
template containing photoproducts. Mol. Cell. Biol., 13, 4276–4283.
6. Tung,B.S., McGregor,W.G., Wang,Y.-C., Maher,V.M. and McCormick,J.J.
(1996) Comparison of the rate of excision of major UV photoproducts in
the strands of the human HPRT gene of normal and xeroderma
pigmentosum cells. Mutat. Res., 362, 65–74.
7. van Zeland,A.A. and Filon,A.R. (1982) Post-replication repair: elongation
of daughter strand DNA in UV-irradiated mammalian cells in culture.
Prog. Mutat. Res., 4, 375–384.
8. Boyer,J.C., Kaufmann,W.K., Brylawski,B.P. and Cordeiro-Stone,M. (1990)
Defective postreplication repair in xeroderma pigmentosum variant
fibroblasts. Cancer Res., 50, 2593–2598.
9. Cordeiro-Stone,M., Zaritskaya,L., Price,L.K. and Kaufmann,W.K. (1997)
Replication fork bypass of a pyrimidine dimer blocking leading strand
synthesis. J. Biol. Chem., 272, 13945–13954.
10. Svoboda,D.L., Briley,L.P. and Vos,J.-M.H. (1998) Defective bypass
replication of a leading strand cyclobutane thymine dimer in xeroderma
pigmentosum variant cell extracts. Cancer Res., 58, 2445–2448.
11. Ensch-Simon,I., Burgers,P.M.J. and Taylor,J.-S. (1998) Bypass of a sitespecific cis-syn thymine dimer in an SV40 vector during in vitro replication
by HeLa and XPV cell-free extracts. Biochemistry, 37, 8218–8226.
12. Masutani,C., Araki,M., Yamada,A., Kusumoto, R., Nogimori,T.,
Maekawa,T., Iwai,S. and Hanaoka,F. (1999) Xeroderma pigmentosum
variant (XP-V) correcting protein from HeLa cells has a thymine dimer
bypass DNA polymerase activity. EMBO J., 18, 3491–3501.
13. Masutani,C., Kusumoto,R., Yamada,A., Dohmae,N., Yokoi, M., Yuasa,M.,
Araki,M., Iwai,S., Takio,K. and Hanaoka,F. (1999) The XPV (xeroderma
pigmentosum variant) gene encodes human DNA polymerase η. Nature,
399, 700–704.
14. Johnson,R.E., Kondratick,C.M., Prakash,S. and Prakash,L. (1999) hRAD30
mutations in the variant form of xeroderma pigmentosum. Science, 285,
263–265.
15. Johnson,R.E., Prakash,S. and Prakash,L. (1999) Efficient bypass of a
thymine-thymine dimer by yeast DNA polymerase, polη. Science, 283,
1001–1004.
16. Gibbs,P.E., McGregor,W.G., Maher,V.M., Nisson,P. and Lawrence,C.W.
(1998) A human homolog of the Saccharomyces cerevisiae REV3 gene,
which encodes the catalytic subunit of DNA polymerase ζ. Proc. Natl
Acad. Sci. USA, 95, 6876–6880.
17. Gibbs,P.E.M., Wang,X.-D., Li,Z., McManus,T.P., McGregor,W.G.,
Lawrence,C.W. and Maher,V.M. (2000) The function of the human
homolog of Saccharomyces cerevisiae REV1 is required for mutagenesis
induced by UV light. Proc. Natl Acad. Sci. USA, 97, 4186–4191.
18. Woodgate,R. (1999) A plethora of lesion-replicating DNA polymerases.
Genes Dev., 13, 2191–2195.
19. Johnson,R.E., Washington,M.T., Prakash,S. and Prakash,L. (1999) Bridging
the gap: a family of novel DNA polymerases that replicate faulty DNA.
Proc. Natl Acad. Sci. USA, 96, 12224–12226.
20. Maher,V.M., Ouellette,L.M., Curren,R.D. and McCormick,J.J. (1976)
Frequency of ultraviolet light-induced mutations is higher in xeroderma
pigmentosum variant cells than in normal human cells. Nature, 261,
593–595.
21. McGregor,W.G., Wei,D., Maher,V.M. and McCormick,J.J. (1999)
Abnormal, error-prone bypass of photoproducts by xeroderma
pigmentosum variant cell extracts results in extreme strand bias for the
kind of mutations induced by UV light. Mol. Cell. Biol., 19, 147–154.
22. Painter,R.B. and Young, B.R. (1980) Radiosensitivity in ataxiatelangiectasia: a new explanation. Proc. Natl Acad. Sci. USA, 77, 7315–
7317.
23. Houldsworth,J. and Lavin,M.F. (1980) Effect of ionizing radiation on DNA
synthesis in ataxia telangiectasia cells. Nucleic Acids Res., 8, 3709–3720.
UVC-induced S phase responses in XP-V
24. Morgan,S.E., Lovly,C., Pandita,T.K., Shiloh,Y. and Kastan,M.B. (1997)
Fragments of ATM which have dominant-negative or complementing
activity. Mol. Cell. Biol., 17, 2020–2029.
25. Kaufmann,W.K., Cleaver,J.E. and Painter,R.B. (1980) UV inhibits replicon
initiation in S phase human cells. Biochim. Biophys. Acta, 608, 191–195.
26. Sarkaria,J.N., Busby,E.C., Tibbetts,R.S., Roos,P., Taya,Y., Karnitz,L.M.
and Abraham,R.T. (1999) Inhibition of ATM and ATR kinase activities
by the radiosensitizing agent, caffeine. Cancer Res., 59, 4375–4382.
27. Liu,Q., Guntuku,S., Cui,X.-S., Matsuoka,S., Cortez,D., Tamai,K., Luo,G.,
Carattini-Rivera,S., DeMayo,F., Bradley,A., Donehower,L.A. and
Elledge,S.J. (2000) Chk1 is an essential kinase that is regulated by Atr
and required for the G2/M DNA damage checkpoint. Genes Dev., 14,
1448–1459.
28. Beamish,H., Williams,R., Chen,P. and Lavin,M.F. (1996) Defect in multiple
cell cycle checkpoints in ataxia-telangiectasia postirradiation. J. Biol.
Chem., 271, 20486–20493.
29. Xie,G., Habbersett,R.C., Jia,Y., Peterson,S.R., Lehnert,B.E., Bradbury,E.M.
and D’Anna,J.A. (1998) Requirements for p53 and the ATM gene product
in the regulation of G1/S and S checkpoints. Oncogene, 16, 721–736.
30. Stoeber,K., Mills,A.D., Kubota,Y., Krude,T., Romanowski,P., Marheineke,
K., Laskey,R.A. and Williams,G.H. (1998) Cdc6 protein causes premature
entry into S phase in a mammalian cell-free system. EMBO J., 17,
7219–7229.
31. Yan,Z., DeGregory,J., Shohet,R., Leone,G., Stillman,B., Nevins,J.R. and
Williams,R.S. (1998) Cdc6 is regulated by E2F and is essential for DNA
replication in mammalian cells. Proc. Natl Acad. Sci. USA, 95, 3603–3608.
32. Jiang,W., Wells,N.J. and Hunter,T. (1999) Multistep regulation of DNA
replication by Cdk phosphorylation of HsCdc6. Proc. Natl Acad. Sci. USA,
96, 6193–6198.
33. Jiang,W. and Hunter,T. (1997) Identification and characterization of a
human protein kinase related to budding yeast Cdc7p. Proc. Natl Acad.
Sci. USA, 94, 14320–14325.
34. Bousset,K. and Diffley,J.F. (1998) The Cdc7 protein kinase is required for
origin firing during S phase. Genes Dev., 12, 480–490.
35. Kumagai,H., Sato,N., Yamada,M., Mahony,D., Seghezzi,W., Lees,E.,
Arai,K. and Masai,H. (1999) A novel growth- and cell cycle-regulated
protein, ASK, activates human Cdc7-related kinase and is essential for
G1/S transition in mammalian cells. Mol. Cell. Biol., 19, 5083–5095.
36. Shay,J.W., West,M.D. and Wright,W.E. (1992) Re-expression of senescent
markers in deinduced reversibly immortalized cells. Exp. Gerontol., 27,
477–492.
37. King,S.A., Wilson,S.J., Farber,R.A., Kaufmann,W.K. and CordeiroStone,M. (1995) Xeroderma pigmentosum variant: generation and
characterization of fibroblastic cell lines transformed with SV40 large T
antigen. Exp. Cell Res., 217, 100–108.
38. Prelich,G. and Stillman,B. (1988) Coordinated leading and lagging strand
synthesis during SV40 DNA replication in vitro requires PCNA. Cell, 53,
117–126.
39. Roberts,J.D. and Kunkel,T.A. (1988) Fidelity of human cell DNA
replication complex. Proc. Natl Acad. Sci. USA, 85, 7064–7068.
40. Painter,R.B. and Young,B.R. (1976) Formation of nascent DNA molecules
during inhibition of replicon initiation in mammalian cells. Biochim.
Biophys. Acta, 418, 146–153.
41. Kaufmann,W.K., Levedakou,E.N., Grady,H.L., Paules,R.S. and Stein,G.H.
(1995) Attenuation of G2 checkpoint function precedes human cell
immortalization. Cancer Res., 55, 7–11.
42. Hirt,B. (1967) Selective extraction of polyoma DNA from infected mouse
cell cultures. J. Mol. Biol., 26, 365–369.
43. Wang,Y.-C. and Hsu,M.-T. (1996) Inhibition of initiation of simian virus
40 DNA replication during acute response of cells irradiated by ultraviolet
light. Nucleic Acids Res., 24, 3149–3157.
44. Levine,A.J. (1997) p53, the cellular gatekeeper for growth and division.
Cell, 88, 323–331.
45. Ford,J.M. and Hanawalt,P.C. (1997) Expression of wild-type p53 is required
for efficient global genomic nucleotide excision repair in UV-irradiated
human fibroblasts. J. Biol. Chem., 272, 28073–28080.
47. Chang,T.H., Ray,F.A., Thompson,D.A. and Schlegel,R. (1997)
Disregulation of mitotic checkpoints and regulatory proteins following
acute expression of SV40 large T antigen in diploid human cells. Oncogene,
14, 2383–2393.
48. DeGregori,J., Kowalik,T. and Nevins,J.R. (1995) Cellular targets for
activation by the E2F1 transcription factor include DNA synthesis- and
G1/S-regulatory genes. Mol. Cell. Biol., 15, 4215–4224. [Erratum, Mol.
Cell. Biol., 1995, 15, 5846–5847.]
49. Ejima,Y., Oshimura,M. and Sasaki,M.S. (1990) Establishment of a novel
immortalized cell line from ataxia telangiectasis fibroblasts and its use for
the chromosomal assignment of radiosensitivity gene. Int. J. Radiat. Biol.,
58, 989–997.
50. Lamb,J.R., Petit-Frere,C., Broughton,B.C., Lehmann,A.R. and Gree,M.H.
(1989) Inhibition of DNA replication by ionizing radiation is mediated by
a trans-acting factor. Int. J. Radiat. Biol., 56, 125–130.
51. Cleaver,J.E., Rose,R. and Mitchell,D.L. (1990) Replication of chromosomal
and episomal DNA in X-ray-damaged human cells: a cis- or trans-acting
mechanism? Radiat. Res., 124, 294–299.
52. Limoli,C.L., Giedzinski,E., Morgan,W.F. and Cleaver,J.E. (2000)
Polymerase η deficiency in the xeroderma pigmentosum variant uncovers
an overlap between the S phase checkpoint and double-strand break repair.
Proc. Natl Acad. Sci. USA, 97, 7939–7946.
53. Haber,J.E. (1998) The many interfaces of Mre11. Cell, 95, 583–586.
54. Petrini,J.H. (1999) The mammalian Mre11-Rad50-nbs1 protein complex:
integration of functions in the cellular DNA damage response. Am. J.
Hum. Genet., 64, 1264–1269.
55. Cleaver,J.E., Afzal,V., Feeney,L., McDowell,M., Sadinski,W., Volpe,J.P.G.,
Busch,D.B., Coleman,D.M., Ziffer,D.W., Yu,Y., Nagasawa,H. and
Little,J.B. (1999) Increased ultraviolet sensitivity and chromosomal
instability related to p53 function in the xeroderma pigmentosum variant.
Cancer Res., 59, 1102–1108.
56. Liu,V.F. and Weaver,D.T. (1993) The ionizing-radiation induced replication
protein A phosphorylation response differs between ataxia telangiectasia
and normal human cells. Mol. Cell. Biol., 13, 7222–7231.
57. Carty,M.P., Zernik-Kobak,M., McGrath,S. and Dixon,K. (1994) UV lightinduced DNA synthesis arrest in HeLa cells is associated with changes in
phosphorylation of human single-stranded DNA-binding protein. EMBO
J., 13, 2114–2123.
58. Shao,R.G., Cao,C.X., Zhang,H., Kohn,K.W., Wold,M.S. and Pommier,Y.
(1999) Replication-mediated DNA damage by camptothecin induces
phosphorylation of RPA by DNA-dependent protein kinase and dissociates
RPA:DNA–PK complexes. EMBO J., 18, 1397–1406.
59. Zernik-Kobak,M., Vasunia,K., Connelly,M., Anderson,C.W. and Dixon,K.
(1997) Sites of UV-induced phosphorylation of the p34 subunit of
replication protein A from HeLa cells. J. Biol. Chem., 272, 23896–23904.
60. Niu,H., Erdjument-Bromage,H., Pan,Z.Q., Lee,S.H., Tempst,P. and
Hurwitz,J. (1997) Mapping of amino acid residues in the p34 subunit of
human single-stranded DNA-binding protein phosphorylated by DNAdependent protein kinase and Cdc2 kinase in vitro. J. Biol. Chem., 272,
12634–12641.
61. Morgan,S.E. and Kastan,M.B. (1997) Dissociation of radiation-induced
phosphorylation of replication protein A from the S-phase checkpoint.
Cancer Res., 57, 3386–3389.
62. Gately,D.P., Hittle,J.C., Chan,G.K.T. and Yen,T.J. (1998) Characterization
of ATM expression, localization, and associated DNA-dependent protein
kinase activity. Mol. Biol. Cell, 9, 2361–2374.
63. Dutta,A. and Stillman,B. (1992) cdc2 family kinases phosphorylate a
human cell DNA replication factor, RPA and activate DNA replication.
EMBO J., 11, 2189–2199.
64. Cordeiro-Stone,M., Makhov,A.M., Zaritskaya,L.S. and Griffith,J.D. (1999)
Analysis of DNA replication forks encountering a pyrimidine dimer in
the template to the leading strand. J. Mol. Biol., 289, 1207–1218.
65. Cox,M.M., Goodman,M.F., Kreuzer,K.N., Sherratt,D.J., Sandler,S.J. and
Marians,K.J. (2000) The importance of repairing stalled replication forks.
Nature, 404, 37–41.
66. Kaufmann,W.K. and Paules,R.S. (1996) DNA damage and cell cycle