Nucleotide excision repair-induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response
Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)-dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ubiquitination by Ubc13 and RNF8. Depletion of these enzymes causes UV hypersensitivity without affecting NER, which is indicative of a function for Ubc13 and RNF8 in the downstream UV-DDR. RNF8 is targeted to damaged DNA through an interaction with the double-strand break (DSB)-DDR scaffold protein MDC1, establishing a novel function for MDC1. RNF8 is recruited to sites of UV damage in a cell cycle-independent fashion that requires NER-generated, single-stranded repair intermediates and ataxia telangiectasia-mutated and Rad3-related protein. Our results reveal a conserved pathway of DNA damage-induced H2A ubiquitination for both DSBs and UV lesions, including the recruitment of 53BP1 and Brca1. Although both lesions are processed by independent repair pathways and trigger signaling responses by distinct kinases, they eventually generate the same epigenetic mark, possibly functioning in DNA damage signal amplification.
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 186 No. 6 835–847
Correspondence to Wim Vermeulen: W.Vermeulen@erasmusmc.nl
Abbreviations used in this paper: 6-4PP, 6-4 photoproduct; ATM, ataxia tel-
angiectasia mutated; ATR, ATM and Rad3-related; CPD, cyclobutane pyrimi-
dine dimer; DDR, DNA damage response; DSB, double-strand break; FHA,
forkhead associated; IF, immunoﬂuorescence; IR, ionizing radiation; IRIF,
IR-induced foci; LUD, local UV damage; NER, nucleotide excision repair; RPA,
replication protein A; shRNA, short hairpin RNA; Ub, ubiquitin; UDS, unsched-
uled DNA synthesis.
Endogenous and environmental agents continuously damage
DNA, compromise its normal functioning, and are associated
with accelerated ageing and malignant transformation. DNA
damage response (DDR) mechanisms, including diverse repair
and cell cycle control pathways, protect organisms against the
adverse effects of genomic insults (Hoeijmakers, 2001). DDR-
associated chromatin modications play an important role in
regulating both DNA repair and checkpoints (Bennett and
Harper, 2008), as illustrated by the involvement of the ataxia
telangiectasia–mutated (ATM) kinase in DNA double-strand
break (DSB)–induced DDR. ATM is the upstream kinase
responsible for the phosphorylation of H2AX on serine 193
(H2AX) in response to DSBs (Rogakou et al., 1998). This
early damage marker subsequently recruits MDC1 (mediator of
DNA damage checkpoint protein 1), which is an important step
for the subsequent recruitment of 53BP1 and BRCA1 at the
damaged chromatin (Stucki et al., 2005), thereby mediating the
checkpoint signaling toward the effector kinases CHK1 and
CHK2 (Kim and Chen, 2008). Additional ATM recruitment
results in enhanced accumulation of DNA repair factors. The col-
lective association of a large number of diverse DDR factors at
the damaged chromatin results in microscopically detectable
structures referred to as ionizing radiation (IR)–induced foci
(IRIF; Bekker-Jensen et al., 2006).
hromatin modiﬁcations are an important com-
ponent of the of DNA damage response (DDR)
network that safeguard genomic integrity. Re-
cently, we demonstrated nucleotide excision repair
(NER)–dependent histone H2A ubiquitination at sites of
ultraviolet (UV)-induced DNA damage. In this study, we
show a sustained H2A ubiquitination at damaged DNA,
which requires dynamic ubiquitination by Ubc13 and
RNF8. Depletion of these enzymes causes UV hyper-
sensitivity without affecting NER, which is indicative of
a function for Ubc13 and RNF8 in the downstream
UV–DDR. RNF8 is targeted to damaged DNA through
an interaction with the double-strand break (DSB)–DDR
scaffold protein MDC1, establishing a novel function
for MDC1. RNF8 is recruited to sites of UV damage in
a cell cycle–independent fashion that requires NER-
generated, single-stranded repair intermediates and ataxia
telangiectasia–mutated and Rad3-related protein. Our
results reveal a conserved pathway of DNA damage–
induced H2A ubiquitination for both DSBs and UV
lesions, including the recruitment of 53BP1 and Brca1.
Although both lesions are processed by independent
repair pathways and trigger signaling responses by
distinct kinases, they eventually generate the same
epigenetic mark, possibly functioning in DNA damage
Nucleotide excision repair–induced H2A
ubiquitination is dependent on MDC1 and RNF8
and reveals a universal DNA damage response
Jurgen A. Marteijn,
Audrey M. Gourdin,
Nico P. Dantuma,
and Wim Vermeulen
Department of Genetics, Center for Biomedical Genetics, Erasmus Medical Center, 3015 GE Rotterdam, Netherlands
Center for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen, Denmark
Department of Cell and Molecular Biology, Karolinska Institute, S-17177 Stockholm, Sweden
© 2009 Marteijn et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the ﬁrst six months after the publica-
tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
THE JOURNAL OF CELL BIOLOGY
JCB • VOLUME 186 • NUMBER 6 • 2009 836
837UV-INDUCED HISTONE H2A UBIQUITINATION • Marteijn et al.
UV-induced DNA damage results in helix-distorting DNA
lesions predominantly consisting of cyclobutane pyrimidine
dimers (CPDs) and 6-4 photoproducts (6-4PPs). In mammals,
these DNA damages are removed by nucleotide excision re-
pair (NER) that eliminates a wide spectrum of helix-distorting
lesions in a multistep “cut and patch”–type reaction. The
damage is excised as a 25–30 oligonucleotide DNA fragment
followed by gap lling through DNA repair synthesis and
restoration of an intact DNA duplex by a nal ligation step
(Hoeijmakers, 2001; Gillet and Schärer, 2006). Although the
function of the various proteins essential for the core DNA
repair process is well understood, its connection with the UV-
induced DNA damage signaling is less well characterized. In
contrast to DSB repair, NER does not take place in sub-
nuclear structures like IRIF. These IRIF are linked with DNA
damage–induced large-scale chromatin modications. Although
a variety of chromatin modications have been associated with
NER (Dinant et al., 2008), the biological signicance of these
changes is poorly understood. However, the best-described
UV-induced damage signaling, involving RAD17, the 9-1-1
complex, ATM and Rad3-related (ATR), and Chk1, is linked
to replication stress rather than to the repair process itself
(Niida and Nakanishi, 2006). However, Giannattasio et al. (2004)
identied a clear NER-dependent signaling pathway in yeast
and mammalian cells (Giannattasio et al., 2004). One of the
currently best-known players in the UV-induced DDR is the
phosphatidylinositol 3-kinase ATR, which is activated upon UV-
induced replication stress (Zou and Elledge, 2003; Falck et al.,
2005). This activation is caused through recruitment of ATR by
ATR-interacting protein to replication protein A (RPA)–coated
single-stranded DNA, which occurs at stalled replication
forks (Cortez et al., 2001). It has recently become clear that
UV damage also induces ATR activation and H2AX in a cell
cycle–independent fashion (O’Driscoll et al., 2003; Hanasoge and
Ljungman, 2007; Matsumoto et al., 2007; Stiff et al., 2008),
which may trigger similar large-scale chromatin modication as
observed after DSB. Similar to the observed NER-dependent
Chk1 activation, this activation is also dependent on active NER.
During NER, single-stranded DNA repair intermediates are
responsible for RPA recruitment, resulting in ATR activation
(Hanasoge and Ljungman, 2007).
Recently, we have identied a novel UV-induced, NER-
dependent chromatin modification, ubiquitination of H2A
(Bergink et al., 2006), although the molecular mechanism and
its function in UV– remain elusive. These ndings were recently
conrmed in another study by Zhu et al. (2009). In addition,
other core histones are also ubiquitinated upon UV damage like
H2B (Robzyk et al., 2000; Giannattasio et al., 2005), H3, and
H4 (Wang et al., 2006), although to a much lower extent in
absolute number of histone molecules being modied. A similar
chromatin mark on H2A (Mailand et al., 2007; Nicassio et al.,
2007) and H2AX (Huen et al., 2007; Ikura et al., 2007) was
later identied in response to DSB. In contrast to DSB–DDR,
within UV-induced DDR, the connection between NER, chro-
matin modications, and signaling is less well established.
Next to the molecular mechanism and function of the observed
UV-induced H2A ubiquitination, an appealing question is to
which extent this epigenetic DDR is mechanistically conserved
between these two types of DNA damage that are repaired by
entirely different repair machineries.
UV-induced H2A ubiquitination is a
continuous and dynamic process and
depends on Ubc13 and RNF8
H2A ubiquitination can be visualized using antibodies rec-
ognizing specically ubiquitinated H2A on Western blot or
immunouorescence (IF; Baarends et al., 2005) or indirect
in living cells studying GFP-ubiquitin (Ub; Dantuma et al.,
2006). Previous experiments in our laboratory (Bergink et al.,
2006) have identied a UV-induced H2A ubiquitination as
shown by IF (Fig. 1 a) and Western blot analysis (Fig. S1 a).
GFP-Ub was found to be accumulated after local UV dam-
age (LUD) iniction at subnuclear regions that were enriched
for Ub-H2A and colocalize with the core NER factors XPC
(Xeroderma pigmentosum group C) and XPA (Fig. 1 a) and
with the UV damage marker anti-CPD (antibody that speci-
cally recognizes the major UV-induced DNA lesion, CPD;
Fig. 1 b; Bergink et al., 2006). These experiments indicate
that GFP-Ub can be used as a sensitive live cell marker for
DNA damage–induced H2A ubiquitination. In this study, we
analyzed the kinetics of this UV-induced H2A ubiquitination
using cells stably expressing GFP-Ub (Fig. 1 b; Bergink
et al., 2006). GFP-Ub accumulation at LUD slowly increases
after irradiation and reaches a maximum after 3 h. Surpris-
ingly, local recruitment of GFP-Ub remains detectable up
to at least 24 h after UV when most NER proteins do not
accumulate anymore at the site of damage (see Fig. 4 f;
Hoogstraten et al., 2008), suggesting that this histone modication
Figure 1. RNF8 and Ubc13 knockdown inhibits the continuous UV-induced H2A ubiquitination. (a) GPF-Ub–expressing HeLa cells (left) or nontransfected
HeLa cells were locally UV irradiated (60 J/m
) and stained with an antibody-recognizing Ub conjugated to H2A (uH2A). uH2A colocalizes with GFP-Ub
(left) and with the damage markers XPC (middle) or XPA (right). (b) HeLa cells stably expressing GFP-Ub were locally UV irradiated (60 J/m
) and ﬁxed
after the indicated times. The local UV-irradiated area was visualized using CPD counterstaining. The GFP-Ub accumulation at LUD is visible up to 24 h.
(c) The mobility of GFP-Ub in HeLa cells was determined by FRAP. 3.5 h after local UV exposure (60 J/m
), GFP-Ub–expressing HeLa cells were subject
to live cell imaging. A nucleus containing a local Ub accumulation was photobleached inside the indicated white box for three iterations at 100% of
laser. Pictures were acquired at the indicated times after photobleaching. 9 min after photobleaching, the GFP-Ub was almost completely redistributed.
(d) GFP-Ub–expressing HeLa cells were transfected with the indicated siRNA oligonucleotides. 48 h after transfection, the cells were locally UV exposed
with 60 J/m
and 3 h later were stained for CPD. Knockdown of RNF8 or Ubc13 results in an almost absence of GFP-Ub accumulation at LUD. The per-
centage of colocalization of GFP-Ub with CPD is plotted for the different siRNA transfections. (e) HeLa cells were similarly treated as in d and stained for
ubiquitinated H2A together with XPA as a damage marker. siRNA-mediated depletion of RNF8 or Ubc13 caused a severely reduced accumulation of uH2A
at LUD. The percentage of colocalization of uH2A with XPA after LUD is plotted in the graph for the different siRNA transfections. Arrows and arrowheads
indicate local damage sites. Error bars indicate SEM.
JCB • VOLUME 186 • NUMBER 6 • 2009 838
RNF8 is recruited to LUD by MDC1
The observed dynamic damage-induced H2A ubiquitination
argued for a continuous presence of the responsible E3 Ub ligase
and E2 Ub–conjugating enzyme at the damaged DNA. The re-
cruitment of these enzymes to LUD was tested in cells express-
ing full-length GFP-tagged RNF8 or Ubc13 (Fig. S2 a). We
found a clear accumulation of both GFP-RNF8 and GFP-Ubc13
at LUD, colocalizing with CPD (Fig. 2 c) and Ub (Fig. S2 b).
Endogenous RNF8 was also found to accumulate at UV lesions
(Fig. 2 d) and remains, like Ub, 24 h after UV enriched at LUD.
This supports a model of continuous H2A ubiquitination at
DNA damage. Importantly, RNF8 recruitment to UV lesions
occurs in a cell cycle–independent manner, as shown by EdU
incorporation (Fig. 2 e; Salic and Mitchison, 2008) and cyclin A
costaining (Fig. S2 c).
RNF8 harbors an N-terminal forkhead-associated (FHA)
domain (Ito et al., 2001; Plans et al., 2006), which is commonly
implicated in phosphorylation-dependent protein–protein inter-
actions (Hammet et al., 2003), and a C-terminal RING nger
(Ito et al., 2001; Plans et al., 2006), which is known to interact
with E2-conjugating enzymes and is essential for its Ub ligase
activity (Fang and Weissman, 2004). We observed that the
RING nger point mutant (C403S; Mailand et al., 2007) local-
izes to LUD similar as wild-type RNF8, whereas the FHA
domain point mutant (R42A; Mailand et al., 2007) is unable to
accumulate at lesions (Fig. 3 a). These data suggest that RNF8
is recruited to UV-damaged DNA via a phospho-specic protein–
protein interaction involving its FHA domain. After IR, RNF8
is recruited to DSBs through binding of its FHA domain to
phosphorylated MDC1 (Huen et al., 2007; Kolas et al., 2007;
Mailand et al., 2007). Therefore, we tested whether the scaffold
protein MDC1 is also involved in loading RNF8 and Ub to LUD
using cells that stably express MDC1 short hairpin RNA
(shRNA) (Fig. S3 a). In the absence of MDC1, both GFP-Ub
(Fig. 3 b) and GFP-RNF8 (Fig. S3 b) failed to accumulate at
LUD. These results were conrmed by immunostaining with
an antibody specic for ubiquitinated H2A in cells transfected
with siRNA targeting MDC1 (Fig. 3 c). Together, these data
argue that MDC1 is required for UV-induced H2A ubiquitination.
Surprisingly, thus far, MDC1 was only found to be implicated in
DSB–DDR, and its implication in UV–DDR is unprecedented.
To rule out the possibility that its recruitment during UV–DDR
was caused by replication stress–induced DSBs after collapsed
replication forks, we showed that MDC is recruited to LUD in-
dependent of replication using MDC1-GFP–expressing cells in
combination with staining for EdU incorporation (Fig. S3 c; Salic
and Mitchison, 2008). Finally, in noncycling conuent primary
broblasts conrmed by Ki-67 staining (Fig. 3 d), endogenous
MDC1 accumulated at LUD (Fig. 3 e). This uncovers a novel
function for this DSB–DDR protein in UV–DDR.
H2A ubiquitination induced by UV requires
ATR and NER intermediates
Within DSB–DDR, recruitment of RNF8 and H2A ubiquitina-
tion are early events and occur with almost similar kinetics as the
loading of the primary DSB-recognizing proteins (Mailand et al.,
2007). In contrast, H2A ubiquitination within UV–DDR is a
represents a persisting chromatin mark. We tested the bind-
ing dynamics of Ub at LUD 3.5 h after irradiation. A swift
recovery of GFP-Ub was observed upon photobleaching both
at the damaged and nondamaged site (Fig. 1 c). To test
whether this is caused by exchange of histone H2A at the
nucleosome or by a continuous cycle of ubiquitination and
deubiquitination of H2A while residing in the nucleosome,
we compared the mobility of GFP-tagged H2A with the mo-
bility of GFP-Ub (Fig. S1 b). Although GFP-Ub is fully re-
covered within 12 min after photobleaching (both within and
outside of the LUD), only a minor part of GFP-H2A is recov-
ered within 12 min, suggesting that H2A is constantly ubiq-
uitinated and deubiquitinated within the context of an intact
nucleosome. These data indicate that local UV-induced H2A
ubiquitination is a dynamic process and further suggest that
Ub ligases are constantly targeted to chromatin containing
damage to maintain the UV-induced H2A ubiquitination up
to 24 h after the initial damage.
Recently, it was found that DSB-induced H2A and
H2AX ubiquitination is mediated by the E3 Ub ligase RNF8
and the E2-conjugating enzyme Ubc13 (Huen et al., 2007;
Kolas et al., 2007; Mailand et al., 2007; Nicassio et al., 2007).
We tested whether these enzymes were also responsible for
the GFP-Ub accumulation at LUD by depleting RNF8 or
Ubc13 in GFP-Ub–expressing HeLa cells. siRNA-mediated
knockdown of RNF8 and Ubc13, as conrmed by immuno-
blotting (Fig. S1 c), resulted in a strongly decreased accumu-
lation of GFP-Ub (Fig. 1 d) and ubiquitinated H2A (Fig. 1 e)
at LUD. These results were conrmed by immunostaining for
endogenous conjugated Ub (Fig. S1 d). In mammalian nuclei,
5–15% of H2A is monoubiquitinated, explaining the relative
abundant nuclear localization of GFP-Ub (Dantuma et al.,
2006). Depletion of RNF8 does not affect the total pool of nu-
clear conjugated Ub (mainly H2A-Ub), suggesting that RNF8
is a DNA damage–specic E3 ligase.
Role of RNF8 and Ubc13 in
We tested the involvement of RNF8 and Ubc13 in UV-induced
DDR by determining the UV sensitivity of HeLa cells depleted
for these proteins. RNF8- and Ubc13-depleted cells were more
UV sensitive than control siRNA–transfected cells (Fig. 2 a)
but not as sensitive as cells depleted for the essential NER endo-
nucleases XPF or XPG. These results suggest that UV-induced
H2A ubiquitination plays an important role in UV survival but
is not as essential as depletion of the NER factors XPF or XPG.
To further investigate the role of RNF8 and Ubc13 in NER,
we measured the activity of the nal step of the NER process
(Hoeijmakers, 2001), the gap-lling DNA synthesis. This un-
scheduled DNA synthesis (UDS) was analyzed in cells treated
with siRNA against XPF, RNF8, and Ubc13 (Fig. 2 b). In con-
trast to the severe UDS reduction by XPF depletion, knockdown
of RNF8 or Ubc13 does not affect UDS despite their function
in UV survival, as revealed by UV hypersensitivity in RNF8-
or Ubc13-depleted cells. These results indicate that RNF8 and
Ubc13 play a role in UV–DDR but do not directly affect the
core NER repair process.
839UV-INDUCED HISTONE H2A UBIQUITINATION • Marteijn et al.
Figure 2. RNF8 and Ubc13 depletion sensitizes cells to UV.
(a) UV survival using HeLa cells transiently transfected with
siRNA targeting RNF8, Ubc13, XPF, XPG, or control siRNA.
The percentage of surviving cells is plotted against the ap-
plied UV-C dose (J/m
). RNF8 and Ubc13 depletion sensi-
tizes cells to UV. (b) Gap-ﬁlling DNA repair synthesis (UDS)
was measured by autoradiography. Wild-type primary ﬁbro-
blasts (C5RO) were transfected with the indicated siRNA.
48 h after transfection, cells were pulse labeled with
H]thymidine after exposure to 16 J/m
UV-C. UDS was
quantiﬁed by counting autoradiographic grains in 50 nuclei,
and the number of counts in control-transfected cells was set
at 100%. RNF8 and Ubc13 have no effect on UDS. (c) HeLa
cells stably expressing Ubc13-GFP or Mrc5 cells expressing
RNF8-GFP were locally UV irradiated (60 J/m
) and immuno-
stained for CPD. Both Ubc13 and RNF8 accumulate at
LUD. (d) Localization of endogenous RNF8 before and after
local UV exposure (60 J/m
). Immunostainings were per-
formed using RNF8 and CPD antibodies. RNF8 accumulation
was found up to 24 h after UV damage. (e) RNF8-GFP–
expressing cells were exposed to LUD and directly after dam-
age, were incubated for 2 h in medium containing EdU (BrdU
analogue). Cells that were in S phase after the UV exposure
stain positive for EdU visualized using Alexa Fluor 594. CPD
was used as a damage marker. The graph adjacent to the
images shows a similar (100%) colocalization of RNF8 with
CPDs in S phase and non–S phase cells. Arrows indicate
local damage sites. Error bars indicate SEM.
JCB • VOLUME 186 • NUMBER 6 • 2009 840
point during UV–DDR RNF8 becomes implicated, we monitored
GFP-RNF8 binding to LUD in NER-decient cells. We found
that in two independent cell lines derived from Xeroderma pig-
mentosum patients mutated in the damage-recognizing and NER-
initiating protein XPC, RNF8-GFP fails to bind to LUD (Fig. 4 a).
This indicates that the RNF8 recruitment, similar to the subsequent
GFP-Ub accumulation (Bergink et al., 2006), is dependent on
relatively slow process, reaching a maximum only 3 h after UV
(Fig. 1 b), whereas lesion binding of UV damage–recognizing NER
proteins is very quick (Luijsterburg et al., 2007; Hoogstraten et al.,
2008). Moreover, depletion of the responsible E2-conjugating
enzyme or E3 ligase does not directly affect NER efciency
(Fig. 2 b). Together, these data suggest that UV-induced H2A
ubiquitination occurs late in UV–DDR. To investigate at which
Figure 3. MDC1 is essential for Ub accumulation at local damage. (a) U20S cells stably expressing wild-type, *RING (C403S), or *FHA (R42A) forms of
GFP-tagged RNF8 were locally UV irradiated followed by CPD immunostaining. The FHA domain of RNF8 is essential for the observed RNF8 accumula-
tion at the damaged area. (b) Wild-type or stably expressing shRNA targeting MDC1 U2OS cells were transiently transfected with GFP-Ub. 36 h after
transfection, cells were locally UV exposed and were immunostained for CPDs 2 h later. The percentage of cells in which GFP-Ub colocalizes with CPD
after LUD is plotted in the graph. MDC1 is essential for the Ub accumulation at the damaged DNA. (c) HeLa cells were transfected with control siRNA or
siRNA targeting MDC1. 36 h after siRNA transfection, cells were UV irradiated (60J/m
) and stained for ubiquitinated H2A and XPA. The percentage of
cells in which uH2A colocalizes with XPA after LUD is plotted in the graph. (d) Proliferation status of primary human C5RO ﬁbroblasts was checked using
the Ki-67 proliferation marker. Proliferating cells are positive for Ki-67 (right), whereas C5RO cells grown conﬂuent for 7 d are negative for Ki-67 (left),
indicating that all cells are in G0. (e) These nonproliferating human primary C5RO ﬁbroblasts were locally UV exposed (60 J/m
), and after 2 h, they were
immunostained for endogenous MDC1 and the essential NER protein XPA. MDC1 accumulates at the local damage in nonproliferating cells. Arrows and
arrowheads indicate local damage sites. Error bars indicate SEM.
841UV-INDUCED HISTONE H2A UBIQUITINATION • Marteijn et al.
active NER. Surprisingly, both RNF8 and Ub accumulation to
LUD was still visible 24 h after UV (Fig. 1 b and Fig. 2 d), whereas
presence of NER factors at LUD faded within 4–6 h after UV
(Fig. 4 f; Hoogstraten et al., 2008). Within this time frame, the bulk
of the UV-induced 6-4PPs are removed by the NER machinery
(Fig. S4 a; Hoogstraten et al., 2008), indicating that detectable
accumulation of NER factors at LUD follows the repair kinetics
of 6-4PP. This triggers the question of why this epigenetic mark
remains far beyond removal of 6-4PP but still requires functional
NER. However, it should be noted that in the majority of UV-
induced lesions, CPDs (60%) are slowly repaired by NER, and
a significant fraction remains unrepaired even 24 h after UV
(Mitchell et al., 1985). To test whether the repair of photolesions in
general causes this chromatin response after UV, we removed these
lesions by photoreactivation. We expressed GFP-Ub in mouse
embryonic broblasts derived from a transgenic mouse model
expressing both 6-4PP and CPD photolyases (Jans et al., 2005).
These photolyases directly reverse photolesions after exposure to
visible light (Fig. S4 b) independent of NER (Jans et al., 2005). In
photoreactivated cells, no accumulation of GFP-Ub at LUD could
be detected 6 h after UV irradiation (Fig. 4 b, right), whereas in
nonphotoreactivated control cells, GFP-Ub clearly accumulated
at LUD (Fig. 4 b, left). These data indicate that H2A is continu-
ously ubiquitinated as long as DNA lesions, including the poorly
repaired CPDs, are present.
Having established that active NER is required to trigger
H2A ubiquitination, the molecular mechanism remains un-
known. Within DSB–DDR, H2A ubiquitination depends on the
DNA damage signaling kinase ATM (Mailand et al., 2007). How-
ever, ATM does not become directly activated by UV irradiation
in nonreplicating cells (Stiff et al., 2006). Thus far, the best-
characterized damage signaling kinase in UV–DDR is ATR
(Abraham, 2004; Hanasoge and Ljungman, 2007). We found that
in broblasts carrying a hypomorphic ATR mutation that causes
severely reduced ATR expression (O’Driscoll et al., 2003), both
endogenous MDC1 and conjugated Ub accumulation at LUD
were signicantly attenuated as compared with wild-type cells
(Fig. 4 c). In addition, we found that when UV-irradiated cells
were treated with the ATM- and DNA-PK–specic inhibitors
(KU-55933 and KU-57788, respectively), MDC1 and RNF8
loading to LUD was not affected (Fig. 4 d). Addition of both inhibi-
tors to cells exposed to IR completely abolished H2AX phos-
phorylation and formation of MDC1 or RNF8 foci, indicating
that the inhibitors efciently down-regulated the activity of both
kinases. These results indicate that the UV-induced MDC1 and
RNF8 recruitment depends on ATR.
Besides the involvement of different kinases, a clear
difference also exists in the time of RNF8 pathway activation
between DSB– and UV–DDR. The RNF8 pathway is activated
before DSB repair (Huen et al., 2007; Mailand et al., 2007);
however, after UV damage, it is activated as a consequence
of NER. This implies that ATR becomes activated in an NER-
dependent fashion. It is established that ATR activation after
UV damage is caused by replication stress that creates single-
stranded DNA, which is a substrate for RPA/ATR-interacting
protein, resulting in the subsequent ATR activation (Shechter
et al., 2004). Nonetheless, we found that MDC1 and RNF8
binding also occurs outside S phase. It was recently described
that in non–S phase cells, single-stranded repair intermedi-
ates generated during NER by the excision of the damaged
strand activate ATR (Hanasoge and Ljungman, 2007; Stiff
et al., 2008). To study whether these NER-mediated repair
intermediates are responsible for the observed RNF8 path-
way activation, we increased the amount of single-stranded
NER intermediates (and consequently increased ATR activa-
tion) by blocking the DNA repair synthesis using the DNA
polymerase inhibitor aphidicolin. To visualize a possible in-
crease of RNF8 activation, we exposed the cells to a relative
low dose of local UV damage (20 J/m
) and shortly after UV
(1.5 h) as compared with the previously used conditions, i.e.,
suboptimal conditions for visualizing H2A-Ub and GFP-
RNF8 accumulation at LUD. As expected, without repair
replication inhibition, only a faint accumulation could be
detected (Fig. 4 e, left). However, a very clear LUD accumu-
lation of GFP-RNF8 was observed (Fig. 4 e, right) in cells
treated with aphidicolin for 1 h.
This indicates that MDC1, RNF8, and the subsequent
GFP-Ub accumulation are relatively late events triggered by the
DNA repair intermediates, which subsequently activate ATR.
This would suggest that the MDC1–RNF8 pathway is activated
at a hierarchical step in the UV–DDR, occurring after the initial
incision step mediated by the core NER machinery. To test this
hypothesis in more detail, we have analyzed the time-dependent
accumulation of several proteins involved in different steps
during UV–DDR by IF at different time points after LUD
(Fig. 4 f and Fig. S4 e). Core NER factors like the damage-
recognizing protein XPC or the DNA helicase of the TFIIH com-
plex XPB completely colocalize with the used damage markers
15 min after UV damage. This colocalization remains for a few
hours and gradually decreases to background levels at 4 h after
UV damage, basically after the repair kinetics of 6-4PPs. In
contrast, RPA, which is known to interact with single-stranded
DNA repair intermediates (Zou and Elledge, 2003), followed
an entirely different kinetic profile. Shortly after LUD, only
20% colocalized with a damage marker and reached a maxi-
mum of almost-full colocalization 4 h after LUD that per-
sisted until 8 h after UV. Similar kinetic proles were found for
phosphorylated H2AX, MDC1, and Ub, indicating that these
events represent late, postincision events of the UV–DDR path-
way. To further analyze the order of accumulation, we have
measured the real-time live cell accumulation of XCP-GFP,
RPAp70-GFP, and Ub-GFP by confocal imaging. After apply-
ing LUD using a UV-C laser (Dinant et al., 2007), we measured
the assembly kinetics of GFP-tagged proteins at LUD (Fig. 4 g).
A striking difference in the assembly kinetics was observed be-
tween these proteins in which XPC reaches equilibrium the
fastest, representative for an early event (damage detecting),
followed by the recruitment of RPA, which represents the for-
mation of single-stranded DNA repair intermediates, and nally
GFP-Ub, which reaches its plateau much later. Together, these
data provide insight into the hierarchy of the UV–DDR re-
sponse: (1) initiation of the NER machinery results in (2) repair
intermediates that nally activate the (3) MDC1–RNF8 path-
way, resulting in the recruitment of Ub at the LUD.
JCB • VOLUME 186 • NUMBER 6 • 2009 842
UV-INDUCED HISTONE H2A UBIQUITINATION • Marteijn et al.
mutagenesis (Huen et al., 2007). To test whether the described
MDC1–RNF8 pathway might also play a role in the amplica-
tion of the checkpoint after UV damage, we have studied the
phosphorylation status of Chk1 in control or MDC1 siRNA–
transfected cells after UV damage (Fig. 5 d). siRNA-mediated
depletion of MDC1 results in a decrease of Chk1 phosphoryla-
tion after UV damage. These results were confirmed by IF
staining of global UV-irradiated cells (Fig. S5 c). Together, these
data indicate that one of the main functions of the UV-induced
activation of the MDC1–RNF8 pathway at the damaged DNA is
to potentiate the cell cycle regulation via Chk1.
In this study, we present novel insight into the molecular
mechanism of UV-induced H2A ubiquitination. Our data show
that after UV damage, the Ub E3 ligase RNF8, in concert with
the E2-conjugating enzyme Ubc13, is essential for the NER-
dependent Ub modication of histone H2A. In a previous study,
we reported that UV-induced ubiquitination of histone H2A re-
quired Ring2 (Bergink et al., 2006). Notably, Ring2 knockdown
also results in a severe reduction of basal H2A ubiquitination as
well as the nuclear Ub pool. Thus, Ring2 knockdown is likely
to disturb the cellular Ub equilibrium (Groothuis et al., 2006)
and may affect downstream-specic ubiquitination reactions
such as the RNF8-dependent H2A ubiquitination. In contrast,
RNF8 knockdown inhibits only UV-induced and not basal H2A
ubiquitination (Fig. 1 c; Huen et al., 2007; Mailand et al., 2007).
Furthermore, we show that RNF8 is recruited to the damaged
DNA, suggesting that RNF8 is the DNA damage–specic H2A
Intriguingly, a signicant proportion of Ubc13 was found
to accumulate at the site of DNA damage, indicating that a large
part of the pool of Ubc13 proteins plays a role during UV-
induced DNA repair. Recently, it was also observed that a large
part of the total amount of Ubc13 is involved during the DSB-
induced DDR, as was shown by the accumulation of Ubc13
at laser-induced DSB damage (Ikura et al., 2007). This indicates
that Ubc13 plays an important role during DNA repair in addi-
tion to its involvement in many other biological processes
RNF8 is essential for 53BP1 and BRCA1
accumulation after UV damage
Despite important mechanistic and kinetic differences, our data
suggest that both DSB and UV lesions trigger a similar epi-
genetic mark, H2A ubiquitination. During DSB–DDR, the
RNF8-dependent H2A or H2AX ubiquitination eventually pro-
motes the recruitment of 53BP1 and BRCA1 to chromatin near
breaks (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007).
It is suggested that ubiquitinated H2A/H2AX is recognized by the
Ub-interacting motifs of RAP80, which through its interaction
with CCDC98 recruits BRCA1/BARD1 to chromatin (Kim
et al., 2007; Sobhian et al., 2007; Wang et al., 2007). It is
currently unknown how 53BP1 is recruited in an RNF8- and
MDC1-dependent way. To study whether the observed UV-induced
H2A ubiquitination also triggers the recruitment of 53BP1 and
BRCA1, we tested whether these thus-far DSB-specic factors
also accumulate at LUD. Both endogenous 53BP1 and BRCA1
(Fig. 5, a and b) accumulate at LUD in an RNF8- and MDC1-
dependent fashion. As in the DSB response, the UV damage–
induced accumulation of BRCA1 at LUD most likely occurs
via its interaction with RAP80, which also accumulates at UV-
induced LUD, colocalizing with CPD or H2AX (Fig. S5 a). Both
the accumulation of 53BP1 and conjugated Ub also take place
in nonproliferating cells (Fig. 5 c), ruling out the possibility that
these proteins accumulate at DSBs originating from stalled rep-
lication forks. The 53BP1 accumulation in primary nonprolifer-
ating cells was further studied using different UV doses. Even
at a relative low dose of 5 J/m
, the 53BP1 colocalization with
CPD was still clearly visible (Fig. S5 b). Together, these results
show that like DSBs, UV-induced DNA damage results in a strong
increase in local concentration of checkpoint mediators like MDC1,
RNF8, and ubiquitinated H2A, suggesting that this robust epi-
genetic mark is a conserved DNA damage signal–amplifying
process between DSB processing and NER.
MDC1 is involved in UV-dependent
One of the proposed functions of this MDC1–RNF8 pathway
after, for example, IR-induced DSBs, is to induce and maintain
cell cycle checkpoint control, thereby protecting cells from
Figure 4. NER intermediates trigger RNF8 accumulation in a DNA damage– and ATR-dependent manner. (a) RNF8-GFP stably expressing NER-proﬁcient
(MRC5) or NER-deﬁcient (XPC-negative cell lines XP20MA and XP4PA) were locally UV exposed (60 J/m
). 2 h later, damaged cells were immunostained
for CPDs. The percentage of cells in which RNF8-GFP colocalizes with CPD after LUD is plotted in the graph. RNF8 does not accumulate in NER-deﬁcient
cells. (b) Mouse embryonic ﬁbroblasts expressing both CPD and 6-4PP photolysases were stably transfected with GFP-Ub. 1.5 h after local UV exposure
), cells were cultured either in the dark (left) or were photoreactivated with visible light for 2 h (right). 6 h after initial UV damage, cells were ﬁxed
and immunostained for CPDs. After UV lesion removal, GFP-Ub does not accumulate at LUD. (c) ATR hypomorphic human cells (Seckel) and C5RO cells
with a wild-type ATR status were locally UV irradiated (25 J/m
) and immunostained after 2 h with antibodies recognizing MDC1 or conjugated Ub (FKII).
XPA was used as a damage marker, indicating that MDC1 and Ub accumulate in an ATR-dependent manner. (d) Stably expressing MDC1-GFP (left) or
RNF8-GFP (right) cells were incubated for 90 min with ATM and DNA-PK inhibitors (KU-55933 and KU-57788) or with an equal volume of DMSO. Cells
were exposed to IR (10 Gy) or UV (60 J/m
), and after 2 h, they were immunostained for H2AX or CPD. Although the addition of these inhibitors clearly
inhibits the IR-induced foci formation of MDC1, MDC1 still accumulates after LUD. (e) Stable MRC5 RNF8-GFP–expressing cells were locally exposed to
UV with or without a pretreatment for 1 h with 1 µg/ml aphidicolin. 1 h after UV damage, cells were ﬁxed and immunostained for CPDs. A strong
increase of RNF8 accumulation at the damaged DNA after aphidicolin pretreatment was observed. (f) The percentage of GFP-MDC1, XPB, RPA, H2AX,
GFP-Ub, and XPC-GFP colocalization with a damage marker (either CPD or XPA) at LUD (irradiated with 45 J/m
) is plotted for the different time points
at 15 min, 1 h, 4 h, and 8 h after LUD. Although XPC and XPB are colocalizing in almost all cells 15 min after UV damage, the other factor colocalizes
signiﬁcantly with the used damage markers 1 h after LUD. (g) Cells stably expressing the XPC-GFP, GFP-Ub, and GFP-RPAp70 proteins were UV damaged
using UV-C (266 nm) laser irradiation. GFP ﬂuorescence intensities at the site of UV damage were measured by real time imaging until they reached a
maximum. Assembly kinetic curves were derived from at least six cells for each protein. Relative ﬂuorescence was normalized on 0 (before damage) and
100% (maximum level of accumulation). Arrows and arrowheads indicate local damage sites. Error bars indicate SEM.
JCB • VOLUME 186 • NUMBER 6 • 2009 844
during UV-induced DDR. Ubc13 is, for the majority, not in-
volved in the core NER machinery because Ubc13 depletion has
no detectable effect on UDS (Fig. 2 b) or XPA accumulation
(Fig. S1 d). Most likely, Ubc13 plays a role in the downstream
UV-induced DDR together with RNF8.
Our data (Fig. 5 e) show that the molecular mechanism
that induces ubiquitination of H2A after genotoxic stress, in-
cluding the recruitment of downstream factors 53BP1 and
(Pickart, 2000). Because RNF8 recruitment to damaged DNA is
independent on Ubc13 (unpublished data), we assume that
Ubc13 depends on RNF8 recruitment, which is in line with the
described interaction of Ubc13 with the RING nger domain of
RNF8 (Plans et al., 2006). The strong accumulation of Ubc13 at
the site of damage, which is surprising for such a usually less-
specic functioning, E2-conjugating enzyme (in contrast to the
more substrate-specic E3 ligases), indicates its important role
Figure 5. 53BP1 and BRCA1 accumulate at LUD in an RNF8- and MDC1-dependent manner. (a and b) U2OS cells conditionally expressing doxycycline
(Dox)-inducible shRNA targeting RNF8 were induced or not with doxycycline for 48 h, and cells stably expressing shRNA targeting MDC1 were locally UV
damaged and stained for 53BP1 and CPD (a) or for BRCA1 and XPA (b). Both 53BP1 and Brca1 are recruited to the site of UV damage in an RNF8- and
MDC1-dependent manner. The percentage of cells in which 53BP1 or BRCA1 colocalize with the used damage marker after LUD is plotted in the graphs.
(c) Nonproliferating human primary ﬁbroblasts (C5RO), as determined by a negative Ki-67 staining (not depicted), were stained for endogenous 53BP1
and conjugated Ub accumulation after local UV exposure (60 J/m
). CPD and XPA were used as a damage marker. (d) HeLa cells were transfected with
siRNA targeting MDC1 or control siRNA. 36 h after transfection, cells were UV exposed (10 J/m
) and lysed at the indicated time points after UV damage.
Chk1 phosphorylation status (Ser317) was analyzed using a phospho-speciﬁc antibody, and tubulin staining was used as a loading control. Arrow, phos-
phorylated Ser317 Chk1; asterisk, -speciﬁc band. The relative amount of phosphorylated Chk1 is plotted in the graph (two blots were quantiﬁed, relative
phospho-Chk1 levels were normalized, and the highest phospho-Chk1 level was set at 100%), indicating that MDC1 knockdown result is a reduction of
the UV-induced Chk1 phosphorylation. (e) Model for the UV-induced DDR. UV-induced lesions are repaired by the core NER machinery, thereby generat-
ing single-stranded DNA repair intermediates, which subsequently activate ATR. This results in recruitment of MDC1 at the chromatin, which is essential
for the RNF8 accumulation at the DNA damage. In concert with Ubc13, RNF8 ubiquitinates H2A, which triggers the recruitment of 53BP1 and BRCA1.
From MDC1 recruitment onwards, the IR- and UV-induced DDR are similar. DSBs are recognized and bound by the Mre11–Rad50–Nbs1 (MRN) complex.
Arrows and arrowheads indicate local damage sites. Error bars indicate SEM.
845UV-INDUCED HISTONE H2A UBIQUITINATION • Marteijn et al.
antibiotics and 10% fetal calf serum at 37°C with 5% CO
. Cell lines sta-
bly expressing ﬂuorescent-tagged proteins (GFP-RNF8, Ubc13-GFP, and
RFP-Ub) were isolated by FACS sorting and neomycin selection. Other cell
lines were used as described previously: GFP-Ub (Dantuma et al., 2006),
MDC1-GFP (Bekker-Jensen et al., 2006), RNF8-GFP (wild type, RING,
and FHA; Mailand et al., 2007), shRNF8 (Mailand et al., 2007), and
shMDC1 (Bekker-Jensen et al., 2006). Aphidicolin was used at 5 µg/ml,
and ATM inhibitor (KU-55933) and DNA-PK inhibitor (KU-57788) were
used at a concentration of 10 µM. For the local UV irradiation, cells were
treated with a UV-C germicidal lamp (254 nm; Phillips) through a 5-µm
microporous ﬁlter at the indicated dose (Moné et al., 2001). Cells were
exposed to IR using a
Cs source at the speciﬁed dose. siRNA oligo-
nucleotides were synthesized (Thermo Fisher Scientiﬁc) to the following se-
quences: MDC1, 5-GUCUCCCAGAAGACAGUGA-3; RNF8, 5-GGAC-
AAUUAUGGACAACAA-3; Ubc13, 5-GGGACUUUUAAACUUGAAC-3;
XPF SMARTpool, 5-UGACAAGGGUACUACAUGA-3, 5-GUAGGAUA-
CUUGUGGUUGA-3, 5-ACAAGACAAUCCHCCAUUA-3, and 5-AAG-
AXGAGCUCACGAGUAU-3; and XPG SMARTpool, 5-CAUGAAAUCUU-
GACUGAUA-3, 5-GAACGCACCUGCUGCUGUA-3, 5-GAAAGAAG-
AUGCUAAACGU-3, and 5-GAACGAACUUUGCCCAUAU-3. siRNA
transfections were performed using Lipofectamine2000 (Invitrogen) ac-
cording to the manufacturer’s protocol. DNA transfections were performed
using Fugene (Roche).
IF microscopy and Western blotting
Cells were ﬁxed using 2% paraformaldehyde in the presence of 0.1% Triton
X-100. Samples were processed as described previously (Rademakers et al.,
2003). For uH2A staining, cells were permeabilized with 0.5% Triton
X-100 for 5 min before ﬁxation with 4% paraformaldehyde for 15 min at
4°C. Immunoﬂuorescent images were obtained using the Aristoplan Flu
(134795; Leitz) or confocal microscope (LSM 510 META; Carl Zeiss, Inc.)
equipped with a 63× 1.4 NA Plan Apochromat oil immersion lens (Carl
Zeiss, Inc.). LSM image browser acquisition software (version 4.0; Carl
Zeiss, Inc.) was used. The following antibodies were used: anti-MDC1 (Ab-
cam), anti-RNF8 (Abcam), anti-53BP1 (H-300; Santa Cruz Biotechnology,
Inc.), anti-BRCA1 (D9; Santa Cruz Biotechnology, Inc.), anti-Ub (FK2; BIO-
MOL International L.P.), anti–cyclin A (GNS; Santa Cruz Biotechnology,
Inc.), anti-H2AX (07-164; Millipore), anti–Ki-67 (Abcam), anti-XPB (clone
IB3; Giglia-Mari et al., 2006), anti-RPA32 (clone 9H8; Abcam), anti-
ubiquitinated H2A (uH2A; Abcam), RAP80 (rabbit polyclonal; Bethyl Labo-
ratories, Inc.), phospho–S317-Chk1 (Western blotting, Bethyl Laboratories,
Inc.; IF, Cell Signaling Technology), and anti-GFP (rabbit polyclonal; Ab-
cam) in combination with the corresponding secondary antibodies labeled
with Alexa Fluor 350, 488, or 594 as indicated (Invitrogen; The Jackson
Laboratory). DNA was stained using DAPI Vectashield (Vector Laborato-
ries). As marker for detecting LUD, anti-hXPA (rabbit polyclonal anti–human
XPA) or mouse anti-CPD (TDM-2; MBL International) were used, depending
on the species in which the other used antibody was raised in. Colocaliza-
tion was quantiﬁed by counting at least 50 cells per experiment in different
ﬁelds. Colocalization was deﬁned as a more than twofold increase in in-
tensity at the LUD deﬁned by the presence of the used damage marker (XPA
or CPD). EdU (5-ethynyl-2’-deoxyuridine) incorporation was visualized
using Click-iT Alexa Fluor 594 according to the manufacturer’s protocol
(Invitrogen). Western blotting was performed with the indicated primary
antibody according to the manufacturer’s protocol. Alexa Fluor 680 goat
anti–mouse or –rabbit (Invitrogen) were used to visualize the Western blot-
ting using an infrared imaging system (Odyssey; LI-COR Biosciences). H2A
ubiquitination after UV damage was studied using HeLa cells stably ex-
pressing His-tagged Ub (Choudhury et al., 2004). Ubiquitinated proteins
were isolated as described previously (Marteijn et al., 2007) and subject
for immunoblotting using uH2A and FKII antibodies.
Live cell confocal laser-scanning microscopy
Confocal laser-scanning microscopy images were obtained using a confocal
microscope (LSM 510 META) with a 63× oil Plan Apochromat 1.4 NA oil im-
mersion lens (Carl Zeiss, Inc.) equipped with a cell culture microscopy stage.
GFP ﬂuorescence imaging was recorded after excitation with a 488-nm argon
laser and a 515–540-nm band-pass ﬁlter. FRAP was performed as described
previously (Houtsmuller and Vermeulen, 2001). The indicated areas in Fig. 1 c
and Fig. S1 b were photobleached by three iterations using 100% 488-nm
laser power. The cytoplasm of the GFP-Ub cell line was photobleached (three
iterations; 100% 488-nm laser power) preceding the photobleaching of the
indicated areas (Fig. 1 c and Fig. S1 b). The recovery of ﬂuorescence in the
photobleached box was followed by imaging every minute. Kinetics of GFP-
tagged RPA, Ub, and XPC accumulation were performed using a UV-C (266 nm)
laser irradiation for 1.5 s as described previously (Dinant et al., 2007).
BRCA1, is highly conserved between DSB- and UV-induced
DDR. This is remarkable considering the important mechanistic
differences between DSB and UV damage repair, which may
explain the variation in observed timing. MDC1 and RNF8 re-
cruitment are early events in the DSB-induced DDR (Mailand
et al., 2007), although it is a relatively late event after UV-
induced DNA damage. It is possible that this difference is
caused by a much faster processing of DSB breaks, resulting in
a fast ATM activation compared with a slower ATR activation
by UV lesion processing.
Importantly, we found factors (e.g., MDC1, 53BP1, and
Brca1) to be recruited to UV-damaged DNA that were previ-
ously known only to play a role in the DSB response. Until now,
their involvement in UV–DDR was unanticipated, although re-
cently, some were found to be phosphorylated by ATR after UV
damage, such as MDC1 (Stewart et al., 2003), RAP80 (Yan et al.,
2008), and 53BP1 (Jowsey et al., 2007). Although these pro-
teins are maximally phosphorylated within minutes upon IR via
ATM, after UV, their phosphorylation becomes apparent after
60 min and keeps increasing in time, showing that the UV–DDR
response via this RNF8 pathway has indeed slower kinetics.
The question of why these DSB–DDR factors (including
MDC1, 53BP1, and Brca1) are also recruited to chromatin after
UV damage is more difcult to answer. Genetic insults may ring
a “general alarm bell,” resulting in the assembly of a toolbox of
diverse DDR proteins near the lesion irrespective of the type of
DNA damage. This enables the cell to facilitate different re-
sponse pathways, but only a specic subset will be used if needed
(Harper and Elledge, 2007). This might also explain why RNF8
is observed to accumulate at stalled replication forks (Sakasai
and Tibbetts, 2008). The recruitment of the DDR proteins such
as MDC1, 53BP1, and BRCA1 may represent an extra line of
defense against the UV-induced damage. Normally, only the
NER-specialized DNA repair enzymes will be used. However, in
the rare event that a DSB originates from a UV lesion during
replication, the DSB-involved proteins that were present “just in
case” at the damaged chromatin can swiftly be used.
Alternatively, the same pathways, and thus the same proteins,
are used as cells use similar crisis management strategies triggered
by different repair machineries. H2A ubiquitination increases the
local concentration of 53BP1 and BRCA1 that enhances activation
of the cell cycle checkpoint kinases Chk1 and Chk2 (Stucki and
Jackson, 2006). siRNA-mediated down-regulation of MDC1 re-
sults in a lower UV-induced Chk1 phosphorylation, indicating that
this pathway is involved in the checkpoint induction and mainte-
nance (Fig. 5 d). Chk1 and Chk2 play a crucial role in DDR by, for
example, regulating the S to G2 and G2/M checkpoints (Niida and
Nakanishi, 2006). Both checkpoints are implicated in the cellular
survival of IR and UV, which may explain the use of a common
Materials and methods
Cell culture and transfection
HeLa, U2OS, MRC5, and the human primary ﬁbroblasts wild-type control
(C5RO), XPC-deﬁcient patient cell lines XP4PA and XP20MA, and ATR hypo-
morphic GM18366 ﬁbroblasts (obtained from The Coriell Institute,
Camden, NJ) were cultured in Ham’s F10 (Invitrogen) supplemented with
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UDS and UV survival
3 d before the UDS assay, C5RO cells were siRNA transfected. UDS was
performed as described previously (Vermeulen et al., 1986). Cells were
UV irradiated with 16 J/m
and incubated for 2 h in culture medium con-
taining 10 µCi (methyl[
H]) thymidine (110 Ci/mmol/ml; GE Healthcare).
Repair capacity was quantiﬁed by counting autoradiographic grains. Cel-
lular survival of HeLa cells was determined using a colony assay. Cells
were plated in 6-cm dishes at various dilutions. After 16 h, cells were ex-
posed to different doses of UV-C (254 nm; TUV lamp; Phillips) and left to
grow for 7 d, ﬁxed, and stained, and colonies were counted to assess the
Online supplemental material
Fig. S1 shows the UV-induced H2A ubiquitination, the mobility of H2A-GFP,
and the siRNA-mediated knockdown efﬁciency of RNF8 and Ubc13.
Fig. S2 shows the characterization of the stable RNF8-GFP and Ubc13-GFP
cell line, the colocalization of RNF8-GFP with RFP-Ub, and the cell cycle–
independent accumulation of RNF8 at the site of UV damage. Fig. S3 shows
the MDC1 dependency of RNF8 accumulation and the accumulation of
MDC1 in both replicating and nonreplicating cells. Fig. S4 shows the effect
of the 6-4PP and CPD photolyases, the effect of aphidicolin, and the kinetics
of different DDR-involved proteins using IF. Fig. S5 shows the RAP80 accu-
mulation at the site of damage, the 53BP1 accumulation in nonproliferating
C5RO cells at different UV doses, and the role of MDC1 on the UV-induced
Chk1 phosphorylation using IF. Online supplemental material is available
We thank Dr. T. Thomsom for sharing the GFP-RNF8 plasmid and Dr. Ikura for
the GFP-Ubc13 construct. We thank Professor J. Hoeijmakers for critically reading
this manuscript, Mr. D. Warmerdam for helpful discussions, Mrs. A. Fazalalikhan,
Mr. N. Wijgers, and Mr. A.F. Theil for technical assistance, and Drs. A.B.
Houtsmuller and W.A. van Cappellen for providing the imaging facility. We
also thank Dr. G. Smith of KuDOS Pharmaceuticals for providing ATM and
DNA-PK inhibitors and Dr. Baer for providing us with the stably expressing
His-Ub cell line.
This study was ﬁnanced by the Dutch Organization for Scientiﬁc Re-
search (grants ZonMW 917-46-364 to W. Vermeulen and ZonMW Veni
917-96-120 to J.A. Marteijn), the Human Frontiers in Science Program (grant
RGP0007/2004-C to H. Lans), the American Institute for Cancer Research
(grant 09-0084 to H. Lans), the European Union Integrated Project DNA
repair FP6 program (grant LSHG-CT-2005-512113 to J.A. Marteijn), the
Swedish Cancer Society, and the Swedish Research Council (N.P. Dantuma).
Submitted: 27 February 2009
Accepted: 13 August 2009
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