Nucleic Acids Research, 2009, Vol. 37, No. 18 Published online 11 August 2009
Ionizing radiation-dependent and independent
phosphorylation of the 32-kDa subunit of
replication protein A during mitosis
Holger Stephan1, Claire Concannon1, Elisabeth Kremmer2, Michael P. Carty3and
1Cell Cycle Control Laboratory, School of Natural Sciences, National University of Ireland, Galway, Galway, Ireland,
2Helmholtz Zentrum Mu ¨nchen-Deutsches Forschungszentrum fu ¨r Gesundheit und Umwelt (GmbH), Marchioninistr.
25, 81377 Mu ¨nchen, Germany and3DNA Damage Response Laboratory, School of Natural Sciences, National
University of Ireland, Galway, Galway, Ireland
Received November 21, 2008; Revised June 30, 2009; Accepted July 2, 2009
The human single-stranded DNA-binding protein,
replication protein A (RPA), is regulated by the
N-terminal phosphorylation of its 32-kDa subunit,
RPA2. RPA2 is hyperphosphorylated in response
to various DNA-damaging agents and also phos-
during S- and M-phase, primarily at two CDK con-
sensus sites, S23 and S29. Here we generated two
monoclonal phospho-specific antibodies directed
against these CDK sites. These phospho-specific
RPA2-(P)-S23 and RPA2-(P)-S29 antibodies recog-
nized mitotically phosphorylated RPA2 with high
specificity. In addition, the RPA2-(P)-S23 antibody
recognized the S-phase-specific phosphorylation
of RPA2, suggesting that during S-phase only
S23 is phosphorylated, whereas during M-phase
both CDK sites, S23 and S29, are phosphorylated.
Immunofluorescence microscopy revealed that the
mitotic phosphorylation of RPA2 starts at the
onset of mitosis, and dephosphorylation occurs
during late cytokinesis. In mitotic cells treated
with ionizing radiation (IR), we observed a rapid
hyperphosphorylation of RPA2 in addition to its
mitotic phosphorylation at S23 and S29, asso-
ciated with a significant change in the subcellular
localization of RPA. Our data also indicate that the
RPA2 hyperphosphorylation in response to IR is
facilitated by the activity of both ATM and DNA-
PK, and is associated with activation of the Chk2
DNA in cells is challenged by various environmental and
cellular stresses causing DNA lesions. Therefore, mechan-
isms to maintain genome stability are important for cell
viability and survival. DNA damage induces numerous
cellular responses and leads to cell-cycle arrest, DNA
repair or the induction of programmed cell death (1). In
mammalian cells, the phosphatidylinositol 3-kinase-like
kinase (DNA-PK), Ataxia-telangiectasia-mutated protein
(ATM) and Ataxia-telangiectasia and Rad3-related pro-
tein (ATR) play important roles in the DNA damage
checkpoint regulation following DNA damage. They
phosphorylate several key proteins involved in the DNA
damage response such as the tumor suppressor protein
p53, checkpoint kinases Chk1 and Chk2, histone H2AX
and replication protein A (RPA) (1–4).
RPA, the human single-stranded DNA (ssDNA)-
binding protein, is a stable heterotrimer consisting of
three subunits with apparent molecular masses of 70, 32
and 14kDa (RPA1, RPA2 and RPA3, respectively) (5).
RPA is one of the key players in various processes of
DNA metabolism including the initiation and elongation
of DNA replication, homologous recombination (HR),
nucleotide excision repair (NER) and long-patch base
excision repair (BER) (6–8). Studies in yeast and mamma-
lian systems indicate that RPA is also involved in DNA
damage recognition and checkpoint activation (9–11). The
RPA–ssDNA complex generated in response to DNA
lesions is implicated in localization of ATR–ATRIP to
sites of DNA damage and Rad9-Hus1-Rad1 together
with TopBP1 to sites of DNA damage for the activation
of ATR (10,12–15). Following this, the RPA2 subunit
*To whom correspondence should be addressed. Tel: +353 91 49 2430; Fax: +353 91 49 5504; Email: firstname.lastname@example.org
? 2009 The Author(s)
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undergoes hyperphosphorylation in response to DNA-
damaging agents, such as UV- and g-irradiation, DNA-
alkylating agents and replication stress (4,16–18). Various
members of the PIKK family such as DNA-PK, ATM and
ATR have been found to phosphorylate the N-terminal
residues of RPA2 in vitro and putatively in vivo (17–21).
A number of different possible phosphorylation sites in
the N-terminus of RPA2 were identified using mass spec-
trometry analysis and 2D phosphopeptide mapping,
which revealed four phosphorylation sites (S4, S8, T21
and S33) and a fifth site at either S11, S12 or S13
(18,19,22). It has been demonstrated in vivo that an
RPA2 mutant that mimics the hyperphosphorylation at
the N-terminus of RPA2 is unable to localize to the rep-
lication centers in cells, but is capable of association with
DNA damage foci (23,24). This is consistent with the find-
ing that RPA2 hyperphosphorylation after DNA damage
disrupts RPA interaction with DNA polymerase a in vitro
(25). Previous reports suggested that in response to DNA
damage, hyperphosphorylation of RPA2 disrupts its asso-
ciation with replication centres during S-phase and
contributes to the inhibition of DNA replication (23,24).
RPA2 is also phosphorylated in a cell-cycle-dependent
manner during S- and M-phase primarily at two CDK
consensus sites, S23 and S29, by Cdk1-cyclin B or
Cdk2-cyclin A (26–29). Replacement of the CDK consen-
sus sites S23 and S29 by alanine abolishes RPA2 phos-
phorylation during S-phase (17,28). Although RPA is
phosphorylated during initiation of DNA replication
(30), N-terminal deletion (residues 2–30), or alanine sub-
stitutions at S23 and S29 of RPA2 had no significant effect
on the ability of RPA to bind ssDNA or to support SV40
DNA replication in vitro (31,32). In contrast, recent
findings suggested that phosphorylated RPA has a signifi-
cantly decreased ability to bind and destabilize duplex
DNA compared to the unphosphorylated form of RPA
(22,29). Additional data showed further that interactions
of the N termini of RPA1 and RPA2 are probably impor-
tant to prevent interference of the phosphorylated RPA2
with the functions of the core DNA-binding domain of
RPA (33). Moreover, RPA purified from mitotic cells
showed a reduced binding to ATM, DNA polymerase a,
and DNA-PK as compared to unphosphorylated recom-
binant RPA (29).
Although the response of RPA to various DNA damag-
ing agents has been investigated for more than a decade,
our knowledge relates to cells in interphase and far less
is known about the DNA damage response of RPA in
mitosis. To investigate the role of RPA2 phosphorylation
in response to DNA damage in mitosis, we have estab-
lished two novel monoclonal phospho-specific antibodies,
RPA2-(P)-S23 and RPA2-(P)-S29, and examined the
localization of RPA throughout mitosis. Here, we
demonstrate that when DNA damage occurs in mitosis,
mitotically phosphorylated RPA2 is additionally hyper-
phosphorylated and re-localizes to damaged chromosomal
DNA. In addition, our results show that ATM and DNA-
PK are required for RPA2 hyperphosphorylation in mito-
sis and that this is associated with an activation of the
Chk2-pathway. On the basis of these observations, we
propose that hyperphosphorylation of RPA2 may play a
role in DNA repair during mitosis.
MATERIALS AND METHODS
Cell culture and cell lines
Cells were maintained at 378C in a humidified atmosphere
containing 5% CO2. Human HeLa S3 cells were grown in
Dulbecco’s minimal essential medium (DMEM; Lonza
Ltd). EBV-transformed lymphoblastoid cells from A-T
(GM O1525) and Seckel syndrome (GM 18367) patients
and control cells (GMO7521), abbreviated as LC, were
obtained from ATCC and cultured in RPMI-1640
medium (Lonza Ltd). DMEM and RPMI-1640 medium
were supplemented with 10% fetal bovine serum (FBS;
(Sigma-Aldrich; 100IU/ml and 100mg/ml, respectively).
Expression vectors and transfection
The full-length ATR cDNA (pcDNA3-ATR) vector and
the full-length ATM cDNA expression vector (pMEP4)
under the control of heavy metal-inducible metallothio-
nein promoter were kind gifts from Drs P. A. Jeggo and
M. F. Lavin, respectively, and were described previously
(34–37). ATM-deficient cells stably expressing full-length
ATM were generated as described previously (37). To
obtain Seckel cells stably expressing full-length ATR,
2?106exponentially growing Seckel cells were transfected
using FuGene-HD (Roche) with 6mg of pcDNA3-ATR
DNA according to the manufacturer’s instructions. Then
cells were cultivated over 3–4 weeks in media containing
G-418 (Sigma-Aldrich; 600mg/ml) starting 48h after
transfection and monitored by immunoblot. Stable
clones were maintained in RPMI-1640 medium supple-
mented with G-418 (400mg/ml).
Synchronization of cells
To obtain mitotically arrested cells, exponentially growing
HeLa S3 cells were treated with nocodazole (100ng/ml
final concentration; Sigma-Aldrich) for 16h (38). Mitotic
cells were separated from interphase cells by shaking the
loosely attached cells mitotic cells off the dish (‘shake-off’)
and collected by centrifugation at 160?g for 5min. To
release mitotic cells from nocodazole block, cells were
washed once with pre-warmed phosphate-buffered saline
(PBS) for 2min and twice with pre-warmed serum-free
medium for 2min and then plated into serum-containing
medium. To obtain a cell population enriched in S-phase,
HeLa S3 cells were synchronized by a double thymidine-
block as previously described by Bauerschmidt et al. (39).
To achieve highly enriched mitotic populations of
lymphoblastoid cells including stably transfected cell
lines two consecutive cell-cycle blocks were performed.
A thymidine treatment for 19h with 2mM thymidine
was followed by a release for 3h in thymidine-free
medium. Then the cells were incubated with nocodazole
(100ng/ml) for 12h.
Nucleic Acids Research, 2009,Vol.37, No. 18 6029
Cells were treated with 10 Gray (Gy) of ionizing radiation
(IR) (dose rate of 2.75Gy/min) in the presence of serum-
containing medium, using a137Cesium source (Mainance
Engineering Ltd, UK) at room temperature. PIKK
inhibitor wortmannin (Sigma-Aldrich), ATM-inhibitor
provided by KuDOS Pharmaceuticals Ltd, Cambridge,
UK) were dissolved as stock solution of 10mg/ml in
DMSO. Where indicated, mitotically arrested HeLa S3
cells were pretreated, 1h prior to IR, with wortmannin,
KU-55933, NU7441 or with DMSO as solvent control to
analyze RPA2 phosphorylation. NU7441 was derived
from NU7026 and has been shown to be a potent radio-
sensitiser by specifically inhibiting DNA-PK (40–42) while
KU-55933 is a specific and very potent small molecule
inhibitor of ATM (40,43). Bleomycin, a DNA-damaging
agent, and Roscovitine, a CDK inhibitor (44), (both
Calbiochem) were solubilized in DMSO and used in the
Cells were harvested by trypsination or mitotic shake off,
washed in ice-cold PBS, and fixed in 70% ethanol
at ?208C. The fixed cells were then collected by centrifu-
gation at 300?g for 5min, washed once in PBS and resus-
pended in 1ml propidium iodide/RNase staining solution
(BD Pharmingen) followed by incubation for 30min at
room temperature in darkness. The cell-cycle stage was
determined by flow cytometry using a FACS Calibur
(BD Pharmingen). Data were analyzed using Cell
QuestTMSoftware (BD Pharmingen).
Purification of RPA and Cdk1-cyclin B kinase
Recombinant human RPA heterotrimer was expressed
and purified from Escherichia coli BL21 (DE3) cells trans-
formed with p11d-tRPA vector (kindly provided by
Dr Marc Wold)as described
Cdk1-cyclin B kinase was expressed in insect cells and
purified as described previously (47).
In vitro kinase assays
The in vitro kinase reactions were carried out as previously
described (48,49) using 100ng of purified RPA as a sub-
strate and 2mg purified Cdk1-cyclin B.
Kinase assays with immunoprecipitated kinase were
carried out as described (38). The Cdk1 were immunopre-
(Calbiochem) using 1000mg of total protein in 1ml of
HeLa S3 cell extracts, 4mg of anti-Cdk1 antibody
([C-19], Santa Cruz Biotechnology, Inc.) and 1mg purified
recombinant histone H1 (kindly provided by Dr Andrew
Flaus) per reaction.
To analyze the mitotic phosphorylation of RPA2, rat
monoclonal phospho-specific anti-RPA2-(P)-S23 [clone
antibodies were raised against two synthetic RPA2
phosphopeptides containing phosphor-S23 or phosphor-
S29, respectively. The anti-RPA1 [RAC-4D9] antibody
was previously described (50,51). In addition, rat mono-
clonal anti-RPA2 [clone RBF-4E4] and anti-RPA3 [clone
RCF-7H5] antibodies were raised against the respective
recombinant full-length proteins. Hybridoma cell lines
according to standard procedures (52).
Cells were washed once in ice-cold PBS and lysed in lysis
buffer [PBS containing 1% Triton X-100, 0.5% sodium
deoxycholate (DOC), 0.1% sodium dodecyl sulphate
(SDS)] supplemented with 10mM NaF, 1mM Na3VO4,
10mM b-glycerophosphate, 1? Phosphatase Inhibitor
Cocktail I and 1? Protease Inhibitor Cock-tail (both
Sigma-Aldrich). Whole cell lysates were then clarified by
centrifugation at 14000?g for 10min at 48C. Equal
amounts of cell lysates (20mg) were separated on 12%
mide), transferred to PVDF membranes, and analyzed
by antibodies as described earlier (53). As indicated
membranes were probed with anti-RPA2 (1/4000 [9H8],
(1/4000, Bethyl Laboratories), phospho-specific anti-
Inc.), anti-Chk1 (1/1500, Cell Signaling Technology,
Inc.), phospho-specific anti-Chk2-(P)-T68 (1/1500, Cell
Signaling Technology, Inc.),
[2CHK01], Neomarkers), phospho-specific anti-H3-(P)-
S10 (1/2500, Sigma-Aldrich), anti-ATR (1/2000 [N-19],
Santa Cruz Biotechnology, Inc.), anti-Cdk1 (1/1500
[C-19], Santa Cruz Biotechnology, Inc.), anti-ATM
(1/1000 [ab2631], Abcam), phospho-specific anti-ATM-
BubR1-(S)-676, [1/1000, kindly provided by Drs S.
Elowe and E. Nigg (54)], or anti-GAPDH (1/5000
[mAbcam 9484], Abcam) overnight at 48C. Western
blots were then probed with horseradish peroxidase-
Immuno Research) and visualized using the ECL or
ECL Plus chemoluminescent solution (GE Healthcare).
The subcellular fractionation protocol was adapted from
(55). Briefly, 1?107mitotically arrested HeLa S3 cells
were washed with PBS, collected at 160?g, and resus-
pended in 1ml of pre-chilled hypotonic buffer [20mM
Hepes (pH 7.5), 10mM KCl, 1mM MgCl2, 0.5mM
EDTA supplemented with 1mM dithiothreitol (DTT),
10mM NaF, 1mM Na3VO4, 10mM b-glycerophosphate,
1? Phosphatase Inhibitor Cocktail I and 1? Protease
Inhibitor Cocktail], and incubated on ice for 10min.
All procedures were carried out at 48C. Cells were then
Dounce-homogenized (with loose fitting pestle) gently by
15–20 strokes. The homogenate was centrifuged at
2000?g for 10min. The supernatant was then carefully
removed, clarified by centrifugation at 14000?g for
10min and referred to as ‘soluble fraction’. The pellet of
Nucleic Acids Research, 2009, Vol. 37,No. 18
the first centrifugation was then resuspended in 1ml of
hypotonic buffer supplemented with 0.025% Triton-
X100 and the tube was rotated for 5min at 48C at low
speed. Samples were subsequently centrifuged at 2000?g
for 5min at 48C and the supernatant referred to as ‘wash
fraction’. The wash step was repeated once again, but after
centrifugation this supernatant was discarded. Finally, the
remaining pellet was resuspended in 1ml homogenization
buffer [20mM HEPES (pH 7.5), 1% SDS 150mM NaCl,
1mM MgCl2, 0.5mM EDTA supplemented with 1mM
DTT, 10mM NaF, 1mM Na3VO4, 10mM b-glyceropho-
sphate, 1? Phosphatase Inhibitor Cocktail I and 1?
Protease Inhibitor Cocktail] and completely solubilized
by brief sonication (10s with 50% amplitude; Digital
Sonifier?S-250D, Branson,UK) on ice. This sample was
referred to as the ‘chromosomal-bound fraction’.
Mitotic cells were allowed to attached on poly-L-lysine-
coated glass slides and then washed in PBS and fixed
with 4% para-formaldehyde in PBS for 15min at room
temperature. After permeabilization with 0.2% Triton
X-100 in PBS for 10min at room temperature, non-
specific binding sites were blocked with 10% goat serum
20 (0.02%) solution for 30min at 378C. Primary antibod-
RPA2-Ser23 (RBP-8H3), anti-RPA2-Ser29 (RBP-8C7)]
and monoclonal mouse anti-a-tubulin [1/2000 (B-5-1-2),
Sigma-Aldrich]} were incubated in 0.05% PBS-Tween 20
overnight at 48C. The following day, the cells were washed
and stained for 1h at room temperature with Cy2-
conjugated anti-mouse or Cy3-conjugated anti-rat second-
ary antibodies (1/500, Jackson ImmunoResearch). DNA
was counterstained with ToPro3 (1/500, Molecular
Probes) in mounting buffer [20mM Tris–HCl (pH 8.0),
90% glycerol,200mM 1,4-diazabicyclo(2.2.2)octane
(DABCO)]. Confocal 12-bit images (single stacks in
Z-dimension, stack size 512?512 pixel) were captured
using a Zeiss LSM 510 confocal laser scanning microscope
system equipped with a Zeiss Axiovert 200 microscope
with a Plan-Apochromat 63?/1.4 oil objective and ana-
lyzed with LSM 5 Image Browser software (Carl Zeiss
GmbH; Jena, Germany)
(Pierce) in PBS-Tween
Phosphorylation of RPA2 in mitosis
In human and yeast cells it has been reported that RPA2 is
phosphorylated in a cell-cycle-dependent manner during
S- and M-phase. In human cells, two CDK consensus
sites, S23 and S29, were phosphorylated in a cell-cycle-
dependent manner (26,28,29). To examine phosphoryla-
tion of RPA2 in mitosis, monoclonal phospho-specific
antibodies recognizing either RPA2 phosphorylation
at Ser23 or Ser29 were produced. Their specificity was
verified by immunoblot analysis of asynchronous (AS),
mitotically (M) and S-phase (S) arrested cells and purified
recombinant human RPA2 (R) (Figure 1A) and by
Cdk1-cyclinB in vitro kinase assay(Figure 1B).
The quality of cell extracts was assessed using indicated
cell-cycle markers. As shown in both figures (Figure 1A
and B), phospho-specific RPA2-(P)-S23 [RBP-8H3] and
RPA2-(P)-S29 [RBP-8C7] antibodies recognized a band
(marked as mp). In addition, very little reactivity of
RPA2-(P)-S23 and RPA2-(P)-S29 was observed in asyn-
chronous control cells (Figure 1A, lane AS), whereas none
was detected with purified recombinant human RPA2 (R),
which represented the basal (no mobility shift) isoform
of RPA2 (marked as b in Figure 1A, lane R). The
RPA2-(P)-S23 antibody, but not RPA2-(P)-S29, recog-
nized an RPA2 isoform marked as sp [Figure 1A (lanes
AS and S) and B]. This isoform is characterized by a
small reduction in RPA2 mobility and is mostly present
in S-phase-arrested cells
only a single CDK site is phosphorylated during
S-phase. To exclude cross reactivity of both phospho-
specific RPA2 antibodies and their recognition sites (phos-
phorylated S23 and S29), an in vitro kinase assay was
performed using Cdk1-cyclin B and three different
RPA2 CDK phosphorylation site mutants. As shown in
Supplementary Figure S1, no cross reactivity with the
unphosphorylated RPA2 or between the phosphorylated
sites was detected.
Analysis of in vitro CDK-phosphorylated recombinant
RPA2 and S-phase phosphorylated RPA2 (Figure 1B)
revealed that anti-RPA2-(P)-S23 antibody detected both
phosphorylated forms marked as sp to a similar extent as
anti-RPA2 antibody. However, it is important to note that
the S-phase-phosphorylated RPA2 (sp) comprises only
about 10% of the total RPA2 in the analyzed S-phase-
enriched cell population (Figure 1B). Altogether, these
findings let us suggest that during S-phase only S23 is
phosphorylated whereas during M-phase both CDK
sites, S23 and S29, are phosphorylated.
Since both newly generated phospho-specific RPA2
antibodies were successfully employed in immunoblot,
they were also used to investigate the localization of mito-
tically phosphorylated RPA2 throughout different stages
of M-phase by immunofluorescence microscopy. It has
been previously reported that mitotic phosphorylation
appears at the G2- to M-phase transition (29), but how
mitotically phosphorylated RPA is distributed in compar-
ison to heterotrimeric RPA, and, moreover, when the
dephosphorylation of RPA2 takes place are still under
discussion. As shown in Figure 1C, RPA2 was excluded
from the mitotic chromosomes throughout mitosis (for
comparison see a-tubulin staining in Supplementary
Figure S2). The two other subunits of RPA, RPA1 and
RPA3, were also excluded from the chromosomes
(Supplementary Figure S3A). In addition, using both
phospho-specific RPA2 antibodies, a sharp decrease of
mitotically phosphorylated RPA2 was observed at the
end of cytokinesis when chromosomal DNA de-condenses
(Figure 1C). Moreover, these data reveal that in early
G1-phase RPA2 is dephosphorylated and re-enters the
newly reformed nucleus (Figure 1C). These findings
indicate that all three events, dephosphorylation of
mitotic RPA2, de-condensation of mitotic chromosomes
andre-localization of RPA,
Nucleic Acids Research, 2009,Vol.37, No. 186031
Figure 1. Characterization of phospho-specific antibodies anti-RPA2-(P)-S23 and anti-RPA2-(P)-S29. (A) Immunoblots showing the reactivity of
phospho-specific anti-RPA2-(P)-S23 and anti-RPA2-(P)-S29 antibodies to RPA2 in asynchronous (AS), mitotically (M), and S-phase (S) arrested
cells. In addition, 100ng of purified human recombinant RPA (R) and the reactivities of anti-RPA2 (total, RBF-4E4) antibody served as controls.
Detection of phospho-specific H3-(P)-S10, cyclin B1 and cyclin A by the appropriate antibodies were used as cell-cycle markers. Detection of GAPDH
in different extracts served as a loading control. Abbreviation used in the figure: hp=hyperphosphorylated RPA2, mp=mitotically phosphorylated
RPA2, sp=phosphorylated at a single CDK-site of RPA2, b=basal RPA2 (no mobility shift). (B) Reactivity of phospho-specific anti-RPA2-(P)-S23,
anti-RPA2-(P)-S29, and total anti-RPA2 antibodies to in vitro phosphorylated RPA2 by Cdk1-cyclin B. Totally 100ng of purified, recombinant RPA2
was phosphorylated by 2mg purified Cdk1-cyclin B. Then in vitro phosphorylated RPA2 and cell extracts obtained from cells arrested in M- and
S-phase were analyzed by western blot. The membrane was probed with phospho-specific anti-RPA2-(P)-S29 and horseradish peroxidase coupled
secondary antibody and reactivity was detected with ECL. Then the membrane was stripped with Restore Western Blot Stripping Buffer (Pierce) and
incubated a second time with anti-RPA2-(P)-S23 antibody to detect RPA2 phosphorylation at S23. After stripping the membrane a second time, it was
analyzed with total RPA2 antibody RBF-E4E to detect all forms of RPA2. Detection of Cdk1 and cyclin B with specific antibodies served as controls
for active kinase. Phosphorylated RPA2 bands in S-phase cell extracts of the immunoblot were quantified using Image Gauge software (Raytest,
Germany) yielding five arbitrary units (AU) of RPA2 sp form in comparison to 51 AU of b form with the anti-RPA2 antibody (total, RBF-4E4).
Additionally the antibodies RBF-4E4 and anti-RPA2-(P)-S23 recognized biochemically phosphorylated and in vivo phosphorylated RPA2 (sp forms)
with similar sensitivity (RBF-4E4: 28 AU and 5 AU, anti-RPA2-(P)-S23: 25 AU and 3.5 AU). (C) Immunolocalization of total and mitotically
phosphorylated RPA2at different stages of M-phase. RPA2 was detected using anti-RPA2-(P)-S23, anti-RPA2-(P)-S29 and total anti-RPA2 [RBF-
4E4] primary antibodies and Cy3-labeled secondary antibodies and analyzed by confocal microscopy. DNA was counterstained with ToPro3.
Nucleic Acids Research, 2009, Vol. 37,No. 18
However, the correlation between the RPA2 dephosphor-
ylation and the de-condensation of mitotic chromosomes
needs to be examined further.
Mitotic RPA is hyperphosphorylated and changes
its localization in response to DNA damage
The role of RPA in DNA replication and DNA damage
response during interphase has been investigated for more
than a decade (9,11). However, far less is known about the
response of RPA when the DNA damage occurs in mito-
sis. To investigate the response of RPA after DNA
damage in mitosis, asynchronous or mitotically arrested
HeLa S3 cells were either mock- or IR treated. As shown
in Figure 2, treatment of asynchronous and mitotic cells
with IR leads to RPA2 hyperphosphorylation, detected as
a slower migrating RPA2 isoform (marked as form hp)
and with an anti-RPA2-(P)-S4/8 antibody. Interestingly,
both newly generated anti-RPA2-(P)-S23 and anti-
RPA2-(P)-S29 antibodies recognized RPA2 hyperpho-
sphorylation only in mitotic cells exposed to IR. These
findings indicate that, in addition to its mitotic phospho-
rylation at S23 and S29, RPA2 becomes hyperphospho-
rylated when DNA damage is induced in mitosis. Since
earlier reports showed alterations of RPA localization in
response to DNA damage during interphase, we wanted to
determine whether RPA re-localization takes place in
mitosis. Therefore, we carried out immunofluorescence
microscopyanalysis of mitotic
g-irradiation. In unirradiated mitotic cells, RPA2 was
cells subjected to
excluded from prometaphase chromosomes, as deter-
mined by staining with anti-RPA2-(P)-S23, anti-RPA2-
(P)-S29 and anti-RPA2 antibodies (Figure 3A, mock-
treated panels). In contrast, in cells exposed to IR, mito-
tically phosphorylated RPA2 changes its distribution
and co-localizes with chromosomes (Figure 3A, IR
treated panels), which was observed within 20min post-
irradiation (data not shown).
To provide additional evidence for these findings, we
performed subcellular fractionations of mitotic cells that
were either mock-treated or exposed to IR. The purity
of the soluble and chromatin-bound protein fractions
was verifiedby immunoblot
and anti-H3-(P)-S10 antibodies, respectively. Chromatin-
bound proteins were solubilized by sonication (Figure 3B),
or alternatively by DNase I treatment (Supplementary
Figure S3B). In the case of mock-treated cells, mitotically
phosphorylated RPA2 was present mainly in the soluble
fraction and with much lower intensity in the wash frac-
tion (Figure 3B and Supplementary Figure S3B). In addi-
tion, trace amounts of ‘basal’ RPA2 was detected in the
chromosomal fraction. In contrast to unirradiated cells,
the distribution of RPA2 between the different fractions
was altered after IR treatment, with hyperphosphorylated
RPA2 being detected predominantly in the chromatin-
bound fraction. In the remaining insoluble fraction after
DNAse I treatment, no RPA subunit was detected either
in mock- or IR treated cells (Supplementary Figure S3B).
These observations further support our findings that
RPA binds to chromosomal DNA in mitotic cells in
response to IR whereas in unirradiated mitotic cells
RPA does not. In addition, we found that the RPA1
and RPA3 subunits were redistributed in a pattern similar
to RPA2 (Figure 3B and Supplementary S3A and S3B),
which suggests that RPA in mitosis is present as a hetero-
trimeric complex. The localization data on all three RPA
subunits contradict previous study (57) but are consistent
with the finding that RPA could be biochemically purified
as a stable heterotrimeric complex from mitotic cells by
others and by us (29,58,59). The results obtained here
indicate that DNA damage in mitotic cells leads to an
association of RPA with chromatin. In addition to its
mitotic phosphorylation, RPA2 is also hyperphosphory-
lated after exposure of cells in M-phase to ionizing
S23 and S29 are not phosphorylated in response to DNA
damage caused by IR in asynchronous cells
Recently published results indicate that CDKs and PIKKs
are involved in RPA2 phosphorylation in response to gen-
otoxic stress (26). To investigate whether IR treated asyn-
chronous cells exhibit a similar response, we analyzed
RPA2 phosphorylation status in mock- or IR treated
cells in the presence or absence of the CDK inhibi-
tor roscovitine. As shown in Figure 3C, no RPA2 phos-
phorylation at S29 was detected in asynchronous cells.
The low S23 phosphorylation signal of the sp migrating
RPA2 form was due to the presence of S-phase cells and
was not perturbed by IR or roscovitine treatment. RPA2
hyperphosphorylation and phosphorylation of ATM,
Figure 2. RPA2 is hyperphosphorylated in response to IR in mitosis.
Immunoblots showing RPA2 hyperphosphorylation in response to IR
as detected by phospho-specific RPA2-(P)-S23 and anti-RPA2-(P)-S29
antibodies. Asynchronous (AS) and mitotically arrested (M) HeL-S3
cells were mock- or IR treated (10Gy) and analyzed 1-h post-
treatment. Total anti-RPA2 and anti-RPA2-(P)-S4/8 antibodies were
employed as control. Recognition of GAPDH served as a loading
control. Abbreviations used in the figure: hp=hyperphosphorylated
RPA2, mp=mitotically phosphorylated RPA2, sp=RPA2 phosphory-
lated at a single CDK site, b=basal RPA2 (no mobility shift).
Nucleic Acids Research, 2009, Vol.37, No. 186033
Figure 3. RPA2 co-localizes with chromosomal DNA in response to IR in mitotic HeLa S3 cells. (A) Images showing changes in the localization
pattern of RPA2 in mitotic HeLa S3 cells, which were mock- or IR treated (10Gy) and fixed 1-h post-irradiation. RPA2 was detected using a total
anti-RPA2 [RBF-4E4], phospho-specific anti-RPA2-(P)-S23 and anti-RPA2-(P)-S29 antibodies. The DNA was counterstained with ToPro-3.
(B) Immunoblot showing subcellular fractionation of mitotic cells mock treated or exposed to IR (10Gy). Subcellular localization of RPA subunits
was detected using RPA antibodies as indicated. Anti-H3-(P)-S10 and anti-GAPDH antibodies were used as controls. Abbreviations used in the
figure: T=whole cell lysates, SF=soluble fraction, WF=wash fraction and CF=chromosomal bound fraction. (C) RPA2 hyperphosphorylation
in response to IR treatment in asynchronous cells. Immunoblot showing RPA2 hyperphosphorylation response of asynchronous HeLa S3 cells in the
presence or absence of CDK inhibitor roscovitine after IR treatment. Asynchronous HeLa S3 cells were preincubated for 30min with 25, 50 and
100mM of roscovitine or DMSO as solvent control, followed by mock- or IR treatment (10Gy) and 1-h incubation in the continued presence or
absence of roscovitine or in the presence of DMSO as solvent control. Cells were harvested and analyzed by immunoblot using an total RPA2,
phosphopecific RPA2-(P)-S4/S8, phosphopecific RPA2-(P)-S23 or phosphopecific RPA2-(P)-S29 antibodies. The activation of the ATM-Chk2 check-
point pathway was monitored using phosphopecific antibodies ATM-(P)-S1981 and Chk2-(P)-T68. The anti-gH2X antibody was employed as marker
for DSBs. Anti-ATM, anti-Chk2 and anti-GAPDH antibody served as loading controls. Abbreviations used in the figure: AS=asynchronous cells,
M=mitotic cells, D=DMSO solvent only, hp=hyperphosphorylated RPA2, mp=mitotically phosphorylated RPA2, b=basal RPA2 (no mobility
shift). To verify the Cdk1 inhibition by roscovitine, Cdk1 was immunoprecipitated, and its kinase activity was measured using a histone H1 kinase
assays. After SDS–PAGE, the gel was stained with Coomassie blue and incorporation of [32P] phosphate into histone H1 was analyzed using a
phosphor imager system (Fuji LA 5000, Fuji Europe, Germany) and quantification with ImageGauge software (Fuji Europe, Germany). The Cdk1
activity of the mock-treated sample in absence of roscovitine was arbitrarily defined as 100% Cdk1 activity. The Coomassie blue stain of the gel and
the immunoblot with anti-Cdk1 antibody demonstrate that equal amount of H1 and Cdk1, respectively, were present in these reactions (asterisk
marks the antibody light chain).
Nucleic Acids Research, 2009, Vol. 37,No. 18
Chk2 and H2AX (at S1981, T68 and S139, respectively)
were seen only in IR treated cells (Figure 3C). Strikingly,
hyperphosphorylation of RPA2 and phosphorylation
of H2AX, but not ATM and Chk2, exhibited elevated
levels with increasing doses of roscovitine after IR treat-
ment. In addition, inhibition of Cdk1 activity by roscov-
itine was monitored using immunoprecipitated Cdk1 as a
kinase source and histone H1 as a substrate (Figure 3C,
bottom). The Cdk1-associated incorporation of [32P]
phosphate into histone H1 was strongly reduced when
cells were treated with increasing doses of roscovitine.
This result is in agreement with previous findings that
roscovitine inhibits CDKs and forms a tight complex,
which can be immunoprecipitated, and that the CDK
inhibition can be measured in a kinase assay in vitro
(60,61). In contrast, IR treatment itself did not display
any noticeable effect on the Cdk1 activity in vitro
(Figure 3C). These findings suggest that CDKs are not
involved in RPA2 hyperphosphorylations in response of
interphase cells to IR, which is in agreement with the
knowledge that CDKs are not activated after IR (62).
DNA damage during mitosis leads to delay in mitotic
progression and checkpoint activation
To further investigate the response of human RPA to
DNA damage in mitosis, mitotically arrested HeLa S3
cells were either mock- or IR treated with 10Gy (see
Supplementary Figure S4 for cell viability) and then
released from nocodazole arrest to allow cells to progress
throughout mitosis (Figure 4A). In the case of mock-
treated cells, dephosphorylation of mitotically phosp-
horylated RPA2 was detected 1-h post-release. By 3-h
post-nocodazole release the majority of RPA2 was
found in the dephosphorylated RPA2 form (form b) con-
sistent with the cells having exited mitosis. Mitotic cells
exposed to IR showed a delay in dephosphorylation
of mitotically phosphorylated RPA2 in comparison to
Figure S5, quantification of the mitotic RPA2 phosphory-
lated form). IR treatment of mitotic cells resulted in the
appearance of RPA2 hyperphosphorylation 1-h post-
release, which decreased over time and was not detected
after 4-h post-release (Figure 4A). Interestingly, a similar
pattern of dephosphorylation was observed for the mito-
tically phosphorylated RPA2, suggesting that both phos-
phorylation signals are abolished when cells exit mitosis.
The latter is in agreement with immunofluorescence
microscopy analyses performed with these antibodies
(Supplementary Figure S2) showing the phosphorylation
of RPA2 at sites S23 and S29. The phosphorylation of
histone H3 at position S10 also declined with similar
kinetics, as determined by western blotting (Figure 4A).
To monitor the activation of the spindle assembly check-
point in mock- and IR treated mitotic cells, anti-BubR1
and anti-BubR1-(P)-S676 antibodies were used. Cells
exposed to IR showed extended checkpoint activation
with BubR1 phosphorylation detected up to 4-h post-
BubR1 phosphorylation was abolished 2-h post-release
To support the hypothesis that the prolonged mitotic
RPA2 phosphorylation was associated with a delay in
mitotic progression caused by DNA damage, we also mon-
itoredcell-cycle progression byflowcytometry (Figure 4B).
FACS analysis revealed that by 3-h post-nocodazole
release the majority of unirradiated cells exited mitosis,
with only ?33% of cells remaining in mitosis (Figure 4B
and C). In contrast, cells subjected to IR were delayed in
mitotic exit, e.g. at 3-h post-release ?52% of cells still
remained in mitosis (Figure 4B and C). These results
revealed that DNA damage generated by IR leads to
delay in mitotic progression.
To directly compare the DNA damaging effect of IR
and bleomycin, mitotically arrested HeLa cells were
exposed to both DSBs agents and than released from the
nocodazole block. Immunoblot analyses revealed that the
extent of RPA2 hyperphosphorylation was very similar
However, the mitotic and S4/S8 phosphorylation were
abolished after 4h in case of IR treated mitotic cells, indi-
cating that cells entered G1-phase, whereas RPA2 from
bleomycin-treated cells still showed a significant mitotic
and S4/S8 phosphorylation at a time point of 8-h post-
treatment and nocodazole release of the cells (Figure 4D).
Since we observed that hyperphosphorylation of RPA2
in response to DNA damage occurs within the first hour
(Figure 4A), we examined RPA2 hyperphosphorylation
in nocodazole-released cells every 15min during the
dazole block were subjected to a similar treatment to inves-
tigate the effect of sustained mitotic arrest on the RPA2
hyperphosphorylation response. As shown in Figure 5A,
RPA2 hyperphosphorylation occurred already in the
first 15min post-irradiation in both arrested and released
cells, which indicates a rapid DNA damage response.
In mammalian cells, DNA damage results in the activa-
tion of DNA damage checkpoints, which are regulated by
checkpoint kinases ATM and ATR, and subsequently
phosphorylate the two signal-transducing kinases Chk1
and Chk2 (1). We assessed the activation of the DNA
damage checkpoint in mitosis by analyzing phosphoryla-
tion of Chk1 and Chk2 with phospho-specific S317 and
T68 antibodies, respectively (Figure 5B). Interestingly,
prometaphase-arrested cells showed only very low levels
of Chk1 activation in response to IR, whereas cells
released from the mitotic block displayed rapid phospho-
rylation of the kinase. In contrast, Chk2 phosphorylation
following IR treatment was observed at similar levels in
both mitotically arrested cells and cells released from
nocodazole arrest. The only difference applied to the
60-min time-point when the level of Chk2 phosphoryla-
tion was noticeably decreased in cells kept under mitotic
arrest in comparison to cells released from nocodazole
arrest (Figure 5B, compare left and right panel).
The results obtained here reveal that IR treated
cells released from a mitotic block show Chk1 and Chk2
activationand RPA2 hyperphosphorylation.
nocodazole-arrested cells exhibit only Chk2 activation
and RPA2 hyperphosphorylation, our findings suggest
that the RPA2 hyperphosphorylation is associated with
Nucleic Acids Research, 2009,Vol.37, No. 186035
ATM and DNA-PK are involved in hyperphosphorylation
of RPA2 in mitosis
ATM and DNA-PK have been implicated in the hyper-
phosphorylation of RPA2 that follows DNA damage
whether these PIKKs also play a role in the hyperpho-
sphorylation of RPA2 in mitosis, we first investigated
the effect of wortmannin, an inhibitor of PIKKs (65), on
RPA2 hyperphosphorylation. HeLa S3 cells arrested in
mitosis were incubated with different doses of wortmannin
or DMSO as solvent control 1h prior to g-irradiation.
It has been shown that wortmannin at a dose of 20mM
efficiently inhibits ATM and DNA-PK, whereas ATR
is only partially affected at this concentration (65). As
shown in Figure 6A, DNA damage-induced RPA2
hyperphosphorylation was observed within 1-h post-
irradiation in IR treated cells but was not seen in mock-
treated cells. Wortmannin doses of 10 and 20mM
induced by IR to a level below detection but did not
have any impact on the mitotic phosphorylation. These
findings indicate that ATM, DNA-PK or both kinases
are involved in RPA2 hyperphosphorylation in mitosis
after IR treatment.
Figure 4. IR treatment of human cells in mitosis leads to delay in mitotic progression and RPA2 hyperphosphorylation. (A) Immunoblot showing
RPA2 phosphorylation patterns in response to IR treatment in mitosis. Mitotic HeLa S3 cells were obtained by nocodazole arrest for 16h followed
by mitotic shake off. Cells were mock- or IR treated (10Gy) and subsequently released from the arrest. Cells were harvested at the indicated time
points. Whole cell lysates were analyzed by immunoblot as indicated. The proteins detected by the phospho-specific anti-H3-(P)-S10 antibody and the
anti-GAPDH antibody served as mitosis marker and loading control, respectively. (B) Flow cytometry profiles showing analysis of the cell-cycle
progression in HeLa S3 cells after release from nocodazole arrest and mock or IR treatment (10Gy). Representative flow cytometry profile of mock-
or IR treated (10Gy) HeLa S3 cells over time following nocodazole release. (C) Diagram showing the quantified average (n=3) of flow cytometry
results present in Figure 4B. Cells in G2/M- and G1-phase are represented as percentage of the total cell population. Results are expressed as
mean?S.D. and differences between mock- and IR treated cell populations were significant according to a Student’s t-test (P?0.01).
(D) Immunoblot showing RPA2 hyperphosphorylation in response to IR or bleomycin treatment in mitosis. Mitotically arrested HeLa S3 cells
were either mock-treated, exposed to IR (10Gy) or incubated for 1h with bleomycin (1mg/ml) and released from the arrest into fresh medium lacking
any agents. Cells were harvested at the indicated time points and whole cell lysates were analyzed by immunoblot as indicated. Abbreviations used in
the figure: hp–hyperphosphorylated RPA2, mp–mitotically phosphorylated RPA2, b–basal RPA2 (no mobility shift), BubR1-P–phosphorylated
BubR1, BubR1–unphosphorylated BubR1.
Nucleic Acids Research, 2009, Vol. 37,No. 18
To further elucidate the role of these kinases, we used
the specific inhibitors for ATM and DNA-PK, KU-55933
and NU7441, respectively (see Supplementary Figure S6
for cell viability after drug treatment). Both drugs were
applied to cells in a manner similar to wortmannin. The
IC50 values for inhibition of ATR kinase activity by
NU7441 or KU-55933 are greater than 100mM, and
ATR activity should not be inhibited under the present
conditions (42,43). As shown in Figure 6B, inhibition of
ATM by KU-55933 and DNA-PK by NU7441 caused a
strong reduction of RPA2 hyperphosphorylation but did
not affect the mitotic phosphorylation. Similar results
were obtained when cells were incubated with caffeine
(data not shown). An amount of 20mM of DNA-PK
and ATM inhibitors did not completely inhibit hyperpho-
sphorylation of RPA2 in IR treated cells whereas the same
doses of wortmannin reduced the hyperphosphorylation
of RPA2 to a level below detection.
To investigate the involvement of the ATR kinase,
Seckel, A-T and control (LC) cells were compared for
their ability to hyperphosphorylate RPA2 in response to
IR in mitotic cells. The expression levels of both PIKKs
were monitored using anti-ATM and anti-ATR antibo-
dies. Examination of RPA2 in mitotic cells after exposure
to IR revealed an impaired hyphosphorylation in A-T cells
whereas Seckel and LC cells showed a robust RPA2
response (Figure 6C). Introduction of exogenic ATM
into A-T cells fully restores their ability to hyperphos-
phorylate RPA2 after IR treatment to the levels similar
to those seen in Seckel and LC cells. Seckel cells stably
expressing transgenic ATR showed no significant increase
in the RPA2 hyperphosphorylation in response to IR.
Following IR treatment, phosphorylation of ATM at
S1981 was observed in Seckel, LC and A-T cells stably
transfected with ATM. Our results suggest that the mitotic
hyperphosphorylation of RPA2 in response to IR is
mediated by ATM and DNA-PK rather than ATR.
The role of the cell-cycle-dependent phosphorylation of
RPA has been in the centre of interest in the fields of
DNA replication, DNA repair and DNA damage signal-
ing for more than a decade (9,11). The establishment of
phospho-specific antibodies has previously provided a
better understanding of DNA damage-dependent RPA
phosphorylation. To enhance the knowledge of the cell-
cycle regulation of RPA, two novel monoclonal RPA2
Figure 5. IR treatment of cells in mitosis yields a different checkpoint response in nocodazole-arrested and cells released from nocodazole block.
Mitotic HeLa S3 cells were mock- or IR treated (10Gy) and released from the mitotic arrest or kept in mitotic arrest. Cells were harvested at
indicated time points. (A) Immunoblot showing RPA2 as detected by total anti-RPA2, phospho-specific anti-RPA2-(P)-S4/8, anti-RPA2-(P)-S23 and
anti-RPA2-(P)-S29 antibodies. (B) Immunoblot showing the checkpoint activation in mitotic cells in response to IR as detected with phospho-specific
Chk1-(P)-S317 and Chk2-(P)-T68 antibodies. Antibodies against total Chk1, Chk2 and anti-GAPDH served as loading controls. Abbreviations used
in the figure: hp=hyperphosphorylated RPA2, mp=mitotically phosphorylated RPA2, b=basal RPA2 (no mobility shift).
Nucleic Acids Research, 2009, Vol.37, No. 186037
antibodies were produced against two characterized CDK
sites, S23 and S29, RBP-8H3 and RBP-8C7, respectively.
Both phospho-specific antibodies recognized mitotically
phosphorylated RPA2, confirming that mitotic phosphor-
ylation of RPA2 includes phosphorylation on S23 and S29
(26,28,29). The RPA2-(P)-S23 antibody also detected
a characteristic RPA2 isoform present during S-phase,
however with lower intensity. This would be consistent
with a smaller fraction of RPA2 being phosphorylated
in S-phase cells compared to mitotic cells (9). In vitro
phosphorylation using Cdk1 and various purified RPA2
phosphorylation site mutants indicated that S23 or S29
are phosphorylatedby this
Figure S1), which supports the findings of other research-
ers (17,19), and that both monoclonal antibodies pre-
sented here effectively discriminate between the two sites.
Our results suggest that S23 is phosphorylated in S- and
M-phase, whereas S29 phosphorylation takes place only
during M-phase. These findings are in agreement with
results of Fang and Newport showing that RPA2 shares
phosphorylation sites in S-phase and mitosis (30). They
also identified an additional site specifically phosphory-
lated in mitosis by Cdk1, but did not determine the
exact site in vivo (30).
Our analysis revealed that both antibodies are very suit-
able for microscopic analyses. The mitotic phosphoryla-
tion of RPA2 lasts throughout mitosis until late stages of
M-phase suggesting a specific dephosphorylation mecha-
nism of RPA2 late in mitosis as supported by the analyses
of mitotic spindles in parallel (for a-tubulin staining see
Supplementary Figure S2). Mitotically phosphorylated
RPA is excluded from chromosomes and the nuclear scaf-
fold and is maintained within the soluble cellular fraction.
This might be necessary to avoid possible interference
of RPA with mitotic processes such as condensation of
chromosomes. It has been shown that mitotically phos-
phorylated RPA binds to dsDNA with lower affinity
than non-phosphorylated RPA (29). Late in cytokinesis
during chromosome de-condensation, and RPA2 dephos-
phorylated at S23 and S29 re-enters the newly formed
nucleus. Our findings and studies of other researchers
have shown that the RPA expression levels do not fluctu-
ate during the cell cycle, especially during mitosis
(28,29,66). These data lead us to hypothesize that, most
likely at the end of cytokinesis, a rapid dephosphorylation
of RPA2 takes place, rather than proteolysis and new
synthesis of RPA2 and its subunits. Since RPA2 is phos-
phorylated during the entire M-phase (see Supplementary
Figure S2 for details), we propose that the phospho-
specific antibodies RPA2-(P)-S23 and RPA2-(P)-S29
described here should be excellent M-phase markers.
The response of RPA to various DNA-damaging events
is well established in interphase cells, but very little is
known about its response to DNA damage that occurs
during M-phase. Using phospho-specific RPA2 antibo-
dies, we observed, in addition to phosphorylation at S23
and S29, a rapid RPA2 hyperphosphorylation response
following IR treatment during mitosis. Moreover, RPA
changes its subcellular localization in response to IR,
from chromatin-excluded to chromatin-associated. In the
Figure 6. Involvement of ATM and DNA-PK in hyperphosphorylation
of RPA2 after IR treatment of mitotic cells. (A) Immunoblot showing
RPA2 hyperphosphorylation response of mitotic HeLa S3 cells in the
presence of PIKK inhibitor wortmannin. Mitotically arrested HeLa S3
cells were incubated for 1h with 5, 10 and 20mM of wortmannin or
DMSO as solvent control prior to mock or IR treatment. At 1-h post-
irradiation cells were harvested and analyzed by immunoblot using
an total RPA2 or phosphopecific RPA2-(P)-S4/S8 antibodies. Anti-
GAPDH antibody served as loading control. (B) Immunoblot showing
the RPA2 hyperphosphorylation response to IR treatment in mitosis in
the presence of specific ATM or DNA-PK inhibitors. Mitotically
arrested HeLa S3 cells were incubated for 1h with 5, 10 and 20mM
of ATM-inhibitor (ATMi) KU-55933, DNA-PK-inhibitor (DNA-PKi)
NU7441 or DMSO as solvent control alone. Following this treatment,
cells were mock- or IR treated (10Gy). At 1h post-irradiation cells
were analyzed by immunoblot using a total RPA2 or phosphopecific
RPA2-(P)-S4/S8 antibodies. Anti-GAPDH antibody served as loading
response of mitotic Seckel, A-T and control (LC) cells after IR treat-
ment. Seckel, A-T and LC cells were enriched in mitosis using two
consecutive cell-cycle arrests (thymidine followed by nocodazole
block), followed by mock or IR treatment (10Gy). Cell extracts were
prepared 1-h post-irradiation. RPA2 was analyzed by immunoblot
using the indicated antibodies. The expression levels of ATM and
ATR were detected with anti-ATM and anti-ATR antibodies. A phos-
pho-specific anti-ATM-(P)-S1981 antibody was employed to monitor
DNA damage-dependent phoshorylation of ATM. Seckel and A-T
cell lines stably transfected with full length ATR (labeled as
‘+ATR’) or ATM (labeled ‘+ATM’) cDNA expression vectors were
established, respectively. An anti-GAPDH antibody was used as load-
ing control. Abbreviations used in the figure: D=DMSO solvent only,
RPA2, b=basal RPA2 (no mobility shift).
Nucleic Acids Research, 2009, Vol. 37,No. 18
case of UV treatment (dose of 5–15J/m2), no RPA2
changes in localization were observed in mitotic cells
(data not shown). These findings indicate that RPA
might be involved in the cellular DNA damage response
and DNA repair processes during mitosis, supporting its
role in DSBs repair pathways found in interphase cells.
Anantha et al. (26) showed that genotoxic stress generated
in or before S-phase by camptothecin and bleomycin
caused RPA2 hyperphosphorylation and phosphorylation
at S29. They suggested that cell-cycle-dependent phos-
phorylation is a requirement for the hyperphosphoryla-
tion in response to DNA damage. In contrast, our
results did not find an involvement of CDKs in the
RPA2 hyperphosphorylation after IR treatment in asyn-
chronous cells. First, the RPA2 hyperphosphorylation
mobility shift observed in asynchronous cells exposed to
IR did not comprise phosphorylation at S23 and S29.
Secondly, the level of RPA2 hyperphosphorylation was
elevated in IR and roscovotine-treated cells further sup-
porting our findings. This is consistent with the knowledge
that the cellular response to DNA damage after IR
includes the degradation of Cdc25A and a lack of activa-
tion of CDKs (62). The apparent contradiction may reflect
different requirements for DNA damage signaling after
the different DNA damaging agents used: IR [this study
and those reviewed in (62)], and camptothecin and bleo-
mycin (26). Contrary to interphase cells, after IR treat-
ment of cells in mitosis, the hyperphosphorylated,
shifted form of RPA2 contained phosphorylated S29
most likely since mitotic RPA2 is already modified by
CDKs. In agreement with our result, S29 phosphorylation
of RPA2 was also observed in bleomycin-treated mitotic
DNA damage occurring during mitosis leads to
checkpoint activation and a delay in mitotic exit (68–70).
We observed an IR-induced delay in dephosphorylation of
mitotic RPA when cells pass through M-phase. This was
due to a prolonged mitotic progression. However, hyper-
phosphorylation and CDK-dependent phosphorylation of
RPA2 was only observed in mitotic cells exposed to IR.
When cells entered the new cell cycle, RPA2 hyperphos-
phorylation, the mitotic shift of RPA2 and phosphoryla-
tion of H3 at position S10 were diminished suggesting
that only cells with repaired DSBs might enter G1. Liu
and Weaver (17) showed that the earliest RPA2 hyperpho-
sphorylation was detected in interphase cells 45min post-
IR treatment with a dose of 50Gy. In contrast, using
lower doses such as 10Gy, RPA2 hyperphosphorylation
was not detected until 2h and peaked at 3–4h after IR
treatment (17,63). Strikingly, we observed IR-induced
hyperphosphorylation of RPA2 already at 15min post-
treatment (starting at 5–10min, data not shown) in both
mitotically arrested cells and cells released from mitotic
block. These results indicate that mitotic cells are able to
induce RPA2 hyperphosphorylation in response to IR
treatment more rapidly than interphase cells. Mitosis is a
very short and vulnerable cell-cycle stage, which may
require a very fast DNA damage response by DNA
repair proteins including RPA. Interestingly, only IR trea-
ted cells released from the mitotic block showed Chk1 and
Chk2 activation, whereas nocodazole-arrested cells exhib-
ited only Chk2 activation. These results suggest that in
order to activate an IR-induced DNA damage response
and subsequently Chk1, cells might have to progress
through mitosis and pass the spindle checkpoint. We
could show the activation of the spindle checkpoint in
progressing mitotic cells treated with IR by monitoring
BubR1 phosphorylation status. In contrast, in response
to IR, Chk2 activation might be triggered independently
from the Chk1 pathway during mitosis. It has been shown
that both downstream checkpoint kinases are differently
regulated in response to DNA damage during the
cell cycle (71,72). Our data suggest that the DNA
damage response of RPA2 is associated with Chk2 acti-
vation in mitotic cells. This is in line with a previous
report, which proposed that mitotic entry after IR is
linked with inactivation and dephosphorylation of Chk1
(73). The results presented here are further supported
by findings of Zachos et al. (74), who examined the mitotic
function of Chk1 using the chicken DT40 system.
They proposed a role for Chk1 in the spindle checkpoint,
specifically in delaying anaphase onset by regulation of
Aurora-B and BubR1. This would suggest that cells
which pass the spindle checkpoint are able to activate
Chk1. Using a Chk1 inhibitor in interphase cells (66),
the inability of Chk1 activity to modulate IR-induced
RPA2 hyperphosphorylation in different cell lines was
observed, which is also in line with our findings.
The results presented here reveal that in mitotic cells,
hyperphosphorylation of RPA2 in response to IR is
mediated through ATM and DNA-PK. In both cases,
the inhibition of either ATM or DNA-PK, but not
ATR, leads to reduction of RPA2 hyperphosphorylation
in M-phase cells after IR treatment. Both members of the
PIKK family are also involved in the cellular response to
DSBs, and have been shown in interphase cells to phos-
phorylate RPA2 in vitro and/or in vivo (17,19,20,63,64).
We propose that IR-induced RPA2 hyperphosphorylation
in mitosis can be mediated by both ATM and DNA-PK
Taken together, our results indicate the involvement
of RPA in a DNA repair process in response to DNA
damage occurring in mitosis. It will be interesting in
future experiments to assess the precise roles of RPA in
response to ionizing radiation in mitotic cells, including a
putative function in promoting DSB repair during mitosis.
Supplementary Data are available at NAR Online.
We wish to thank Drs A. Stephan, C. Morrison and
S. Cruet-Hennequart for the careful reading of this manu-
script and helpful discussions. Furthermore, we thank
Drs P. A. Jeggo, A. Flaus, S. Kozlov, M. F. Lavin,
K. Weisshart, M. Wold, S. Elowe and E. Nigg for provid-
ing reagents and Hemant Kumar (Indian Institute for
Technology, Delhi, India) for assistance and practical
Nucleic Acids Research, 2009,Vol.37, No. 186039
support. We are grateful to Drs M. O’Connor and G.
Smith (KuDOS Pharmaceuticals Ltd, Cambridge, UK)
for generously providing
(NU7441) and ATM inhibitor (KU-55933).
the DNA-PK inhibitor
Irish Research Council for Science Engineering and
Foundation Ireland (SFI). Funding for open access
charge: Science Foundation Ireland.
Conflict of interest statement. None declared.
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