CtIP and MRN promote non-homologous
end-joining of etoposide-induced DNA
double-strand breaks in G1
Verena Quennet1, Andrea Beucher1, Olivia Barton1, Shunichi Takeda2and
Markus Lo ¨brich1,*
1Radiation Biology and DNA Repair, Darmstadt University of Technology, 64287 Darmstadt, Germany and
2Department of Radiation Genetics, Kyoto University, Kyoto, Japan
Received September 15, 2010; Revised November 1, 2010; Accepted November 2, 2010
Topoisomerases class II (topoII) cleave and re-ligate
the DNA double helix to allow the passage of an
intact DNA strand through it. Chemotherapeutic
drugs such as etoposide target topoII, interfere
with the normal enzymatic cleavage/re-ligation
reaction and create a DNA double-strand break
(DSB) with the enzyme covalently bound to the
50-end of the DNA. Such DSBs are repaired by one
of the two major DSB repair pathways, non-
homologous end-joining (NHEJ) or homologous
recombination. However, prior to repair, the cova-
lently bound topoII needs to be removed from the
DNA end, a process requiring the MRX complex and
ctp1 in fission yeast. CtIP, the mammalian ortholog
of ctp1, is known to promote homologous recom-
bination by resecting DSB ends. Here, we show that
human cells arrested in G0/G1 repair etoposide-
induced DSBs by NHEJ and, surprisingly, require
the MRN complex (the ortholog of MRX) and CtIP.
CtIP’s function for repairing etoposide-induced
DSBs by NHEJ in G0/G1 requires the Thr-847 but
not the Ser-327 phosphorylation site, both of
which are needed for resection during HR. This
finding establishes that CtIP promotes NHEJ of
etoposide-induced DSBs during G0/G1 phase with
an end-processing function that is distinct to its
DNA double-strand breaks (DSBs) are highly cytotoxic
lesions, posing a major threat to genomic integrity.
Following DSB induction, cells elicit an orchestrated
DNA damage response which encompasses pathways of
DSB repair, the initiation of cell cycle checkpoints and, in
some cells, the induction of apoptosis (1,2). DSBs can be
repaired by two major pathways, homologous recombin-
ation (HR) and non-homologous end-joining (NHEJ)
(3–5). NHEJ is the predominant repair pathway through-
out the cell cycle and is particularly important in the G1
phase of the cell cycle (6–8). HR, in contrast, is important
for repairing stalled or collapsed replication forks (9,10),
and can also repair two-ended DSBs in S and G2 phase
when the presence of a sister chromatid provides a
template for repair (11).
Mre11 is part of the Mre11-Rad50-Nbs1 (MRN)
complex which is important for HR-mediated DSB
repair and damage signaling (12). The MRN complex,
besides being a target of ATM, is a direct inducer of
ATM kinase activity which is particularly important for
efficient damage signaling (13). Mre11 from human and
yeast possesses nuclease activity and contributes to DSB
end resection to generate single stranded DNA (ssDNA),
the intermediate for HR repair processes (14). The role of
the MRN complex in NHEJ is perhaps less clear (15) but
Mre11 and Nbs1 are required for an end-joining pathway
that repairs a sub-set of ionizing radiation induced DSBs
in G1 (16). This subset represents DSBs localizing to het-
erochromatic DNA regions and also requires ATM (17).
Further, cells synchronized at G0/G1 phase contain
phospho-Nbs1 foci following etoposide treatment, sug-
gesting the involvement of MRN in NHEJ of etoposide-
induced DSBs (18).
CtIP is a critical player in multiple molecular pathways.
It was originally identified as a binding partner of the
transcriptional suppressor CTBP (C-terminal binding
protein) (19) and interacts with the Brca1 BRCT
domains in a manner that is dependent on the phosphor-
ylation of CtIP at serine 327 (20,21). CtIP promotes HR
by initiating DSB end resection and the formation of
ssDNA (22). Mutating the CtIP site threonine 847 to
alanine (T847A) prevents its phosphorylation and results
*To whom correspondence should be addressed. Tel: +49 6151 167460; Fax: +49 6151 167462; Email: email@example.com
Nucleic Acids Research, 2011, Vol. 39, No. 6Published online 17 November 2010
? The Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
in impaired resection (23) but serine 327 phosphorylation
also seems to be required for resection and HR (24). Both
Ser-327 and Thr-847 are CDK1 phosphorylation sites.
Although CtIP promotes HR in S and G2 phase, there
is evidence that it can also function in G1 in a specialized
end-joining (MMEJ) (24). Since MMEJ involves short
regions of sequence homology at the break site, CtIP
may promote MMEJ by initiating (limited) resection
similar to its role in HR.
DNA topoisomerases are responsible for the conversion
of DNA topology via their cleavage/re-ligation equilibrium
(25,26). Topoisomerase II (topoII) is a homo-dimeric
enzyme. Each subunit cleaves one strand of the DNA
double helix creating a transient DSB to allow the passage
of an intact DNA strand through it (27). Chemotherapeutic
drugs such as etoposide target topoII and interfere with the
normal enzyme reaction. Disruption of the cleavage/
re-ligation reaction stabilizes cleavage complexes, intermedi-
ates in the catalytic cycle of the enzyme which can be con-
verted to DSBs with the enzyme covalently bound to the
50-end of the DNA (28,29). Importantly, the covalently
bound enzyme needs to be removed from the DNA end
before repair can ensue, a process requiring the MRX
complex and ctp1 in fission yeast (orthologs of mammalian
MRN and CtIP) (30). Consistent with this requirement,
chicken DT40 cells defective in CtIP are hyper-sensitive
to etoposide treatment (31). However, the repair pathway
Paradoxically, NHEJ seems to play a major role in the re-
sistance to topoII-mediated DNA damage (32–34) raising
the possibility that CtIP and MRN promote the repair of
etoposide-induced DSBs by NHEJ.
Here, we measure the repair of DSBs after etoposide
treatment specifically in G1 phase and show that NHEJ-
deficient cells are unable to repair etoposide-induced
DSBs. Importantly, cells deficient in Mre11 or Nbs1
but not ATM also exhibit a major repair defect.
Furthermore, CtIP depletion leads to a repair defect in
G1 which is epistatic to the Mre11 repair defect and
involves NHEJ. Finally, we show that CtIP’s function in
promoting repair of etoposide-induced DSBs by NHEJ in
G1 requires the Thr-847 but not the Ser-327 phosphoryl-
ation site. Since both CtIP phosphorylation sites are
required for resection during HR, this separates CtIP’s
end-processing from its resection function. Our findings
provide new mechanistic insight into the repair pathways
conferring resistance to the anti-cancer drug etoposide.
MATERIALS AND METHODS
Cells and cell culture
Primary human fibroblasts utilized were HSF1 [wild-type
(wt)], C2886 (wt), AT1BR (ATM deficient), HSC62
(Brca2 deficient) (IVS19-1 G to A) (35), ATLD2 (Mre11
deficient), 180BR (LigIV deficient) (36), CZD82CH and
GM07166A (Nbs1 deficient); immortalized and trans-
formed cell lines utilized were 82-6 hTert (wt) and 2BN
hTert (XLF defective) and HeLa. ATLD2 cells were
grown in Dulbeccos minimal essential medium (DMEM)
supplemented with 20% FCS, 1% non-essential amino
acids (NEAA) and 1% antibiotics (penicillin–strepto-
mycin). AT1BR cells were cultured in HAM’S F10
buffer, supplemented with 15% FCS and 1% antibiotics
and human HeLa cells in DMEM, supplemented with
10% FCS and 1% NEAA. All other cells were cultured
in MEM supplemented with 20% FCS (10% for HSF1),
1% NEAA and with 1% antibiotics (82-6 hTert cells
without antibiotics). All cells were maintained at 37?C in
a 5% CO2incubator.
siRNA transfection of HeLa cells was carried out using
HiPerFect Transfection Reagent (Qiagen) following the
manufacturer’s instructions. Mre11, CtIP and Rad51
siRNAs were used at a final concentration of 20, 50 and
10nM, respectively. Experiments were performed 48h
after transfection (120h for Mre11). The knock-down
analysis or immunoblotting. siRNA sequences were as
follows: Mre11 (ACA GGA GAA GAG ATC AAC T);
CtIP1 (TCC ACA ACA TAA TCC TAA T); CtIP2 (AAG
CTAAAACAGGAACGAATC); Rad51 (AAG GGA
ATT AGT GAA GCC A); control (AAT TCT CCG
AAC GTG TCA CGT).
Random plasmid integration
After 24h incubation with CtIP2 siRNA, HeLa cells were
transfected with Effectene (Qiagen) following the manu-
facturer’s protocol to integrate various GFP-tagged
siRNA-resistant CtIP plasmids. On the following day,
cells were treated with etoposide (Sigma), fixed and
stained for gH2AX foci and GFP. Only GFP-positive
G1 cells were analyzed. 82-6 hTert cells were transfected
by electroporation with siRNA and plasmid in the same
reaction according to the manufacturer’s protocol 48h
prior to etoposide treatment (Amaxa).
Chemical treatment and irradiation
Cells were treated with 20 or 100mM of etoposide (Sigma)
immortalized cells) or half an hour (for HeLa cells).
After incubation, cells were washed with PBS and fresh
medium was added (in case of non-confluent cells with
aphidicolin (Calbiochem) at a concentration of 3mg/ml).
ATM inhibitor (Tocris) and DNA-PK inhibitor (Sigma)
were added at 10mM 1h prior to etoposide treatment,
during etoposide incubation and repair time. Aclarubicin
at 5mM was added immediately before etoposide treat-
ment. X-irradiation at 90kV and 19mA was performed
at a dose rate of 2Gy/min. Dosimetry considered the
increase in dose for cells grown on glass coverslips
relative to plastic surfaces (37).
(for primary andhTert
All cells were grown on glass coverslips for immunofluores-
cence microscopy. HeLa cells and 82-6 hTert cells were fixed
with 2% formaldehyde in PBS for 15min, washed three
times for 10min in PBS, permeabilized in 0.2% Triton
Nucleic Acids Research,2011, Vol.39, No. 6 2145
X-100 in PBS for 10min at 4?C and washed three times for
10min with PBS/1% FCS. All other cells were fixed for
30min with methanol at ?20?C, dipped for 1min in ice
cold acetone for permeabilization and washed three times
for 10min with PBS/1% FCS. Non-specific antigens were
blocked for 30min in 5% BSA (AppliChem) in PBS/1%
FCS. Samples were incubated with primary antibodies in
PBS/1% FCS over night at 4?C, washed three times in
PBS/1% FCS and incubated for 1h at room temperature
with Alexa Fluor 488- or Alexa Fluor 594-conjugated sec-
ondary antibodies (1:500, Invitrogen). After three times of
washing in PBS, cells were DAPI (Sigma) stained and
mounted using Vectashield mounting medium (Vector
Laboratories). All cells were examined using a Zeiss micro-
scope and Metasystems software (Altlussheim, Germany).
Cells were harvested and sonicated three times for 1min in
RIPA lysis buffer (50mM Tris/HCl, pH 8, 150mM NaCl,
0.5% Natriumdesoxycholat, 1% Triton X-100, 0.1% SDS
and fresh added protease inhibitor cocktail 1:25) and
incubated for 30min at 4?C. After centrifugation of the
cell extracts for 30min at 4?C with 15.7g, the protein
concentration was determined and the cell lysates were
boiled with SDS Laemmli loading buffer [4% (w/v)
SDS, 200mM DTT, 120mM Tris/HCl, pH 6.8, 10mM
Bromphenol blue] for 5min at 95?C (target proteins
>200kD at 80?C). Proteins were separated via SDS–
membrane was blocked for 1h in 5% low fat milk in
TBS/0.1% Tween-20 and immunoblotting was carried
out with primary antibody in TBS/0.1% Tween-20/1%
low fat milk over night at 4?C or for 1h at room tempera-
ture, followed by HRP-conjugated secondary antibody in-
cubation in PBS/0.1 % Tween-20/1% low fat milk for 1h.
The immunoblots were developed using ECL (Roche).
Signal detection was carried out with a chemi smart
system (Vilber Lourmat).
Antibodies for immunofluorescence were: mouse mono-
clonal a-gH2AX, 1:1000 (Upstate); rabbit polyclonal
a-gH2AX, 1:2000 (Abcam); mouse monoclonal a-GFP,
1:200 (Roche); rabbit polyclonal a-CENP-F, 1:2000
(Santa Cruz); rabbit polyclonal a-RAD51 (PC130),
1:15000 (Calbiochem). Antibodies for immunoblotting
Cruz); mouse monoclonal a-Mre11, 1:1000 (Abcam);
rabbit polyclonal a-CtIP, 1:1500 (Bethyl Laboratories);
rabbit polyclonal a-RAD51, 1:2000 (Abcam); mouse
monoclonal a-Tubulin, 1:3000 (Santa Cruz).
Repair of etoposide-induced DSBs in G1/G0 involves
NHEJ and Mre11/Nbs1 function
We used confluent primary human fibroblasts to investi-
gate G1/G0 phase cells and scored gH2AX foci as a
marker for DSBs. Etoposide is an established inducer of
DSBs (38). Consistent with this, etoposide-induced foci
formation is abolished in cells treated with specific ATM
and DNA-PK inhibitors, indicating that ATM and
(Figure 1A). Furthermore, pre-treatment with aclarubicin,
an intercalative antibiotic that efficiently inhibits the cata-
lytic activity of topoII (39,40), completely abolishes
etoposide-induced foci formation (Figure 1B). This
establishes that gH2AX foci after etoposide treatment
represent DSBs arising from topoII activity.
Wt cells repair ?90% of the gH2AX foci induced by 20
or 100mM etoposide within 4h post treatment. In contrast,
180 BR cells deficient in the NHEJ factor DNA ligase IV
(LigIV) exhibit a substantial repair defect (Figure 1C;
Supplementary Figure S1), consistent with the hyper-
sensitivity of NHEJ mutant cells to etoposide (32,33).
Further, HSC62 cells deficient in the HR factor Brca2
(35), repair etoposide-induced DSBs similar to wt cells
(Figure 1C). These results establish that etoposide-induced
DSBs in G1/G0 are repaired by NHEJ.
We next investigated the contribution of the MRN
complex to DSB repair after etoposide treatment.
ATLD2 cells defective in Mre11 and two Nbs1 deficient
cell lines show a significant repair defect with unrepaired
DSBs up to 8h post treatment. In contrast, AT1BR cells
defective inATM show
demonstrating that the role of the MRN complex after
etoposide treatment is independent of ATM (Figure 2;
Supplementary Figure S2).
Repair of etoposide-induced DSBs in G1/G0 involves CtIP
To study the role of CtIP in etoposide-induced DSB repair
we treated HeLa cells with CtIP siRNA. Since HeLa cells
do not readily arrest in G0/G1, we utilized cell cycle
markers to distinguish the different cell cycle phases
(11). In short, G2-phase cells show a strong pan-nuclear
CENP-F staining pattern while S-phase cells show weak
and G1-phase cells no CENP-F staining. Aphidicolin is a
specific inhibitor of the replicative DNA polymerases a
and d and was used to prevent S-phase cells from progress-
ing into G2 and G1 during analysis. It causes pronounced
pan-nuclear gH2AX phosphorylation in S-phase cells due
to replication stalling but no damage in G1 and G2 cells
(11,41). G2-phase cells show a very strong punctuate
gH2AX signal after etoposide treatment probably due to
high numbers of etoposide-induced DSBs (Figure 3A).
Thus, G2- and S-phase cells could be clearly identified
and were excluded from analysis.
G1-phase cells depleted for CtIP show a DSB repair
defect after etoposide treatment similar to Mre11-
depleted cells. Importantly, down-regulation of both
factors does not confer a defect greater than inhibition
of each factor alone, suggesting an epistatic relationship
between CtIP and Mre11 for the repair of etoposide-
induced DSBs (Figure 3B). In contrast, down-regulation
of Rad51, a key HR protein (42), does not affect repair
kinetics after etoposide treatment in G1 and depletion of
CtIP does not affect repair of radiation-induced DSBs in
G1 (Supplementary Figure S3A and SB). These data
2146Nucleic Acids Research, 2011,Vol.39, No. 6
suggest that CtIP is involved in etoposide-induced DSB
repair in G1.
To substantiate the notion that CtIP is involved in
etoposide-induced NHEJ, we depleted CtIP in hTert
immortalized human fibroblasts deficient for the NHEJ
factor XLF (2BN hTert cells) (43,44). Repair proficient
hTert cells show a repair defect after siRNA mediated
CtIP depletion similar to CtIP-depleted HeLa cells
(Figure 3C). 2BN hTert cells exhibit a substantial repair
defect similar to that of LigIV-deficient 180BR cells.
Down-regulation of CtIP in 2BN hTert cells does not
further elevate the gH2AX foci level, demonstrating an
(Figure 3C). These data establish that CtIP is involved
in etoposide-induced DSB repair in G1 by NHEJ.
CtIP function during etoposide-induced DSB repair in G1
requires Thr-847 phosphorylation
To gain further mechanistic insight into the role of CtIP in
NHEJ of etoposide-induced DSBs, we analyzed different
CtIP derivatives. We transfected CtIP-depleted HeLa cells
Figure 1. Etoposide-induced DSBs are repaired by NHEJ. (A) HSF1 cells were incubated with a specific ATM and DNA-PK inhibitor 1h prior to
etoposide treatment or irradiation. Foci formation is abolished by combined inhibitor treatment, showing that the kinases ATM and DNA-PK but
not ATR phosphorylate H2AX. (B) gH2AX foci due to etoposide (etopo) treatment require topoII activity. Pre-treatment with aclarubicin (acl.), a
topoII inhibitor, abolishes the formation of etoposide-induced gH2AX foci. Aclarubicin alone does not form gH2AX foci. (C) gH2AX foci kinetics
in primary human fibroblasts. Wt (HSF1 and C2886) and Brca2-deficient cells (HSC62) show similar repair kinetics whereas LigIV-deficient cells (180
BR) exhibit elevated gH2AX foci levels after 20mM etoposide treatment in G0/G1. Background foci numbers were subtracted. Error bars represent
the standard deviation (SD) from at least three different experiments.
Nucleic Acids Research,2011, Vol.39, No. 62147
transiently with different GFP-tagged siRNA resistant
plasmids, each of them carry a certain mutation of CtIP.
The consensus site Thr-847 was mutated to alanine
(T847A) which prevents its phosphorylation and hence
activation. To investigate the effect of CtIP/Brca1
complex formation on the repair of etoposide-induced
DSBs, we expressed a mutated form of CtIP in which
Ser-327 was substituted by alanine (S327A) which also
results in prevention of phosphorylation and the disability
to interact with Brca1. A wt CtIP plasmid and an empty
vector carrying GFP were transfected as positive and
GFP-positive G1-phase cells (Figure 4A) and distin-
guished cell cycle phases on the basis of their DNA
content as described previously (11).
CtIP siRNA treated cells transfected with wt CtIP
repair etoposide-induced DSBs similar to control siRNA
treated cells. CtIP siRNA treated cells transfected with
T847A CtIP show the same repair defect as CtIP-
depleted cells transfected with an empty vector, which
demonstrates the necessity of Thr-847 phoshorylation
for CtIP function in G1. Interestingly, the S327A
mutant form of CtIP shows no repair defect (Figure 4B)
suggesting that CtIP/Brca1 complex formation is dispens-
able for NHEJ of etoposide-induced DSBs in G1. Higher
etoposide concentrations and data obtained with hTert
immortalized human cells substantiate these observations
(Figure 4C and Supplementary Figure S4A). In contrast
to their differential requirement for etoposide-induced
DSB repair in G1, both T847A and S327A mutants are
deficient in Rad51 foci formation after irradiation in G2
(Figure 4D). Thus, CtIP is differentially regulated and
etoposide-induced DSBs by NHEJ in G1 and the repair
of radiation-induced DSBs by HR in G2.
Thr-847, which is important for repair in G1, represents
a CDK1 phosphorylation site but CDK1 activity in G1 is
low (45,46). Therefore, we examined if CDK1 activity is
required for repair of etoposide-induced DSBs in G1 and
analyzed HeLa cells treated with roscovitine, a selective
CDK inhibitor (47). CDK inhibition 3h prior to treat-
ment significantly reduces Rad51 foci formation after
irradiation in G2 but does not affect the repair of
etoposide-induced DSBs in G1, suggesting that Thr-847
phosphorylation and hence CtIP activation is dependent
on other kinases in G1 (Figure 4E). To exclude the possi-
bility that CtIP phosphorylation occurs in G2 and is main-
tained until cells reach G1, we treated cells with
roscovitine 6 and 9h prior to etoposide treatment or
irradiation and obtained the same result (Supplementary
The major finding of our work is that CtIP and the MRN
complex promote NHEJ of etoposide-induced DSBs in
G1. Both CtIP and the MRN complex have important
roles in resecting DSB ends during HR (48,49) and in
the removal of covalently bound topoII from DSB sites
(30,50,51). Cell survival studies suggested that NHEJ is a
major repair pathway for etoposide-induced DSBs;
however, HR also contributes to resistance leaving
unclear how CtIP and the MRN complex interplay with
NHEJ to provide repair of etoposide-induced DSBs (32–
34,52). We have addressed this question by specifically
analyzing G1/G0-phase cells which, we show, repair
etoposide-induced DSBs exclusively by NHEJ with no
contribution of HR. Hence, the uncovered functions of
CtIP and the MRN complex in G1/G0 phase are
distinct to their function in HR. In support of this predic-
tion, CtIP’s roles during removal of topoII from the break
site in G1 and resection of DNA ends during G2 have
distinct phosphorylation requirements. We have used
gH2AX foci analysis to measure DSB repair kinetics
which served in this and several other previous publica-
tions as a highly sensitive, accurate and reliable method
for assessing DSB levels in non-replicating G1/G0-phase
cells (53–56). Although we have previously provided ex-
tensive evidence for a 1:1 relationship between foci
numbers and DSBs [summarized in (57)] we here
confirm that the foci analyzed arise from the enzymatic
property of the topoII enzymes.
Nucleolytic processing by Mre11 is an essential function
of fundamental importance for DNA repair, distinct from
MRN-mediated control of ATM signaling (58). The
nuclease activity is important for DSB end resection
during HR as well as for the removal of topo II from
Similarly, the Saccharomyces cerevisiae Spo11 protein
which initiates meiotic recombination must be removed
before repair can occur, a process performed by the
endonucleolytic activity of the Mre11 subunit of the
MRX complex (49,59). Although the MRN complex is
not a core component of NHEJ (58,60,61), we show
here that it has a clear requirement for the repair of
etoposide-induced breaks by NHEJ which is independent
breaksare repaired in
Figure 2. Etoposide-induced DSB repair by NHEJ involves the MRN
complex. gH2AX foci kinetics were assessed in primary human fibro-
blasts. Mre11-defective (ATLD2) and Nbs1-defective (CZD82CH and
GM07166A) but not ATM-defective primary human fibroblasts
(AT1BR) exhibit elevated foci levels after 20mM etoposide treatment
in G0/G1 phase. Background foci numbers were subtracted. Error bars
represent the SD from at least three different experiments.
2148Nucleic Acids Research, 2011,Vol.39, No. 6
Mre11-deficient cells perhaps reflecting the elimination of
topoII from the break sites via Tdp2 (62).
CtIP controls the initiation of DNA end resection and
was described as an endonuclease that is stimulated by the
MRN complex (48,49). Hartsuiker et al. (30,63) provided
evidence that Ctp1 in S. pombe is responsible for the
removal of 50-linked proteins such as Rec12 (Spo11) and
topoII, confirming functional conservation between Ctp1
and the more distantly related Sae2 protein from
S. cerevisiae (CtIP in humans). Sae2 seems to be particu-
larly important for the initiation of resection at DSBs with
covalently bound proteins since sae2? mutants are defect-
ive in removing Spo11–DNA adducts (48,59). Loss of
CtIP results in a dramatic defect in processing mitotic
DSBs and down-regulation of CtIP decreases HR
frequencies (22,64). Our observed involvement of CtIP
in G1-phase cells is perhaps surprising since CtIP levels
in human cells are highest during S/G2 and low during G1
(65). However, CtIP in chicken cells does function in G1
during MMEJ, a specialized end-joining pathway (24).
Huertas and Jackson (23) showed that the function of
CtIP during HR is activated by CDK-dependent phos-
phorylation on Thr-847. Here, we show that Thr-847
phosphorylationis also needed
etoposide-induced DSBs in G1 by NHEJ. However, in
contrast to its role in G2, Thr-847 phosphorylation in
G1 can occur in the presence of the CDK inhibitor
roscovitine suggesting that phosphorylation on this site
is performed by other kinases in the absence of CDKs.
Ser-327 is another CtIP site which is needed for CtIP
function during HR in G2 but not for MMEJ in G1
(24). However, a more recent paper reported that resection
measured by Rad51 foci formation is independent of
Ser-332 phosphorylation in DT40 cells (Ser-327 in
humans) and that S332A mutants exhibit sensitivity to
etoposide treatment (31). We observed normal repair
phosphorylatable S327A mutant suggesting that repair
of etoposide-induced DSBs in G1 does not involve the
resection function of CtIP in G2. This might also
explain why CDK activity, which is essential for
etoposide-induced DSB repair.
Taken together, our results show that etoposide-
induced DSBs in G1 are repaired by NHEJ with a require-
ment for the MRN complex and CtIP. We further show
that the function of CtIP in this process has a phosphor-
ylation requirement which is distinct to its role in resecting
DSBs during HR. We suggest that the MRN complex and
forthe repair of
in G1inthe non-
is not requiredfor
Figure 3. NHEJ of etoposide-induced DSBs in G1/G0 involves CtIP.
(A) Identification of cell cycle phases in HeLa cells (see text for explan-
ation). (B) gH2AX foci kinetics in siRNA treated HeLa cells analyzed
48h after transfection. Down-regulation of Mre11 alone, CtIP alone or
Mre11 and CtIP in combination results in similarly elevated gH2AX
foci levels after etoposide treatment in G1-phase cells. Background foci
numbers were subtracted. Error bars represent the SD from at least
three different experiments. (C) gH2AX foci kinetics in hTert
immortalized human fibroblasts. CtIP down-regulation in wt cells
(82-6 hTert) results in a modest but significant repair defect.
XLF-deficient cells (2BN hTert) exhibit a substantially higher repair
defect. CtIP depletion in XLF-defective cells has no additional effect.
Efficient CtIP down-regulation was confirmed by the abolishment of
Rad51 foci formation after irradiation (data not shown). Background
foci numbers were subtracted. Error bars represent the SD from at least
three different experiments.
Nucleic Acids Research,2011, Vol.39, No. 62149
Figure 4. CtIP function during repair of etoposide-induced DSBs in G1 requires Thr-847 phosphorylation. (A) HeLa cells were depleted for
endogenous CtIP by siRNA and transfected with various GFP-tagged CtIP plasmids. Only GFP-positive cells in G1 were analyzed. (B) gH2AX
foci kinetics in HeLa cells after 20mM etoposide. Cells transfected with the CtIP mutation T847A but not the mutation S327A exhibit a repair defect.
Background foci numbers were subtracted. Error bars represent the SD from at least three different experiments. (C) gH2AX foci kinetics in HeLa
cells after 100mM etoposide. Background foci numbers were subtracted. Error bars represent the SD from at least two different experiments.
(D) Rad51 foci in CENP-F positive G2-phase HeLa cells after 2Gy X-rays. Cells transfected with the CtIP mutation T847A or the mutation
S327A exhibit a defect in the formation of Rad51 foci. Background foci numbers were subtracted. Error bars represent the SD from at least two
different experiments. (E) gH2AX and Rad51 foci analysis in HeLa cells treated with the CDK inhibitor roscovitine (rosc) for 3h prior to etoposide
treatment or irradiation. CDK inhibition does not affect gH2AX foci levels after etoposide treatment in G1-phase cells but inhibits Rad51 foci
formation after 2Gy X-irradiation in G2-phase cells. Background foci numbers were subtracted. Error bars represent the SD from at least two
2150 Nucleic Acids Research, 2011,Vol.39, No. 6
CtIP remove topoII from the DSB site prior to repair
Supplementary Data are available at NAR Online.
The Deutsche Forschungsgemeinschaft (Lo 677/4-1/2 to
M.L.); Bundesministerium fu ¨ r Bildung und Forschung
(02S8135, 02S8355 and 03NUK001C). Funding for open
access charge: Institutional.
Conflict of interest statement. None declared.
1. Jeggo,P.A. and Lobrich,M. (2007) DNA double-strand breaks:
their cellular and clinical impact? Oncogene, 26, 7717–7719.
2. Lobrich,M. and Jeggo,P.A. (2007) The impact of a negligent G2/
M checkpoint on genomic instability and cancer induction. Nat.
Rev. Cancer, 7, 861–869.
3. van Gent,D.C. and Hoeijmakers,J.H. (2009) DNA double strand
break repair: zooming in on the focus. Cell Cycle, 8, 3813–3815.
4. Mansour,W.Y., Schumacher,S., Rosskopf,R., Rhein,T., Schmidt-
Petersen,F., Gatzemeier,F., Haag,F., Borgmann,K., Willers,H. and
Dahm-Daphi,J. (2008) Hierarchy of nonhomologous end-joining,
single-strand annealing and gene conversion at site-directed DNA
double-strand breaks. Nucleic Acids Res., 36, 4088–4098.
5. Wyman,C. and Kanaar,R. (2006) DNA double-strand break
repair: all’s well that ends well. Annu. Rev. Genet., 40, 363–383.
6. Chen,B.P., Chan,D.W., Kobayashi,J., Burma,S., Asaithamby,A.,
Morotomi-Yano,K., Botvinick,E., Qin,J. and Chen,D.J. (2005)
Cell cycle dependence of DNA-dependent protein kinase
phosphorylation in response to DNA double strand breaks.
J. Biol. Chem., 280, 14709–14715.
7. Weterings,E. and Chen,D.J. (2007) DNA-dependent protein
kinase in nonhomologous end joining: a lock with multiple keys?
J. Cell Biol., 179, 183–186.
8. Rothkamm,K., Kruger,I., Thompson,L.H. and Lobrich,M. (2003)
Pathways of DNA double-strand break repair during the
mammalian cell cycle. Mol. Cell Biol., 23, 5706–5715.
9. Arnaudeau,C., Lundin,C. and Helleday,T. (2001) DNA
double-strand breaks associated with replication forks are
predominantly repaired by homologous recombination involving
an exchange mechanism in mammalian cells. J. Mol. Biol., 307,
10. Helleday,T., Lo,J., van Gent,D.C. and Engelward,B.P. (2007)
DNA double-strand break repair: from mechanistic understanding
to cancer treatment. DNA Repair, 6, 923–935.
11. Beucher,A., Birraux,J., Tchouandong,L., Barton,O., Shibata,A.,
Conrad,S., Goodarzi,A.A., Krempler,A., Jeggo,P.A. and
Lobrich,M. (2009) ATM and Artemis promote homologous
recombination of radiation-induced DNA double-strand breaks in
G2. EMBO J., 28, 3413–3427.
12. Tauchi,H., Kobayashi,J., Morishima,K., van Gent,D.C.,
Shiraishi,T., Verkaik,N.S., vanHeems,D., Ito,E., Nakamura,A.,
Sonoda,E. et al. (2002) Nbs1 is essential for DNA repair by
homologous recombination in higher vertebrate cells. Nature, 420,
13. Uziel,T., Lerenthal,Y., Moyal,L., Andegeko,Y., Mittelman,L. and
Shiloh,Y. (2003) Requirement of the MRN complex for ATM
activation by DNA damage. EMBO J., 22, 5612–5621.
14. Trujillo,K.M. and Sung,P. (2001) DNA structure-specific nuclease
activities in the Saccharomyces cerevisiae Rad50*Mre11 complex.
J. Biol. Chem., 276, 35458–35464.
15. Shrivastav,M., De Haro,L.P. and Nickoloff,J.A. (2008)
Regulation of DNA double-strand break repair pathway choice.
Cell Res., 18, 134–147.
16. Riballo,E., Kuhne,M., Rief,N., Doherty,A., Smith,G.C.,
Recio,M.J., Reis,C., Dahm,K., Fricke,A., Krempler,A. et al.
(2004) A pathway of double-strand break rejoining dependent
upon ATM, Artemis, and proteins locating to gamma-H2AX foci.
Mol. Cell, 16, 715–724.
17. Goodarzi,A.A., Noon,A.T., Deckbar,D., Ziv,Y., Shiloh,Y.,
Lobrich,M. and Jeggo,P.A. (2008) ATM signaling facilitates
repair of DNA double-strand breaks associated with
heterochromatin. Mol. Cell, 31, 167–177.
18. Robison,J.G., Dixon,K. and Bissler,J.J. (2007) Cell cycle-and
proteasome-dependent formation of etoposide-induced replication
protein A (RPA) or Mre11/Rad50/Nbs1 (MRN) complex repair
foci. Cell Cycle, 6, 2399–2407.
19. Schaeper,U., Subramanian,T., Lim,L., Boyd,J.M. and
Chinnadurai,G. (1998) Interaction between a cellular protein that
binds to the C-terminal region of adenovirus E1A (CtBP) and a
novel cellular protein is disrupted by E1A through a conserved
PLDLS motif. J. Biol. Chem., 273, 8549–8552.
20. Chen,L., Nievera,C.J., Lee,A.Y. and Wu,X. (2008) Cell
cycle-dependent complex formation of BRCA1.CtIP.MRN is
important for DNA double-strand break repair. J. Biol. Chem.,
21. Yu,X. and Chen,J. (2004) DNA damage-induced cell cycle
checkpoint control requires CtIP, a phosphorylation-dependent
binding partner of BRCA1 C-terminal domains. Mol. Cell Biol.,
22. Sartori,A.A., Lukas,C., Coates,J., Mistrik,M., Fu,S., Bartek,J.,
Baer,R., Lukas,J. and Jackson,S.P. (2007) Human CtIP promotes
DNA end resection. Nature, 450, 509–514.
23. Huertas,P. and Jackson,S.P. (2009) Human CtIP mediates cell
cycle control of DNA end resection and double strand break
repair. J. Biol. Chem., 284, 9558–9565.
24. Yun,M.H. and Hiom,K. (2009) CtIP-BRCA1 modulates the
choice of DNA double-strand-break repair pathway throughout
the cell cycle. Nature, 459, 460–463.
25. Nitiss,J.L. (1998) Investigating the biological functions of DNA
topoisomerases in eukaryotic cells. Biochim. Biophys. Acta, 1400,
26. Wang,L. and Eastmond,D.A. (2002) Catalytic inhibitors of
topoisomerase II are DNA-damaging agents: induction of
chromosomal damage by merbarone and ICRF-187. Environ.
Mol. Mutagen., 39, 348–356.
27. Champoux,J.J. (2001) DNA topoisomerases: structure, function,
and mechanism. Annu. Rev. Biochem., 70, 369–413.
28. Chen,A.Y. and Liu,L.F. (1994) DNA topoisomerases: essential
enzymes and lethal targets. Annu. Rev. Pharmacol. Toxicol., 34,
29. Chen,G.L., Yang,L., Rowe,T.C., Halligan,B.D., Tewey,K.M. and
Liu,L.F. (1984) Nonintercalative antitumor drugs interfere with
the breakage-reunion reaction of mammalian DNA topoisomerase
II. J. Biol. Chem., 259, 13560–13566.
30. Hartsuiker,E., Neale,M.J. and Carr,A.M. (2009) Distinct
requirements for the Rad32(Mre11) nuclease and Ctp1(CtIP) in
the removal of covalently bound topoisomerase I and II from
DNA. Mol. Cell, 33, 117–123.
31. Nakamura,K., Kogame,T., Oshiumi,H., Shinohara,A.,
Sumitomo,Y., Agama,K., Pommier,Y., Tsutsui,K.M., Tsutsui,K.,
Hartsuiker,E. et al. (2010) Collaborative action of Brca1 and
CtIP in elimination of covalent modifications from double-strand
breaks to facilitate subsequent break repair. PLoS Genet., 6,
32. Adachi,N., Suzuki,H., Iiizumi,S. and Koyama,H. (2003)
Hypersensitivity of nonhomologous DNA end-joining mutants to
VP-16 and ICRF-193: implications for the repair of
topoisomerase II-mediated DNA damage. J. Biol. Chem., 278,
33. Adachi,N., Iiizumi,S., So,S. and Koyama,H. (2004) Genetic
evidence for involvement of two distinct nonhomologous
end-joining pathways in repair of topoisomerase II-mediated
DNA damage. Biochem. Biophys. Res. Commun., 318, 856–861.
34. Malik,M., Nitiss,K.C., Enriquez-Rios,V. and Nitiss,J.L. (2006)
Roles of nonhomologous end-joining pathways in surviving
topoisomerase II-mediated DNA damage. Mol. Cancer Ther., 5,
Nucleic Acids Research,2011, Vol.39, No. 6 2151
35. Howlett,N.G., Taniguchi,T., Olson,S., Cox,B., Waisfisz,Q.,
Die-Smulders,C., Persky,N., Grompe,M., Joenje,H., Pals,G. et al.
(2002) Biallelic inactivation of BRCA2 in Fanconi anemia.
Science, 297, 606–609.
36. Riballo,E., Critchlow,S.E., Teo,S.H., Doherty,A.J., Priestley,A.,
Broughton,B., Kysela,B., Beamish,H., Plowman,N., Arlett,C.F.
et al. (1999) Identification of a defect in DNA ligase IV in a
radiosensitive leukaemia patient. Curr. Biol., 9, 699–702.
37. Kegel,P., Riballo,E., Kuhne,M., Jeggo,P.A. and Lobrich,M.
(2007) X-irradiation of cells on glass slides has a dose doubling
impact. DNA Repair, 6, 1692–1697.
38. Caldecott,K., Banks,G. and Jeggo,P. (1990) DNA double-strand
break repair pathways and cellular tolerance to inhibitors of
topoisomerase II. Cancer Res., 50, 5778–5783.
39. Jensen,P.B., Sorensen,B.S., Demant,E.J., Sehested,M., Jensen,P.S.,
Vindelov,L. and Hansen,H.H. (1990) Antagonistic effect
of aclarubicin on the cytotoxicity of etoposide and
40-(9-acridinylamino)methanesulfon-m-anisidide in human small
cell lung cancer cell lines and on topoisomerase II-mediated DNA
cleavage. Cancer Res., 50, 3311–3316.
40. Petersen,L.N., Jensen,P.B., Sorensen,B.S., Engelholm,S.A. and
Spang-Thomsen,M. (1994) Postincubation with aclarubicin
reverses topoisomerase II mediated DNA cleavage, strand breaks,
and cytotoxicity induced by VP-16. Invest. New Drugs, 12,
41. Shibata,A., Barton,O., Noon,A.T., Dahm,K., Deckbar,D.,
Goodarzi,A.A., Lobrich,M. and Jeggo,P.A. (2010) Role of ATM
and the damage response mediator proteins 53BP1 and MDC1 in
the maintenance of G(2)/M checkpoint arrest. Mol. Cell Biol., 30,
42. Shivji,M.K. and Venkitaraman,A.R. (2004) DNA recombination,
chromosomal stability and carcinogenesis: insights into the role of
BRCA2. DNA Repair, 3, 835–843.
43. Ahnesorg,P., Smith,P. and Jackson,S.P. (2006) XLF interacts with
the XRCC4-DNA ligase IV complex to promote DNA
nonhomologous end-joining. Cell, 124, 301–313.
44. Dai,Y., Kysela,B., Hanakahi,L.A., Manolis,K., Riballo,E.,
Stumm,M., Harville,T.O., West,S.C., Oettinger,M.A. and
Jeggo,P.A. (2003) Nonhomologous end joining and V(D)J
recombination require an additional factor. Proc. Natl Acad. Sci.
USA, 100, 2462–2467.
45. Aylon,Y., Liefshitz,B. and Kupiec,M. (2004) The CDK regulates
repair of double-strand breaks by homologous recombination
during the cell cycle. EMBO J., 23, 4868–4875.
46. Ira,G., Pellicioli,A., Balijja,A., Wang,X., Fiorani,S.,
Carotenuto,W., Liberi,G., Bressan,D., Wan,L.,
Hollingsworth,N.M. et al. (2004) DNA end resection,
homologous recombination and DNA damage checkpoint
activation require CDK1. Nature, 431, 1011–1017.
47. De Azevedo,W.F., Leclerc,S., Meijer,L., Havlicek,L., Strnad,M.
and Kim,S.H. (1997) Inhibition of cyclin-dependent kinases by
purine analogues: crystal structure of human cdk2 complexed
with roscovitine. Eur. J. Biochem., 243, 518–526.
48. Huertas,P., Cortes-Ledesma,F., Sartori,A.A., Aguilera,A. and
Jackson,S.P. (2008) CDK targets Sae2 to control DNA-end
resection and homologous recombination. Nature, 455, 689–692.
49. Lengsfeld,B.M., Rattray,A.J., Bhaskara,V., Ghirlando,R. and
Paull,T.T. (2007) Sae2 is an endonuclease that processes hairpin
DNA cooperatively with the Mre11/Rad50/Xrs2 complex. Mol.
Cell, 28, 638–651.
50. Connelly,J.C. and Leach,D.R. (2004) Repair of DNA covalently
linked to protein. Mol. Cell, 13, 307–316.
51. Montecucco,A. and Biamonti,G. (2007) Cellular response to
etoposide treatment. Cancer Lett., 252, 9–18.
52. Campos-Nebel,M., Larripa,I. and Gonzalez-Cid,M. (2010)
Topoisomerase II-mediated DNA damage is differently repaired
during the cell cycle by non-homologous end joining and
homologous recombination. PLoS One, 5.
53. Deckbar,D., Birraux,J., Krempler,A., Tchouandong,L.,
Beucher,A., Walker,S., Stiff,T., Jeggo,P. and Lobrich,M. (2007)
Chromosome breakage after G2 checkpoint release. J. Cell Biol.,
54. Deckbar,D., Stiff,T., Koch,B., Reis,C., Lobrich,M. and
Jeggo,P.A. (2010) The limitations of the G1-S checkpoint. Cancer
Res., 70, 4412–4421.
55. Krempler,A., Deckbar,D., Jeggo,P.A. and Lobrich,M. (2007) An
imperfect G2M checkpoint contributes to chromosome instability
following irradiation of S and G2 phase cells. Cell Cycle, 6,
56. Kinner,A., Wu,W., Staudt,C. and Iliakis,G. (2008) Gamma-H2AX
in recognition and signaling of DNA double-strand breaks in the
context of chromatin. Nucleic Acids Res., 36, 5678–5694.
57. Lobrich,M., Shibata,A., Beucher,A., Fisher,A., Ensminger,M.,
Goodarzi,A.A., Barton,O. and Jeggo,P.A. (2010) gammaH2AX
foci analysis for monitoring DNA double-strand break repair:
strengths, limitations and optimization. Cell Cycle, 9, 662–669.
58. Buis,J., Wu,Y., Deng,Y., Leddon,J., Westfield,G., Eckersdorff,M.,
Sekiguchi,J.M., Chang,S. and Ferguson,D.O. (2008) Mre11
nuclease activity has essential roles in DNA repair and genomic
stability distinct from ATM activation. Cell, 135, 85–96.
59. Neale,M.J., Pan,J. and Keeney,S. (2005) Endonucleolytic
processing of covalent protein-linked DNA double-strand breaks.
Nature, 436, 1053–1057.
60. Di Virgilio,M. and Gautier,J. (2005) Repair of double-strand
breaks by nonhomologous end joining in the absence of Mre11.
J. Cell Biol., 171, 765–771.
61. Huang,J. and Dynan,W.S. (2002) Reconstitution of the
mammalian DNA double-strand break end-joining reaction
reveals a requirement for an Mre11/Rad50/NBS1-containing
fraction. Nucleic Acids Res., 30, 667–674.
62. Cortes,L.F., El Khamisy,S.F., Zuma,M.C., Osborn,K. and
Caldecott,K.W. (2009) A human 5’-tyrosyl DNA
phosphodiesterase that repairs topoisomerase-mediated DNA
damage. Nature, 461, 674–678.
63. Hartsuiker,E., Mizuno,K., Molnar,M., Kohli,J., Ohta,K. and
Carr,A.M. (2009) Ctp1CtIP and Rad32Mre11 nuclease activity
are required for Rec12Spo11 removal, but Rec12Spo11 removal is
dispensable for other MRN-dependent meiotic functions. Mol.
Cell. Biol., 29, 1671–1681.
64. Zhu,Z., Chung,W.H., Shim,E.Y., Lee,S.E. and Ira,G. (2008) Sgs1
helicase and two nucleases Dna2 and Exo1 resect DNA
double-strand break ends. Cell, 134, 981–994.
65. Yu,X. and Baer,R. (2000) Nuclear localization and cell
cycle-specific expression of CtIP, a protein that associates
with the BRCA1 tumor suppressor. J. Biol. Chem., 275,
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