DNA nucleotide excision repair-dependent signaling
to checkpoint activation
Federica Marini*†, Tiziana Nardo‡, Michele Giannattasio*, Mario Minuzzo*, Miria Stefanini†‡, Paolo Plevani*†,
and Marco Muzi Falconi*†
*Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita ´ degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy; and‡Istituto di Genetica
Molecolare, Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, 27100 Pavia, Italy
Edited by Philip C. Hanawalt, Stanford University, Stanford, CA, and approved September 26, 2006 (received for review June 30, 2006)
Eukaryotic cells respond to a variety of DNA insults by triggering
a common signal transduction cascade, known as checkpoint re-
sponse, which temporarily halts cell-cycle progression. Although
the main players involved in the cascade have been identified,
there is still uncertainty about the nature of the structures that
activate these surveillance mechanisms. To understand the role of
nucleotide excision repair (NER) in checkpoint activation, we ana-
lyzed the UV-induced phosphorylation of the key checkpoint pro-
teins Chk1 and p53, in primary fibroblasts from patients with
xeroderma pigmentosum (XP), Cockayne syndrome (CS), tricho-
thiodystrophy (TTD), or UV light-sensitive syndrome. These disor-
ders are due to defects in transcription-coupled NER (TC-NER)
and?or global genome NER (GG-NER), the NER subpathways re-
pairing the transcribed strand of active genes or the rest of the
genome, respectively. We show here that in G0?G1 and G2?M
phases of the cell cycle, triggering of the DNA damage cascade
requires recognition and processing of the lesions by the GG-NER.
Loss of TC-NER does not affect checkpoint activation. Mutations in
XPD, XPB, and in TTDA, encoding subunits of the TFIIH complex,
involved in both transcription and NER, impair checkpoint trigger-
ing. The only exception is represented by mutations in XPD,
resulting in combined features of XP and CS (XP?CS) that lead to
activation of the checkpoint cascade after UV radiation. Inhibition
of RNA polymerase II transcription significantly reduces the phos-
phorylation of key checkpoint factors in XP?CS fibroblasts on
exposure to UV damage.
cell cycle ? human fibroblasts ? DNA repair ? DNA damage response
suggests that ssDNA, probably coated by replication protein A
(RPA), triggers the checkpoint. In fact recruitment of the
ataxia-telangiectasia mutated- and Rad3-related (ATR)-
interacting protein (ATRIP) protein kinase complex to sites of
RPA-coated ssDNA (1, 2). Others have suggested that check-
point proteins are able to directly bind UV-damaged DNA
without any need for processing of the lesion (3). Moreover, it
is still largely unclear whether NER plays any role in activating
This repair pathway, responsible for removing helix-distorting
lesions from DNA, essentially consists of four sequential steps:
recognition of the DNA lesion, opening of a bubble around the
lesion, incision of DNA upstream and downstream the damage
by the endonucleases XPF-ERCC1 and XPG, and finally DNA
resynthesis and ligation. Global genome NER (GG-NER) de-
pends on recognition of the damage in nontranscribed DNA by
activity (UV-DDB), including two subunits (DDB1 and DDB2?
XPE). Transcription-coupled NER (TC-NER) is initiated by the
arrest of the RNA polymerase at a lesion on the transcribed
strand of an active gene, in a process that requires the CSA and
CSB proteins. Defects in NER lead to various human inherited
syndromes: xeroderma pigmentosum (XP), Cockayne syndrome
he nature of the signal that activates DNA damage response
pathways after UV radiation is still debated. Some evidence
(CS), trichothiodystrophy (TTD), a mild UV light-sensitive
syndrome (UVsS), and a complex pathological phenotype with
combined symptoms of XP and CS (XP?CS). Despite sharing
cutaneous photosensitivity and genetic heterogeneity, these
diseases show distinct clinical features. XP is a cancer-prone
disorder, whereas CS and TTD are multisystem diseases char-
acterized by developmental and neurological abnormalities and
premature aging. The seven NER-deficient complementation
groups identified in XP are defective in both NER subpathways,
except for the XP-C and XP-E groups that are specifically
defective in GG-NER. Conversely, the genes responsible for CS
(CSA and CSB) and a still unidentified gene responsible for four
UVsS cases are involved in TC-NER (4, 5).
Some controversies exist in the literature about the role of
NER in triggering the checkpoint cascade in response to UV
radiation. In yeast, we showed that NER is required to allow
checkpoint complexes to bind stably to UV-damaged chromo-
somes, likely through a physical interaction between NER and
checkpoint proteins. Triggering of the cascade seems to require
initial processing of the lesions. In fact, a variety of yeast NER
mutants fail to activate the G1 and G2 checkpoints after UV
irradiation, whereas loss of TC-NER alone does not affect
checkpoint activation (6). In agreement with our data, UV-
induced H2AX phosphorylation was found defective in resting
human primary fibroblasts lacking functional XPA, a DNA
damage-binding protein involved in the repair of UV-induced
lesions from both active and inactive regions of the genome, but
was evident in fibroblasts from CS patients assigned to the CS-B
group (7). Similarly, H2AX phosphorylation after UV irradia-
tion within the G1phase was found to depend on XPA and XPC
in human fibroblasts immortalized by transfection with the
telomerase gene (8).
Others could not detect a strong activation of the ATR-
dependent checkpoint in normal human immortalized cell lines,
outside the S-phase of the cell cycle (9, 10). XP-C cells, trans-
formed with simian virus 40 were shown to be proficient in the
phosphorylation of key ATR targets in response to UV (9).
Recent results suggested that XPA-deficient immortalized hu-
man cells are defective in activating the DNA damage check-
point in S-phase, but further processing of UV radiation-induced
photoproducts does not seem to be required. Moreover, check-
point signaling does not seem to be compromised by a defect in
Author contributions: F.M. and M.M.F. designed research; F.M. performed research; T.N.,
analyzed data; and F.M., M.S., P.P., and M.M.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations: NER, nucleotide excision repair; TC-NER, transcription-coupled NER; GG-
NER global genome NER; XP, xeroderma pigmentosum; TTD, trichothiodystrophy; CS,
CS; ATR, ataxia-telangiectasia mutated- and Rad3-related.
igm.cnr.it, firstname.lastname@example.org, or email@example.com.
© 2006 by The National Academy of Sciences of the USA
November 14, 2006 ?
vol. 103 ?
no. 46 ?
only one of the two NER subpathways, GG-NER or TC-NER
(10). Despite the available information concerning the first steps
irradiation (11), several issues remain open: (i) Can the ATR-
mediated checkpoint be activated in nonreplicating G0?G1or G2
cells? (ii) Is the lesion by itself sufficient to activate the check-
point, or is the open preincision complex formation required?
(iii) Is further processing of the lesion by the NER machinery,
to activate ATR? (iv) What is the relative contribution of the two
NER subpathways, GG-NER and TC-NER, in checkpoint
To address these issues and to understand the role of NER in
triggering the signal transduction cascade in response to UV
radiation, we analyzed checkpoint activation in primary fibro-
blasts from several NER-defective patients, representative of
distinct clinical, cellular, and molecular alterations. The data we
triggering of the DNA damage cascade after UV radiation
depends on NER. Both recognition and processing of the UV
radiation-induced photoproducts by the GG-NER are required
to activate the checkpoint. Conversely, loss of TC-NER does not
affect checkpoint activation. In S-phase cells, checkpoint acti-
vation does not depend on NER, probably because other kind of
lesions, such as exposed single-stranded regions of DNA at
stalled replication forks or strand breaks, contribute to trigger
the checkpoint cascade. We also found that mutations in XPD,
XPB, and TTDA, encoding subunits of the TFIIH complex,
involved in both transcription and NER, lead to a failure to
activate the checkpoint. Only defects in the XPD gene that result
in XP?CS do not impair checkpoint activation, in response to
UV radiation. Intriguingly, in XP-D?CS fibroblasts, triggering of
the DNA damage cascade depends on ongoing RNA polymerase
Results and Discussion
With the aim of improving our understanding on the functional
relationship between recognition and processing of UV radiation-
induced photoproducts by NER and triggering of surveillance
mechanisms, we analyzed checkpoint activation in primary fibro-
blasts from 27 NER-defective patients (Table 1, which is published
as supporting information on the PNAS web site). It is important
to emphasize that primary human cell lines were chosen for our
study because they have intact surveillance mechanisms, compared
with certain immortalized cell lines. In particular, fibroblast strains
represent an in vitro cell system that still maintains the cell contact
inhibition and the cell density-dependent growth typical of the in
vivo situation. These features are lost in other cell systems, such as
transformed fibroblasts and lymphoblastoid cell lines, and may be
important in the cascade of events leading to checkpoint activation.
analysis was performed at the single-cell level by immunofluo-
rescence by using phosphospecific antibodies, recognizing the
key ATR targets Chk1 and p53 (anti-P-Chk1-Ser-317 and anti-
Checkpoint Activation in G0and G2?M Fibroblasts Depends on NER.
Nonproliferating fibroblasts from normal donors appeared to be
able to activate the checkpoint, because both Chk1 and p53 get
phosphorylated after UV irradiation (Fig. 1). Others could not
detect a strong ATR-dependent checkpoint activation in immor-
talized cells that are not replicating their DNA (9, 10). This
difference may be due to the analysis of primary cell lines
compared with transformed cells. In these immortalized cells,
p53 function is compromised, affecting the removal of cyclobu-
tane pyrimidine dimers from genomic DNA, in addition to other
pathways (12, 13). Cells from CS patients mutated in either the
CSA or CSB gene (14) show no defect in Chk1 and p53
phosphorylation, whereas XP-A fibroblasts (15) are defective in
activating the checkpoint after UV irradiation, compared with
normal and CS fibroblasts (Fig. 1). In XP-A G2?M-synchronized
fibroblasts, the signaling is also drastically compromised (Fig. 2).
In S-Phase Cells, Checkpoint Activation Does Not Depend on NER.
Asynchronously growing XP-A fibroblasts can activate the
checkpoint, but co-staining with either anti-phospho-Chk1 or
p53 and anti-BrdU antibodies revealed that phosphorylation of
Chk1 and p53 is restricted to S-phase cells (Fig. 3). We noticed
that in normal and CS-B proliferating fibroblasts, BrdU-positive
cells (i.e., cells in S phase) show a stronger staining with
anti-phospho-Chk1 or p53 antibodies than BrdU-negative cells
(Fig. 3 and data not shown). The data suggest that, after UV
irradiation, in normal fibroblasts the DNA damage checkpoint
can be activated in all cell-cycle phases; however, the signal is
higher in S phase, when probably other kind of lesions, such as
replication forks stalling in front of UV radiation-induced
photoproducts or strand breaks, may contribute to enhance the
signal activating the checkpoint cascade (16). Thus, the analysis
p-chk1 DAPIDAPI p-p53
20 J?m2UV-C radiation. Conversely, XP-A (XP20PV) primary fibroblasts are
impaired in Chk1 and p53 phosphorylation in response to DNA damage. Cells
were held under low serum conditions for 5 days before mock or UV irradia-
for each cell strain analyzed (data not shown). The activation of the DNA
damage checkpoint was examined 1 h after UV irradiation, by immunofluo-
rescence by using phosphospecific antibodies, anti-P-Ser-317-Chk1 and anti-
P-Ser-15-p53, as described in Materials and Methods.
XPA-dependent and CSA?CSB-independent checkpoint activation in
www.pnas.org?cgi?doi?10.1073?pnas.0605446103 Marini et al.
at the single-cell level enabled us to show that mutations in XPA
do interfere with UV-induced checkpoint activation in G1and
G2?M phases, but not in S phase, of the cell cycle.
Checkpoint Activation Does Not Require Functional TC-NER but De-
pends on GG-NER. We next analyzed a large collection of non-
proliferating primary fibroblasts from XP, CS, TTD, and UV
light-sensitive syndrome patients to understand whether DNA
damage recognition by repair proteins is sufficient for check-
point activation or further processing of the primary lesion is
required and what is the relative contribution of the two NER
subpathways, TC-NER and GG-NER. Human fibroblasts were
were stained with antibodies anti-phospho-Chk1 and anti-
phospho-p53. The nuclear fluorescence intensity of captured
images was quantified, and the percentage of positively stained
cells whose fluorescence intensity was above a fixed threshold
was scored (Fig. 4).
CS-A and CS-B primary fibroblasts are proficient in check-
point activation, as well as fibroblasts derived from two UV
light-sensitive syndrome patients (Fig. 4A) who exhibit a TC-
NER defect but have a proficient GG-NER, like CS cells (5, 17).
These findings demonstrate that TC-NER is not necessary for
triggering the DNA damage cascade, after UV radiation.
Conversely, a total or partial inability to activate the check-
point after UV irradiation was observed in XP-C and XP-E
primary fibroblasts (Fig. 4A) that are defective, respectively, in
the XPC-RAD23B and in the UV-DDB complex, two DNA
damage-binding complexes specifically involved in GG-NER in
mammals. Although in XP-C cells GG-NER is totally defective,
in XP-E only the removal of the most abundant UV radiation-
induced photoproduct (the cyclobutane pyrimidine dimer) from
role of XPC and XPE in GG-NER may explain why the defect
in checkpoint activation is more severe in XP-C than in XP-E
fibroblasts. We showed that a yeast strain exclusively defective in
GG-NER (rad7?) is proficient in checkpoint activation after UV
radiation (6). Rad7 and Rad16 functions in Saccharomyces
cerevisiae have many parallels to UV-DDB functions in mam-
malian cells, even though the two complexes do not show any
structural similarity. Both the DDB subunits and Rad7–16 are
components of E3 ubiquitin ligase complexes, whose candidate
substrate is XPC?Rad4 (4). Mutations in XPE in mammalian
p-chk1DAPI DAPI p-p53
p-chk1 DAPI DAPIp-p53
Normal (C3PV) and XP-A (XP20PV) primary fibroblasts were synchronized in
G2?M, with nocodazole, before mock or UV irradiation (20 J?m2), and immu-
nofluorescence was performed as described. More than 90% of the cells were
in G2?M phases, as determined by flow cytometry analysis (data not shown).
XPA-dependent checkpoint activation in G2?M primary fibroblasts.
p-chk1 DAPI DAPIp-p53
CS-B (CS1PV) fibroblasts were pulsed-labeled for 1 h with 50 ?M BrdU before mock or UV irradiation (20 J?m2). Cells were fixed 1 h later and coimmunostained
with anti-P-Ser-317-Chk1 or anti-P-Ser-15-p53 and anti-BrdU antibodies.
Marini et al. PNAS ?
November 14, 2006 ?
vol. 103 ?
no. 46 ?
cells and in RAD7 in yeast cause similar cellular phenotype;
nevertheless, whereas rad7? cells are proficient in triggering
the checkpoint cascade, XP-E fibroblasts cannot fully activate
the checkpoint after UV radiation. A possible explanation of the
difference found between yeast and human cells in the involve-
ment of GG-NER in checkpoint activation might be related to
the different percentage of transcribed genes relative to the
whole genome in the two species. Because a small percentage of
mammalian genome is transcribed, given a certain amount of
lesions per base pair, it is expected that GG-NER has to deal
with many more lesions in the whole genome in human cells
compared with yeast cells.
Open Complex Formation and Processing of the Lesion Are Necessary
for Checkpoint Activation. Nonproliferating fibroblasts defective
in either XPF or XPG nucleases (22, 23) are unable to phos-
phorylate Chk1 and p53 after UV irradiation (Fig. 4A). The data
are in agreement with the observations we made in yeast, where
checkpoint triggering requires the ability to form an opened
incision complex in G1and G2blocked cells (6).
Mutations in the TFIIH complex, involved in both initiation
of basal transcription and opening of a bubble around the lesion
in NER, can lead to quite different XP, TTD, or combined
TFIIH is made up of a total of 10 subunits, three of which were
found defective in NER syndromes: the helicases XPD and XPB
and TTDA, an 8-kDa protein involved in the stabilization of the
complex. XPB is essential for TFIIH activity in both transcrip-
tion and repair, and this probably accounts for the rarity of XP-B
patients that show either XP?CS or TTD phenotype. XPD
basal transcription. Thus the XPD gene is rather tolerant of
mutations, and XP-D defects have been found in many patients
with TTD or with XP and in rare XP?CS patients (24). The XPD
gene mutation spectrum in patients is consistent with the
hypothesis that the site of the mutation determines the clinical
phenotype (25–27). Mutations in p8?TTDA were found in three
families with TTD-affected members (28).
We analyzed checkpoint activation after UV irradiation in 10
primary fibroblasts carrying mutations in XPD, two in XPB, and
two in TTDA (Fig. 4B; refs. 26–30). With the exception of XP-D
cell strains from patients with the combined XP?CS phenotype
(XP8BR, XPCS2, and XP1NE), all fibroblasts are totally or
partially defective in Chk1 and p53 phosphorylation, 1 h after
UV irradiation (between 10% and 30% positively stained XPD,
XPB, and TTDA mutated cells were scored, compared with
80–90% positive normal cells, by immunofluorescence with
phosphospecific antibodies anti-P-Chk1-Ser-317 and anti-P-p53-
Ser-15). The data suggest that TFIIH opening of the DNA
bubble around the lesion is required to properly activate the
checkpoint in nonproliferating cells after UV radiation. It is
interesting to notice that such a defect in checkpoint activation
can be found both in fibroblasts from patients with XP features
(e.g., XP16PV and XP17PV) and in fibroblasts from patients
with TTD features (e.g., TTD11PV and TTD20PV). TTD-A
cells also show a defect in checkpoint activation (Fig. 4B;
TTD14PV and TTD1BR), in agreement with the recently
demonstrated specific requirement of p8?TTD-A in NER,
where it stimulates the opening of the DNA around the damage
and XPA recruitment (31). TFIIH deficiency can thus lead to a
similar defect in checkpoint activation in both XP and TTD
patients, despite their extremely different clinical features and
% positive cells
XP-E (XP23PV) XP-E (XP25PV)
XP-E (XP23PV)XP-E (XP25PV)
% positive cells
XP-D XP-B TTD-A
nonproliferating primary human fibroblasts derived from 27 patients (Table 1), affected by inherited NER-defective syndromes, by immunofluorescence using
phosphospecific antibodies, recognizing Chk1 and p53 (anti-P-Chk1-Ser-317 and anti-P-p53-Ser-15). Approximately 300 fibroblasts per cell strain were scored,
experiments. (C) Checkpoint activation was analyzed in nonproliferating primary fibroblasts from a normal donor (C3PV) and four NER-defective patients 1 h
after UV radiation (20 J?m2) by immunoblot using the indicated antibodies. In extracts from normal, CS-B (CS1PV), and XP-D?CS (XP8BR) fibroblasts,
P-Ser-317-Chk1 and P-Ser-15-p53 strongly increased after DNA damage. XP-A (XP20PV) and XP-D (TTD8PV) fibroblasts are impaired in checkpoint activation.
Processing of the lesion by GG-NER is required to trigger the signal cascade in response to UV. (A and B) We analyzed checkpoint activation in
www.pnas.org?cgi?doi?10.1073?pnas.0605446103 Marini et al.
incidence of skin cancer. These findings support the hypothesis
that the transcriptional defect present in TTD may somehow
prevent in these patients the full development of a precarcino-
genic lesion into a proper cancer (32).
Triggering of the checkpoint cascade in resting fibroblasts
from a normal donor and from four patients representative of
NER-defective syndromes, XP, CS, TTD, and XP?CS, was also
analyzed by immunoblot by using phosphospecific antibodies,
anti-P-Chk1-Ser-317 and anti-P-p53-Ser-15 (Fig. 4C). In agree-
ment with the immunofluorescence data, whereas CS-B resting
fibroblasts are proficient in checkpoint activation, mutations in
XPA and XPD genes leading to XP and TTD clinical features,
respectively, severely impair phosphorylation of checkpoint fac-
tors. The total amount of p53 protein does not increase after
DNA damage, possibly because we analyzed triggering of the
checkpoint cascade 1 h after UV irradiation, whereas others
reported an increase in p53 only at later time points after UV
radiation (15). A low level of p53 phosphorylation could be
observed both in mock and UV-treated XP20PV and TTD8PV
fibroblasts, probably because of unrepaired endogenous DNA
damage that accumulated in normal growing conditions. UV
radiation did not further increase the amount of P-Ser-15-p53 in
these cell strains.
Transcription-Associated Checkpoint Activation in XP-D Fibroblasts
from XP?CS Patients. Contrary to all of the other TFIIH mutant
cells that we analyzed, fibroblasts from patients XP8BR, XPCS2,
and XP1NE, with a combined XP-D?CS phenotype of different
degrees of clinical severity, are able to activate the checkpoint
after UV irradiation (Figs. 4 B and C and 5). It was shown that
in cells from these patients breaks are introduced into cellular
DNA on exposure to UV damage, but these breaks are not at the
sites of the damage, and it has been proposed that they are
introduced erroneously by the NER machinery at sites of
transcription initiation (33). Interestingly, triggering of the
checkpoint cascade that can be observed after UV irradiation in
these cells is associated with transcription. In fact, inhibition of
RNA polymerase II transcription by actinomycin D significantly
reduced Chk1 and p53 phosphorylation after UV irradiation in
XP-D?CS fibroblasts, but it did not affect checkpoint activation
in either normal or XP-A cells (Fig. 5B). We also analyzed
checkpoint activation in extracts from resting XP8BR fibroblasts
by immunoblot (Fig. 4C). Both Chk1-Ser-317 and p53-Ser-15
were already phosphorylated in mock-treated cells. A high
percentage of positive cells were also scored among XP-D?CS
mock-treated fibroblasts (Fig. 4B). We suppose that the check-
point might be partially activated by oxidative damage that
accumulates in normal growth conditions in XP-D?CS fibro-
blasts. In fact, DNA breaks were shown to be also generated by
the introduction of plasmids harboring oxidative damage in
XP-D?CS cells (33). After UV irradiation, a clear increase in the
In summary, in the present work, the analysis of checkpoint
activation after UV irradiation in a large collection of primary
fibroblasts shows that in nonproliferating cells, NER recognition
and processing of the lesion are required to trigger the signal
transduction cascade that leads to the phosphorylation of key
checkpoint proteins, such as Chk1 and p53. Functional GG-NER
is necessary and sufficient to activate the checkpoint response,
whereas TC-NER may be dispensable. In XP-D fibroblasts from
XP?CS patients, despite the fact that UV lesions are not
processed by NER, the checkpoint might be activated by DNA
breaks that occur at sites of transcription initiation.
Materials and Methods
Cell Culture. Primary human fibroblasts from normal and repair-
deficient individuals (Table 1) were cultured in Ham’s F10
medium containing 15% FCS (Euroclone, Milan, Italy) and kept
at 37°C in a humidified atmosphere with 5% CO2. To bring the
cells into a nonproliferating state, cells were held under low
serum conditions for 5 days (0.5% FCS), and the absence of any
S-phase cells was monitored by BrdU incorporation for each cell
strain analyzed (data not shown). Cells were synchronized in
G2?M, adding 0.3 ?M nocodazole (Sigma-Aldrich, St. Louis,
MO) to Ham’s F10 medium containing 15% FCS for 20 h. To
detect cells that were replicating their DNA, 50 ?M BrdU
(Sigma) was added to the medium of exponentially growing
fibroblasts for 1 h before UV radiation.
UV Irradiation. For UV irradiation, medium was removed, and
cells were washed once with PBS and then irradiated with a
Vilber Lourmat (Marne-la-Vale ´e) 12-W lamp (predominantly
254 nm) at a dose of 20 J?m2(dose rate of ?1 J?m2per s).
Subsequently, the medium was added back to the cells and the
cells returned to culture conditions for 1 h.
Transcription Inhibition. Fibroblasts held under low-serum condi-
tions for 5 days were treated with actinomycin D (Sigma-
Aldrich) at a concentration of 0.5 ?g?ml for 2 h before irradi-
ation. The transcription inhibitor was also added during the
postirradiation 1-h incubation period.
Antibodies. Phosphospecific antibodies anti-P-Ser-317-Chk1 and
anti-P-Ser-15-p53, anti-Chk1, and anti ??? tubulin antibodies
% positive cells
damage, and checkpoint activation is associated with transcription. (A) Al-
though in XP-D fibroblasts from the XP patient XP16PV phosphorylation of
Chk1-Ser-317 and p53-Ser-15 cannot be detected 1 h after UV radiation (20
The indicated nonproliferating cell strains were either mock-treated or UV
irradiated, with or without adding actinomycin D (0.5 ?g?ml) to the medium
for 2 h, before irradiation. The percentage of positively staining cells was
measured, and the results represent the mean and SD of three experiments.
XP-D?CS fibroblasts can phosphorylate Chk1 and p53 after UV
Marini et al. PNAS ?
November 14, 2006 ?
vol. 103 ?
no. 46 ?
(rabbit polyclonal) were purchased from Cell Signaling Tech- Download full-text
Active Motif (Carlsbad, CA). Alexa Fluor 555 goat anti-rabbit
secondary antibodies were from Molecular Probes (Eugene,
OR). FITC-anti-BrdU antibodies were from Beckton Dickinson
(Franklin Lakes, NJ).
Immunofluorescence Microscopy. For the detection of phosphory-
lated Chk1-Ser-316 and p53-Ser-15, cells were grown onto sterile
coverslips. After being rinsed in PBS, they were fixed in 2%
paraformaldehyde in PBS for 10 min at room temperature. After
being rinsed twice in PBS, cells were permeabilized in 0.2%
Triton X-100 in PBS for 2 min at 4°C. This was followed by
another rinse in PBS. Cells were then incubated for 1 h at room
Primary antibody (anti-P-Ser-317-Chk1 or anti-P-Ser-15-p53)
dilutions in PBS supplemented with 2% BSA (Sigma-Aldrich)
and followed by extensive washing in PBS. Incubations with
Alexa Fluor 555 goat anti-rabbit secondary antibodies were
performed for 1 h at room temperature at 1:500 dilution in 2%
BSA in PBS. Nuclei were counterstained with 10 ?g?ml DAPI
(Sigma-Aldrich) in PBS for 2 min at room temperature. After
extensive washing in PBS, coverslips were mounted in Vectash-
ield (Vector Laboratories). For detection of incorporated BrdU,
immunofluorescence was performed essentially as described
above, except that after incubation with secondary antibodies,
cells were fixed again with 2% paraformaldehyde in PBS,
washed, treated with 4N HCl 10 min, and incubated 1 h with
FITC-anti-BrdU antibodies 1:40. Images were taken with Olym-
pus (Melville, NY) BX61 microscope analySIS software. Quan-
tification of fluorescence intensity was performed on blind
captured images by using standardized capture settings; image
processing and quantification were performed with the ImageJ
1.36b software (Wayne Rasband, National Institutes of Health,
Bethesda, MD). Cells were considered positive when the fluo-
The error bars represent the standard deviation of the mean. A
minimum of three independent experiments was carried out
where error bars are shown.
Western Blotting. Protein extracts and immunoblots were per-
formed as suggested by Cell Signaling Technology. Briefly, cells
were lysed in 1? SDS sample buffer (62.5 mM Tris?HCl, pH
6.8?2% wt/vol SDS?10% glycerol?50 mM DTT?0.01% wt/vol
bromophenol blue), sonicated 10 sec, and heated to 95°C for 5
min, and equal amounts of total protein extract were loaded onto
10% SDS?PAGE gels. The blots were incubated overnight with
polyclonal antibodies anti-P-Ser-317-Chk1, anti-Chk1, anti-P-
Ser-15-p53, and anti-??? tubulin (Cell Signaling Technology)
and with anti-p53 (Active Motif) at 1:1,000 dilutions.
We thank Pietro Transidico, Imaging Unit, FIRC Institute of Molecular
Oncology Foundation (Milan, Italy) for support in the quantification of the
immunofluorescence images and all of the members of our laboratory for
from Associazione Italiana Ricerca sul Cancro, Consorzio Interuniversita-
rio per le Biotecnologie, Ministero dell’ Universita ` e della Ricerca, Fonda-
zione Cariplo, and the European Union FP6 Integrated Project DNA
repair. The financial support of Telethon-Italy Grant GGP030406 (to
M.M.F.) is gratefully acknowledged.
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