RNF168 Binds and Amplifies Ubiquitin
Conjugates on Damaged Chromosomes
to Allow Accumulation of Repair Proteins
Carsten Doil,1Niels Mailand,1Simon Bekker-Jensen,1Patrice Menard,1Dorthe Helena Larsen,1Rainer Pepperkok,2
Jan Ellenberg,3Stephanie Panier,4Daniel Durocher,4Jiri Bartek,1Jiri Lukas,1,* and Claudia Lukas1
1Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Strandboulevarden 49,
DK-2100 Copenhagen, Denmark
2Cell Biology/Biophysics Units
3Gene Expression Unit
European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69117 Heidelberg, Germany
4Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada
DNA double-strand breaks (DSBs) not only interrupt
the genetic information, but also disrupt the chro-
matin structure, and both impairments require repair
mechanismsto ensure genomeintegrity. Weshowed
previously that RNF8-mediated chromatin ubiquity-
lation protects genome integrity by promoting the
accumulation of repair factors at DSBs. Here, we
provide evidence that, while RNF8 is necessary to
trigger the DSB-associated ubiquitylations, it is not
sufficient to sustain conjugated ubiquitin in this
compartment. We identified RNF168 as a novel chro-
matin-associated ubiquitin ligase with an ability to
bind ubiquitin. We show that RNF168 interacts with
ubiquitylated H2A, assembles at DSBs in an RNF8-
dependent manner, and, by targeting H2A and
H2AX, amplifies local concentration of lysine 63-
linked ubiquitin conjugates to the threshold required
for retention of 53BP1 and BRCA1. Thus, RNF168
defines a new pathway involving sequential ubiquity-
lations on damaged chromosomes and uncovers
a functional cooperation between E3 ligases in
Following the generation of DNA double-strand breaks (DSBs),
the neighboring chromatin undergoes extensive modifications
initiated by ATM-dependent phosphorylation of histone H2AX
(g-H2AX) and culminating at the generation of a distinct
compartment capable of retaining various genome caretakers
(Fernandez-Capetillo et al., 2004; Lukas et al., 2004b). Several
lines of evidence suggest that the main function of the DSB-
induced chromatin modifications (and the resulting concentra-
tion of proteins in this compartment) is to enhance the efficiency
of repair and signaling reactions, especially under conditions
that pose increased demand on genome surveillance (Celeste
et al., 2003). This concept has gained significant support from
studies showing that a forced tethering of DSB regulators to
chromatin was sufficient to induce checkpoint signaling and
delay cell-cycle progression even without the concomitant
DNA damage (Bonilla et al., 2008; Soutoglou and Misteli, 2008).
Recent work provided important mechanistic insights into the
role of posttranslational modifications in the hierarchical protein
assembly on damaged chromatin. The central coordinating
factor appears to be MDC1, a large adaptor protein, which
through its BRCT domains directly binds phosphorylated S139
of g-H2AX (Stucki et al., 2005) and is among the first proteins
to arrive at the DSB sites (Lukas et al., 2004a). Importantly,
MDC1 notonlysenses themostproximal DSB-associated phos-
phorylation events (g-H2AX), but it also integrates distinct types
of constitutive, as well as DNA damage-induced, signaling and
thereby orchestrates the ensuing protein assemblies at the
DSB sites. Thus, constitutive phosphorylation of the S-D-T clus-
ters by Casein kinase 2 generates binding sites for the FHA and
BRCT domains of NBS1, a core component of the MRE11-
NBS1-RAD50 nuclease complex (Melander et al., 2008; Spycher
et al., 2008). At the same time, MDC1 integrates also the bona
fide DNA damage signaling through the ATM-dependent phos-
phorylation of a cluster of S/T-Q residues, which generate
a landing platform for RNF8, an FHA-containing ubiquitin ligase.
RNF8 catalyzes local histone ubiquitylation and thereby renders
the DSB-flanking chromatin permissive to assemble additional
DSB regulators such as 53BP1 and BRCA1 (Huen et al., 2007;
Kolas et al., 2007; Mailand et al., 2007; Sobhian et al., 2007;
Wang et al., 2007; Wang and Elledge, 2007; Yan et al.,
2007). Finally, in addition to phosphorylations and ubiquityla-
tions, other posttranslational modifications contribute to the
full-scale development of this compartment. It has been shown
that the TRRAP/TIP60 histone acetyltransferase facilitates
53BP1 and BRCA1 retention at the DSB sites (Murr et al.,
2006), and one report suggested that TIP60 is functionally linked
with histone ubiquitylations (Ikura et al., 2007). How all of these
Cell 136, 435–446, February 6, 2009 ª2009 Elsevier Inc. 435
processes are coordinated in space and time is a major chal-
lenge for future research.
To gain new insights into these issues, we performed a high-
content microscopy screen for genes whose downregulation by
RNA interference (RNAi) prevents accumulation of 53BP1 at the
chromatin modifications (H2AX phosphorylation, RNF8-medi-
ated ubiquitylation, and TRRAP/TIP60-dependent acetylation)
induced by DNA damage. Furthermore, accumulation of 53BP1
at DSBs requires also constitutive epigenetic marks (such as di-
H4), which are recognized by the Tudor domain of 53BP1 (Bo-
tuyan et al., 2006; Huyen et al., 2004). Thus, 53BP1 integrates
a multitude of upstream events, which makes it particularly suit-
able to screen for hitherto unknown factors involved in genome
surveillance. Here, we mine this screen and identify RNF168,
a protein that combines E3 ubiquitin ligase activity with an ability
to bind conjugated ubiquitin on damaged chromosomes and
mechanistically defines a molecular pathway involved in the
DNA damage-induced chromatin response.
Identification of RNF168 as a Novel Factor Required
for 53BP1 Accumulation on Damaged Chromosomes
Using previously developed methods for microscopy-based
high-throughput RNAi screening (Erfle et al., 2007) combined
with a custom-designed readout for 53BP1 focus formation
(see Supplemental Experimental Procedures and Figures
S1A–S1G available online), we identified RNF168 as one of the
strongest hits that inhibited focal accumulation of 53BP1 on
chromatin (Figure 1A). This inhibition was achieved by indepen-
dent siRNA oligonucleotides and was as robust as the effect of
MDC1 or RNF8, two known regulators of 53BP1 retention at
Figure 1. RNF168 Regulates Retention of
53BP1 at the DNA Damage-Modified Chro-
(A) Representative fields from the siRNA arrays
showing cell populations cultured for 3 days on
spots containing control (siCon) or RNF168-tar-
geting siRNAs and immunostained with an anti-
body to 53BP1. The siRNA ID number specifies
the oligonucleotide in the Ambion siRNA database
screen for regulators of 53BP1 retention at spon-
taneous nuclear foci. The plotted values show
the area of the least incidence of 53BP1 foci. The
positions of RNF168 as the two high-scoring hits
are indicated (red). MDC1 (yellow) and RNF8
(green) are indicated as positive controls.
(C) A schematic structure of RNF168 (Ensembl
gene ID: ENSG00000163961).
(D) U-2-OS cells were treated with a control siRNA
(siCon) or two siRNAs (si#1 or si#2) targeting
distinct regions of RNF168 mRNA (left). Alterna-
tively, two U-2-OS cell lines (sh#1.1, sh#1.2)
conditionally expressing an RNF168-targeting
shRNA were induced or not with Doxycycline
(Dox) and incubated for 2 days (right). RNA and
proteins were extracted and subjected to RT-
MCM7 immunoblot is a loading control.
(E) U-2-OS cells were treated with the indicated
siRNAs, incubated for 2 days, subjected to IR
(3 Gy), and 1 hr later immunostained with the indi-
geting siRNA (si#1) for 24 hr, transfected with the
RNF168, incubated for 24 hr, irradiated as in (E),
and immunostained with an antibody to 53BP1.
Scale bars, 10 mm.
436 Cell 136, 435–446, February 6, 2009 ª2009 Elsevier Inc.
DSBs (Figures 1B and S1E). The gene encoding RNF168 (RING
finger protein 168) is located on chromosome 3 (3q29) and
displays structural hallmarks of a RING finger ubiquitin ligase
(Figure 1C). In addition to the RING domain, RNF168 possesses
two motifs interacting with ubiquitin (MIU) known to bind polyu-
biquitin chains (Penengo et al., 2006).
Having identified RNF168 in a screen, we tested whether it
regulates 53BP1 after the standard clastogen-induced DNA
breakage. Indeed, the accumulation of 53BP1 in IR-induced
transfection of siRNA (Figures 1D, 1E, and S2A) or by inducing
by several independent siRNAs targeting distinct regions of
RNF168 mRNA (Figures 1E and S2A), and the 53BP1 accumula-
Figure 2. Knockdown of RNF168 Impairs
Cellular Responses to DSBs
(A) U-2-OS cells were treated with the RNF168-
targeting siRNA for 2 days, microirradiated by
the laser, and 1 hr later immunostained with the
indicated antibodies. At least 100 cells were mi-
croirradiated for each condition and yielded
consistent results. Scale bars, 10 mm.
(B) U-2-OS cells were treated with the RNF168-
targeting (#1) or control (siCon) siRNAs for 2
days, exposed to IR (2 Gy), and at the indicated
time points immunostained with an antibody to
MDC1. The nuclei where the number of MDC1
foci exceeded the background level (10 foci per
nucleus) were counted. The graph is a summary
from two experiments and shows the percentage
of cells with IR-induced MDC1 foci relative to the
starting point (1 hr after IR); 400 cells were scored
for each time point. Error bars, SE.
(C) U-2-OS cells were treated as in (B), exposed to
IR (4 Gy), and at the selected time points analyzed
by immunoblotting with the indicated phospho-
specific antibodies. The immunoblots for total
RNF168 and MCM7 are controls for RNAi effi-
ciency and equal loading, respectively.
(D) U-2-OS cells were treated and irradiated as in
mined by flow cytometry. The graph is a summary
from two experiments. Error bars, SE.
tion at DSBs was rescued by the
(Figure 1F). Importantly, the latter experi-
ments revealed that RNF168 itself accu-
mulated at the DSB foci (Figure 1F,
bottompanel). Together,thesedata iden-
tified RNF168 as a candidate regulator of
the DSB-induced chromatin response,
and we set out to investigate the under-
RNF168 Regulates DSB-Associated
Ubiquitylation and Accumulation
of a Subset of Repair Proteins
We and others have recently described
RNF8, a RING domain E3 ligase, which,
by promoting local chromatin ubiquitylation, triggers the accu-
mulation of several repair complexes, including 53BP1 (see
Introduction). To test whether RNF168 is integrated in or oper-
under conditions that generate limited amounts of DNA strand
breaks in defined nuclear volumes (Bekker-Jensen et al., 2006;
Lukas et al., 2003). Under these conditions, knockdown of
RNF168 had no measurable effect on the extent of H2AX phos-
phorylation and accumulation of MDC1 and NBS1 (Figures 2A
and S3), consistent with our previous findings that these initial
steps of the DSB response do not require ubiquitylation (Mailand
et al., 2007). Strikingly, however, although the recruitment of
RNF8 was also normal in RNF168-depleted cells, the accumula-
tion of conjugated ubiquitin at the DSB sites was severely
Cell 136, 435–446, February 6, 2009 ª2009 Elsevier Inc. 437
impaired (Figures 2A and S3). Thus, downregulation of RNF168
created a situation in which RNF8 accumulated at the sites of
DNA damage, yet it was unable to sustain local chromatin ubiq-
uitylation. Consequently, 53BP1 and BRCA1, two repair factors
whose retention on damaged chromosomes requires ubiquitin
(see Introduction), were unable to accumulate at the micro-
laser-generated DSB tracks in the RNF168-deficient cells
(Figures 2A and S3), consistent with the lack of 53BP1 retention
at IR-induced nuclear foci observed earlier in RNF168-deficient
cells (see Figures 1E, 1F, S2A, and S2B). Together, RNF168
emerged from these experiments as a novel factor, which oper-
ates downstream of RNF8 and is required for sustained ubiquity-
lation of the DSB sites and accumulation of a subset of genome
caretakers on damaged chromosomes.
Knockdown of RNF168 Delays Recovery from Genotoxic
To test the significance of the RNF168-controlled events for the
DNA damage response, we silenced endogenous RNF168 in
U-2-OS cells by RNAi, exposed these cells to a moderate dose
of IR, and followed in time the MDC1-decorated DSB foci in indi-
vidual nuclei. Because MDC1 operates upstream of the ubiqui-
tin-dependent steps during
response (Figure 2A), it can serve as a sensitive indicator of the
DSB dynamics even in the absence of the DNA damage-associ-
ated E3 ligases. The number of nuclei with MDC1 foci detected
early (1 hr) after IR was very similar in control and RNF168-defi-
cient cells (92% and 98%, respectively). However, while in
control cells, MDC1 progressively dissociated from DSBs
(consistent with the ongoing DNA repair), knockdown of
RNF168 caused marked persistence of the MDC1 foci even at
the time points when the control cells returned to the predamage
values (Figures 2B and S4). This suggested delayed dynamics of
the DSB repair in RNF168-deficient cells, a notion that was
further supported by a prolonged phosphorylation of NBS1
and SMC1 (two genome caretakers targeted by ATM after
DNA damage) (Figure 2C), and by an extended IR-induced G2
arrest (Figure 2D). Based on these results, we conclude that
RNF168 facilitates restoration of genome integrity challenged
the DSB-induced chromatin
Assembly of RNF168 at DSBs Is Mediated
by Its Ubiquitin-Binding Domains
To mechanistically elucidate RNF168 function, we turned to the
earlier observation that RNF168, when ectopically expressed,
concentrated in the IR-induced nuclear foci (see Figure 1F).
We could extend this result by showing that also endogenous
RNF168 accumulates at DSBs generated either by the laser mi-
croirradiation (Figures 3A, top panel, and 4A) or IR (Figures 4C
and 4D). Of note, RNF168 localized to the broad, g-H2AX-deco-
rated chromosomal regions (see insets in Figures 4A and S5A)
throughout the interphase (Figure S5B), consistent with the
emerging evidence that the formation of the DSB-flanking chro-
matin compartment is cell-cycle independent (Bekker-Jensen
et al., 2006; Mailand et al., 2007).
To understand the structural underpinnings of the RNF168-
chromatin interaction, we silenced endogenous RNF168 by
siRNA, reintroduced into these cells various forms of YFP-
tagged (and siRNA-resistant) RNF168, and tested their ability
to accumulate at the DSB sites. Both wild-type RNF168 and its
variant bearing an inactivating mutation in the catalytic RING
domain (*RING) avidly accumulated at DSBs (Figure 3B). In
contrast, simultaneous mutations of both MIU domains (**MIU)
attenuated retention of RNF168 at the DSB sites (Figure 3B),
especially in cells with low levels of MIU-deficient RNF168
(Figure 3B, ‘‘Low’’). High overexpression of this mutant was still
the protein always remained dispersed in the nucleoplasm, indi-
‘‘High’’). Indeed, a rigorous measurement of protein mobility by
fluorescence recovery after photobleacing (FRAP) revealed
that the mean residence time, which wild-type RNF168 spent
bound to DSB (11.13 ± 0.22 s), decreased nearly 5-fold (to
2.47 ± 0.07 s) in the double-MIU mutant (Figure 3C). These
results suggested that the ubiquitin-binding domains contribute
to the retention of RNF168 at DSBs, and this was further sup-
ported by the observation that a short inhibition of the 26S pro-
teasome under conditions that disrupt chromatin-associated
tion of RNF168 at DSBs (Figure 3A, bottom panel).
Maturation of the DSB-Flanking Chromatin Requires
Catalytic and Ubiquitin-Binding Domains of RNF168
Because the above complementation assays were performed in
cells with silenced endogenous RNF168, they allowed us to test
the impact of RNF168 structural domains on ubiquitylation of the
DSB-flanking chromatin and retention of repair proteins in this
compartment (a process that we have recently dubbed as
‘‘maturation of the DSB-flanking chromatin’’) (Mailand et al.,
2007). Strikingly, whereas cells complemented with wild-type
RNF168 regained the ability to generate ubiquitin conjugates at
the DSB sites (Figure 3B, top panel), both the *RING mutant
(able to assemble at DSBs but impaired in its E3 ligase activity)
(Figure 3B, middle panel) and the moderate levels of the **MIU
mutant (active but deficient in DSB interaction) (Figure 3B,
bottom panels, ‘‘Low’’) failed to rescue DSB ubiquitylation.
In the latter case, we noticed that massive overexpression of
MIU-deficient RNF168 did induce some DSB-associated ubiqui-
tylation (Figure 3B, bottom panels, ‘‘High’’). Because such
extreme levels appear to subvert natural requirements for
RNF168 interaction with damaged chromatin, we wanted to
quantify more exactly, and under more physiological conditions,
the function of the MIU domains. We generated cell lines stably
expressing moderately elevated wild-type or MIU-deficient
versions of RNF168, both tagged with GFP and rendered RNAi
insensitive (Figure S6A). After silencing the endogenous
RNF168, the wild-type GFP-RNF168 avidly accumulated at the
laser-generated DSB sites and induced massive ubiquitylation
in this compartment (Figure 3D). In contrast, the MIU-deficient
RNF168, expressed to a very similar level as the wild-type
protein (Figure S6A), accumulated poorly and was significantly
impaired in generating ubiquitin conjugates at the DSB-flanking
chromatin (Figure 3D). These results were exactly mirrored in an
assay in which we measured retention of 53BP1, a sensitive
surrogate of DSB-associated ubiquitylation (see Introduction).
Also here, the wild-type GFP-RNF168, but not the MIU-deficient
438 Cell 136, 435–446, February 6, 2009 ª2009 Elsevier Inc.
mutant, efficiently restored accumulation of 53BP1, both at the
laser-generated DSB tracks (Figure S7A) and in the IR-induced
nuclear foci (Figure S7B). Thus, we conclude that the maturation
of the DSB-flanking chromatin is facilitated by the ubiquitin-
dependent recruitment of RNF168 into this compartment.
Recruitment of RNF168 to DSBs Requires RNF8
One potential explanation for the MIU-mediated accumulation of
RNF168 at DSBs would be if RNF168 could trigger its own
recruitment by catalyzing local chromatin ubiquitylation. This,
however, seemed unlikely because the RING-deficient form of
RNF168could still accumulate
Figure 3B). Therefore, we tested whether the recruitment of
RNF168 to DSBs requires RNF8 or BRCA1, the other two E3
ligases known to associate with the DSB-flanking chromatin.
Whereas knockdown of BRCA1 had no measurable effect,
downregulation of RNF8 inhibited RNF168 accumulation at
DSBs (Figure 4A). Consistently, knockdown of MDC1, the
upstream regulator of RNF8, also prevented accumulation of
RNF168 in this compartment (Figure 4A). Because the knock-
down of MDC1 or RNF8 did not alter the total levels of endoge-
at the DSBsites(see
nous RNF168 (Figure S6B), we conclude that the MDC1/RNF8
pathway regulates the RNF168 recruitment to the DSB compart-
of GFP-RNF168 in the undamaged nucleoplasm (Figure S8),
indicating that the functional interplay between RNF8 and
RNF168 is restricted to the sites of DNA damage.
To explore the hierarchy with which the distinct E3 ligases
accumulate at DSBs in vivo, we subjected microirradiated cells
stably expressing moderate levels of GFP-tagged RNF8,
RNF168, or BRCA1 (Figures S6A and S9 and see Mailand
et al., 2007) to a time-lapse microscopy and determined the
time (t1/2) when a given protein reached half-maximum of its
saturation in the DSB compartment (Lukas et al., 2004a and
see Supplemental Experimental Procedures). These measure-
ments revealed that each E3 ligase accumulated with distinct
dynamics (Figures 4B and S9). Thus, the fastest protein to
assemble at DSBs was RNF8 (t1/2 = 1.15 min), followed by
RNF168 (t1/2 = 2.21 min), and only then by BRCA1 (t1/2 =
3.40 min). Collectively, these in vivo measurements supported
downstream of RNF8 but upstream of BRCA1.
Figure 3. Assembly and Function ofRNF168
at the DSB Sites Is Facilitated by Its Ubiqui-
(A) U-2-OS cells were microirradiated, incubated
for 1 hr, and immunostained with the indicated
antibodies. Where indicated, the cells were incu-
bated for 90 min with MG132 (5 mM) before micro-
irradiation. The lack of RNF168 accumulation at
DSBs in the Mg132-treated cells was observed
in more than 100 microirradiated cells.
(B) U-2-OS cells were treated with the RNF168-
targeting siRNA (#1) for 24 hr, transfected with
the indicated RNAi-resistant RNF168 constructs,
incubated for 24 hr, microirradiated, incubated
for an additional 1 hr, and coimmunostained with
antibodies to conjugated ubiquitin (FK2) and
consistent results. Arrows mark the cells in which
the transgene expression restored DSB ubiquity-
lation; asterisks indicate cells with inefficient
(C) U-2-OS cells were transfected with the indi-
cated forms of GFP-RNF168, incubated for
24 hr, and microirradiated as in (B). At 1 hr later,
the exchange rate of the GFP-tagged proteins at
the DSB sites was determined by FRAP. The
FRAP curves were derived from ten cells for
each condition. NFU, normalized fluorescence
units. Error bars, SE.
(D) U-2-OS cell lines stably expressing the indi-
cated versions of RNAi-resistant GFP-RNF168
were microirradiated and immunostained as in
(B). The microirradiated cells were scored for the
presence of conjugated ubiquitin at the DSB
tracks. The images (top) show representative
fields; the graph (bottom) is a summary of two
independent experiments in which at least 50 cells
for each category were scored. Error bars, SE.
Scale bars, 10 mm.
Cell 136, 435–446, February 6, 2009 ª2009 Elsevier Inc. 439
Figure 4. Requirements and Dynamics of RNF168 Accumulation at the DSB Sites
(A) U-2-OS cells were treated with the siRNAs indicated on the left for 3 days, microirradiated, and immunostained with the indicated antibodies. At least 100
microirradiated cells were scored for each siRNA treatment and showed consistent results.
(B) U-2-OS cells stably expressing the indicated GFP-tagged proteins were microirradiated, and the GFP-associated fluorescence intensities in the DSB tracks
(C) U-2-OS cells were transfected with the expression plasmid for Myc-USP3 for 24 hr, exposed to IR (4 Gy), and 1 hr later immunostained with the indicated
marks a cell in which Myc-USP3 did not impair RNF8 focus formation.
(D) U-2-OS cells were transfected with plasmids for wild-type (WT) or catalytically inactive (C168S) forms of Myc-USP3, irradiated, and immunostained as in (C).
Arrow marks a cell in which WT Myc-USP3 prevented IR-induced focus formation of RNF168; asterisk marks a cell in which inactive USP3 failed to suppress
RNF168 foci. Scale bars, 10 mm.
440 Cell 136, 435–446, February 6, 2009 ª2009 Elsevier Inc.
Retention of RNF168 at DSBs Is Mediated
by Ubiquitylated H2A
We next set out to elucidate how RNF8 promotes RNF168 chro-
matin retention. We have recently shown that H2A can be
ubiquitylated by RNF8 in vitro and that both basal and DNA
damage-induced H2A ubiquitylations are influenced by RNF8
in cells (Mailand et al., 2007). Another recent study identified
USP3 as a deubiquitylating enzyme capable of reverting H2A
ubiquitylation (Nicassio et al., 2007), suggesting that USP3 and
RNF8 operate, at least in part, as opposing enzymatic activities.
Indeed, we could support this concept by showing that, while
overexpression of USP3 had no effect on the DSB retention of
RNF8 itself, it abolished the IR-induced focus formation of
53BP1 and RAP80, two chromatin-binding factors whose reten-
tion at the DSB sites requires RNF8 (Figure 4C). But most signif-
icantly in thiscontext, overexpression of wild-type USP3, butnot
its catalytically inactive mutant, prevented the IR-induced focus
formation of RNF168 (Figures 4C and 4D), indicating that the
assembly of RNF168 at DSBs requires H2A ubiquitylation.
The latter conclusion was further supported by a biochemical
analysis of chromatin-enriched fractions from irradiated 293T
cells expressing various forms of Strep-tagged RNF168.
Whereas the wild-type RNF168 avidly bound to mono-, di-,
Figure 5. RNF168 Binds and Targets for
Ubiquitylation a Subset of Histones
(A) 293T cells were transfected with the indicated
versions of Strep-RNF168 and after 24 hr exposed
to IR (10 Gy). At 1 hr later, the chromatin fractions
were isolated, and the Strep-containing com-
plexes werepurified and analyzedby immunoblot-
ting with the indicated antibodies.
(B) 293T cells were treated with RNF8 or control
siRNAs for 48 hr, transfected with the Strep-
RNF168 (WT) expression plasmid, incubated for
24 hr, irradiated (10 Gy), and 1 hr later analyzed
as in (A).
(C) U-2-OS cells were cotransfected with the
FLAG histones, Strep-RNF8, and Myc-Ubiquitin
expression plasmids as indicated, incubated for
24 hr, lysed in denaturing buffer, and subjected
to immunoprecipitation with anti-FLAG agarose
beads under denaturing
complexes were analyzed by immunoblotting
with the indicated antibodies.
and, to some extent, even polyubiquity-
lated H2A, the disruption of both MIU
motifs abolished this interaction (Fig-
ure 5A). This was reflected by the ability
of thewild-type, butnotthe MIU-deficient
version of RNF168, to copurify with
nucleosomes (exemplified by the core
histone H2B in Figure 5A). Of note, the
comparison of the abundance of H2A
and H2B in these fractions revealed that
RNF168 bound poorly to unmodified
H2A (Figure 5A), indicating that the
RNF168-containing complexes preferen-
tially contained nucleosomes enriched in ubiquitylated H2A.
Importantly, in a complementary experiment, knockdown of
RNF8 markedly decreased the chromatin-bound fraction of
wild-type RNF168 (Figure 5B). Together, these data suggested
that RNF8-mediated ubiquitylation of H2A generates a docking
signal for RNF168.
RNF168 Ubiquitylates H2A and H2AX and Triggers
Generation of Lysine 63-Linked Ubiquitin Conjugates
Having established the hierarchy in the RNF8 and RNF168
assembly at the DSB sites, we set out to investigate how these
two E3 ligases cooperate in increasing local chromatin ubiquity-
lation. To this end, we tested, by an in vivo ubiquitylation assay,
whether RNF168 can target various histones (Mailand et al.,
2007) and found that RNF168 efficiently ubiquitylated H2A and
H2AX, but not H2B or H3 (Figure 5C). The striking ramification
of this result was that the substrate specificity of RNF168 under
these conditions was very similar to the one described earlier for
RNF8 (Mailand et al., 2007), suggesting that RNF168 may stabi-
lize and/or increase histone modifications initiated by RNF8.
It has been reported that RNF8 forms an active holoenzyme
with UBC13, the only known E2 ubiquitin-conjugating enzyme
capable of generating the lysine 63 (K63)-linked ubiquitin chains
Cell 136, 435–446, February 6, 2009 ª2009 Elsevier Inc. 441
(Plans et al., 2006). Consistently, by means of overexpressing
various lysine-deficient ubiquitin mutants, a recent study re-
ported preferential accumulation of K63-linked ubiquitin poly-
mers at the IR-induced nuclear foci (Sobhian et al., 2007). Stim-
ulated by these findings, we reasoned that, if RNF168
propagated the RNF8 signaling, it should be able to generate
K63-linked ubiquitin chains at the DSB-modified chromatin. To
test this, we immunostained microirradiated cells with the
linkage-specific antibodies that discriminate ubiquitin chains
joined by K63 and K48, respectively (Newton et al., 2008).
Indeed, whereas the anti-K63 linkage antibody robustly deco-
rated the laser-generated DSB tracks (Figure 6A, top panel), no
K48-linked ubiquitin chains were detected under these condi-
tions (Figure 6A, bottom panel), and the accumulation of K63-
linked ubiquitin was completely abrogated by knocking down
endogenous RNF168 (Figure 6A, middle panel). Importantly,
we were able to complement these results by an in vivo ubiqui-
tylation assay, which revealed that the RNF168-induced ubiqui-
Figure 6. RNF168 Binds UBC13 and Cata-
lyzes Formation of K63-Linked Ubiquitin
(A) U-2-OS cells were microirradiated and 1 hr
later immunostained with antibodies to K63- or
K48-linked ubiquitin conjugates as indicated. All
cells we coimmunostained with an antibody to
g-H2AX to detect the DSB tracks. Where indi-
cated, cells were treated with control (siCon)- or
RNF168 (#1)-targeting siRNAs for 2 days before
microirradiation. At least 100 microirradiated cells
were scored for each condition and showed
consistent results. Scale bars, 10 mm.
(B) The 293 cells were cotransfected with the indi-
cated constructs, subjected to anti-FLAG immu-
noprecipitation under denaturing conditions as in
Figure 5C, and analyzed by immunoblotting with
antibodies to the Myc-tag and the K63- or K48-
linked ubiquitin conjugates. The immunoblotting
in the bottom panel is a control for expression of
FLAG-H2A and Strep-RNF168, respectively.
(C) U-2-OS cells were cotransfected with the HA-
UBC13 and Strep-RNF168 constructs. After
24 hr, the Strep complexes were purified and
analyzed by immunoblotting with the indicated
antibodies. WCE, whole-cell extracts.
tylation of H2A largely contained K63-
linked, but not K48-linked, ubiquitin
chains (Figure 6B). Although the latter
results were obtained by overexpressed
proteins, the fact that knocking down of
lation of K63 ubiquitin linkages at DSBs in
cells (see Figure 6A) strongly suggests
that RNF168 catalyzes formation of this
type of ubiquitin chains on damaged
chromatin. Consistently, we could show
that RNF168 and UBC13 physically inter-
acted in cells (Figure 6C).
Interestingly, by a meticulous monitoring of the DSB-associ-
ated ubiquitylations during the first 10 min after DNA damage,
wefound thatthe temporal accumulation of conjugated ubiquitin
at DSBs tightly correlated with the retention of RNF168 in this
compartment (Figure S10A) and that no increase in local ubiqui-
tin concentration was observed in cells with depleted RNF168,
even in the earliest time points (Figure S10B). These data further
supported the hypothesis that the ubiquitin conjugates gener-
ated by RNF8 are transient and/or unstable and require amplifi-
cation and/or stabilization by RNF168 to achieve the threshold
needed for the completion of the DSB-induced chromatin
RNF8 and RNF168 Constitute a Pathway Required
for Efficient Cellular Responses to Genotoxic Stress
Finally, to address whether RNF8 and RNF168 operate along
a common pathway, we combined treatment of cells by ionizing
radiation with a protocol allowing us to genetically manipulate
442 Cell 136, 435–446, February 6, 2009 ª2009 Elsevier Inc.
the ‘‘dosage’’ of RNF168. Specifically, we tested numerous
RNF168-targetting siRNA oligonucleotides until we found
a pair in which one siRNA (labeled as ‘‘#1’’) induced a robust
downregulation of RNF168 at both mRNA and protein levels
(Figure 7A, lane 2), whereas the other (labeled as ‘‘#6,’’ targeting
the 30untranslated region) reduced the RNF168 expression less
efficiently (Figure 7A, lane 3). Importantly, the degree of RNF168
downregulation achieved by either of these siRNAs was suffi-
cient to prevent any cytologically detectable retention of
53BP1 at the IR-induced nuclear foci (Figure 7B, gray bars;
see also Figures 1E and S2A). We then overexpressed in these
cells RNF8 and monitored the 53BP1 focus formation. Strikingly,
whereas the elevated RNF8 was unable to support 53BP focus
formation in cells with strongly depleted RNF168, it effectively
induced 53BP1 foci in cells with a higher residual amount of
RNF168 (Figures 7B and S11). These results suggested that,
although RNF8 cannot ‘‘rescue’’ the near-to-complete absence
of RNF168, the increased level of the former is able to cooperate
with the residual amount of the latter to execute the DSB-
induced chromatin response and promote retention of repair
proteins in this compartment.
To independently test the emerging epistatic relationship
between RNF8 and RNF168, we exploited an earlier observation
that the cell line conditionally expressing the RNF168-targeting
shRNA (shRNA#1.2) also contained more residual amount of
RNF168 when compared to the strong siRNA oligonucleotide
diated cells after induction of the ‘‘milder’’ shRNA was better
than after exposing the cells to the ‘‘stronger’’ siRNA (compare
yellow lines in Figures 7C and 7D). But most importantly, while
Figure 7. RNF168 and RNF8 Cooperate to
Promote Chromatin Retention of 53BP1
and to Protect Cells against DSB-Gener-
(A) U-2-OS cells were treated with a control siRNA
(siCon) or two distinct RNF168-targeting siRNAs
(#1 and #6, respectively). After 2 days, RNA and
proteins were extracted and subjected to RT-
The relative abundance of RNF168 mRNA and
protein for each treatment is indicated in red.
MCM7 is a loading control.
(B) U-2-OS cells were treated by the indicated
siRNAs as in (A) and after 24 hr transfected with
a control (nuclear GFP; nclGFP) or HA-tagged
RNF8 (HA-RNF8) expression plasmids. After
24 hr, the cells were exposed to IR (4 Gy), incu-
bated for 1 hr, immunostained with the indicated
antibodies, and scored for the IR-induced 53BP1
foci. The graph is a summary from two experi-
ments in which at least 100 cells for each category
(C) The U-2-OS cell line conditionally expressing
RNF168-targeting shRNA (U-2-OS/shRNA#1.2)
was transfected with the indicated siRNAs and 1
day later induced or not by Doxycycline (Dox).
tion, incubated for an additional 1 day in the
medium with or without Dox, and exposed to the
indicated doses of IR. After 11 days, the dishes
were stained with crystal violet, and the colonies
with more than 50 cells were counted. Three inde-
pendent experiments were performed, each with
RNF168-depleted cells were highly significant
(see also Supplemental Experimental Procedures
for the statistical analysis).
(D) U-2-OS cells (the same strain as in [C] but
without induction) were transfected with the indi-
cated siRNAs and subjected to the clonogenic
survival assay as in (C). The efficiencies of si/
shRNA downregulation for experiments in (B–D)
are shown in Figures 1D and 7A (RNF168) and
(B–D) Error bars, SE.
(E–G) A hypothetical model of ubiquitylation
cascade within the DSB-modified chromatin
compartment. See Discussion for details.
Cell 136, 435–446, February 6, 2009 ª2009 Elsevier Inc. 443
the concomitant knockdown of RNF8 had no additive effect
on IR sensitivity of cells with strongly depleted RNF168
(Figure 7D, yellow and orange lines), it significantly reduced
survival in cells with less effective (‘‘hypomorphic’’) downregula-
are consistent with a model in which RNF8 and RNF168 operate
on a shared pathway that facilitates survival after genotoxic
Our study reveals an unexpected complexity of how regulatory
ubiquitylation orchestrates the chromatin response to DNA
breakage (Figures 7E–7G). Although RNF8 is the first E3 ligase
to assemble at the DSB-modified chromatin (Figure 7E), it is
not sufficient to support sustained ubiquitylation. Instead,
RNF8 appears to prime the DSB-modified chromatin for recruit-
ment of RNF168, another E3 ligase (Figure 7F), whose activity is
essential to amplify the initial DSB-associated ubiquitylations to
a threshold required for the physiological function of this
compartment (Figure 7G).
We can envisage two scenarios in which RNF168 increases
local ubiquitylation at damaged chromosomes. First, it is
possible that some targets of RNF168 overlap with those of
RNF8. In such a scenario, RNF168 may not only amplify its
own retentionat theDSB-flanking chromatin, but alsopropagate
the initial chromatin ubiquitylations (initiated by RNF8) to the
neighboring nucleosomes. Indeed, our results provide several
lines of evidence in support of such a model. Thus, we were
able to show that both RNF8 and RNF168 target for ubiquityla-
tion the same spectrum of histones (H2A and H2AX) (see
Figure 5).Inaddition, wefound that,like RNF8,RNF168 interacts
Furthermore, we provide evidence that, while neither of the two
levels of the other (RNF168) to trigger ubiquitin-dependent chro-
codepletion of RNF8 (Figure 7C). Collectively, these results are
consistent with an ‘‘epistatic’’ model in which the initial ubiquitin
polymers generated by RNF8 are recognized by the ubiquitin-
binding domain of RNF168 and then stabilized and/or amplified
to generate an ‘‘interaction trap’’ for additional factors required
for restoration of genome integrity (Figure 7E–7G).
There is also a second possibility, namely that RNF168 may
extend the spectrum of the DSB-associated ubiquitylations by
targeting unique substrate(s). Although we do not have direct
evidence in support of this hypothesis, we by no means want
to exclude it. Clearly, the key challenge forthe future isto identify
the spectrum of unique versus overlapping substrates of RNF8
and RNF168 E3 ligases, a task that, in order to solve this issue
in full, would have to include systems biology approaches such
as differential analysis of ubiquitylated proteomes in cells with
genetically disrupted RNF8 and RNF168, respectively. Impor-
tantly, the above scenarios are not mutually exclusive, and
both are compatible with our main conclusion, namely that the
key function of RNF168 in genome surveillance is to increase
chromatin ubiquitylation to the threshold required for retention
of important repair factors such as 53BP1 or BRCA1. Interest-
ingly, BRCA1 is also a RING domain E3 ligase, and there is
evidence that cells with dysfunctional BRCA1 accumulate less-
conjugated ubiquitin at the DSB sites (Polanowska et al.,
2006). Thus, the maturation of the DSB-flanking chromatin can
be viewed as a pathway initiated by RNF8, amplified by
RNF168, and maintained by BRCA1 (Figures 7E–7G).
Such a concept has important ramifications. The sequential
involvement of three ubiquitin ligases and, indeed, the massive
accumulation of conjugated ubiquitin at the DSB-modified chro-
matin suggest that regulatory ubiquitylation represents a crucial
posttranslational modification generated in the vicinity of these
highly cytotoxic chromosomal lesions. Furthermore, the RNF8-
RNF168-BRCA1 pathway can be viewed as an unprecedented
type of functional interplay among the RING domain E3 ligases.
Although the mutual crosstalk within the RING family of enzymes
is well established, the known examples are almost exclusively
based on the assembly of distinct RING domain proteins into
tionalholoenzymes(Buchwald etal.,2006).Incontrast, wecould
not detect direct binding between RNF8 and RNF168, and
BRCA1 did not seem to interact with either of the former proteins
(our unpublished data). Instead, it seems that a ubiquitylation
event catalyzed by the more upstream E3 ligase in this pathway
triggers the recruitment of the downstream component, which
can then amplify and/or maintain a high concentration of conju-
gated ubiquitin near the DSB lesions as long as it is needed for
efficient repair (Figures 7E–7G).
Finally, whereas the sequential involvement of three E3 ligases
provides an opportunity for a more subtle regulation of the DSB-
induced chromatin response, it also broadens the target for
potential mutations and subversion of this pathway in disease.
Most notably in this regard, BRCA1 is an established tumor
suppressor, whose mutations predispose to familial breast and
ovarian cancer (Boulton, 2006). Furthermore, other studies un-
derscored the importance of the ubiquitin-driven pathways for
genomic stability by showing that inhibition of the proteasome
(which, among other effects, impairs regulatory ubiquitylation
in the nucleus) suppresses DNA repair by homologous recombi-
nation (Murakawa et al., 2007). Strikingly, the locus encoding
RNF168, the upstream component of the BRCA1 ‘‘assembly
line,’’ has been found mutated in the RIDDLE (radiosensitivity,
immune deficiency, dysmorphic features, and learning difficul-
ties) syndrome (Stewart et al., 2009 [this issue of Cell]). Thus,
the RNF8-RNF168-BRCA1 cascade emerges as a target for
tant to test whether its alterations can explain the defects in hith-
erto elusive clinical cases with hallmarks of radiation sensitivity
and/or increased incidence of genome instability.
Screen for 53BP1 Regulators
The screening procedure using the siRNA arrays was carried out as described
(Erfle et al., 2007) with modifications specified in Figures S1A–S1G. Briefly,
U-2-OS cells were seeded on the siRNA arrays, incubated for 3 days,
444 Cell 136, 435–446, February 6, 2009 ª2009 Elsevier Inc.
immunostained with antibodies to 53BP1, and analyzed by high-content
microscopy for the retention of 53BP1 in the intranuclear foci. The complete
screening procedure is described in the Supplemental Experimental Proce-
Plasmids, PCR, and RNA Interference
ThecDNA for human RNF168, tagged on the N terminus by YFP,wasobtained
from Imagenes (http://www.imagenes-bio.de/). The RNF168 ORF was
rendered si/shRNA resistant by introducing silent mutations specified in the
Supplemental Experimental Procedures. The point mutations in the RING
domain (*RING; C16S) and the MIU domains (**MIU; A179G-A450G) (Penengo
et al., 2006) were generated using the QuickChange Site-Directed Mutagen-
esis Kit (Stratagene). The GFP-, GST-, and Strep-tagged versions of
RNF168 were generated by subcloning the respective cDNAs into the pAc-
GFP-C1 (Clontech), pGEX-6P (Amersham Biosciences), and pEXPR-103
(IBA BioTAGnology), respectively. The cDNA for USP3 was obtained from Im-
agenes; the mutation in its catalytic site (C168S) was generated by site-
directed mutagenesis and subcloned into pcDNA3-Myc (Invitrogen). The
expression plasmid for HA-RNF8 was described (Mailand et al., 2007). The
plasmid for HA-Ubc13 (pEF-HA-Ubc13) was a gift from Dr. Ze’ev Ronai. For
RT-PCR, RNA was extracted by the RNeasy Mini Kit (Invitrogen), and the
PCR reaction was performed by One Step RT PCR Kit (QIAGEN) with primers
specified in the Supplemental Experimental Procedures. Plasmid transfec-
tions were performed using FuGene 6 (Roche Molecular Biochemicals). All
siRNA transfections were performed with 100 nM siRNA duplexes using Lipo-
fectamine RNAiMAX (Invitrogen) and with oligonucleotide sequences speci-
fied in the Supplemental Experimental Procedures.
Cell Culture and Generation of DSBs
Human U-2-OS osteosarcoma cells and 293T human embryonic kidney cells
were grown in DMEM containing 10% fetal bovine serum (GIBCO). U-2-OS
cell lines expressing GFP-RNF8 and GFP-BRCA1 were described (Mailand
et al., 2007). U-2-OS cell lines expressing various forms of GFP-RNF168 are
characterized in Figure S6A. U-2-OS cell lines expressing the RNF168-target-
ingshRNAinaDoxycycline-inducible mannerwere generated with oligonucle-
otidesspecified inthe SupplementalExperimentalProceduresand induced as
in Mailand et al. (2006). Flow cytometry was performed as in Sorensen et al.
(2000). Survival curves in clonogenic assays were analyzed using the linear
quadratic model for the relationship between cell survival and radiation
dosage: Ln(SF) = aD + bD2, in which SF is the survival fraction, and D is the
radiation dose. Comparison of the survival curves for different treatments
was performed as described (Franken et al., 2006). IR was delivered by the
X-ray generator (Pantak HF160, 150 kV, 15 mA, dose rate 2.18 Gy/min). Laser
microirradiation and conditions for time-lapse microscopy were described
(Bekker-Jensen et al., 2006; Lukas et al., 2003, 2004a).
Immunochemical and Biochemical Methods
Rabbit antibody to RNF168 is characterized in the accompanying paper by
Stewart et al. (2009). The linkage-specific antibodies to K63 (Apu3.A8) or
K48 (Apu2.07) ubiquitin conjugates were provided by Kim Newton and Vishva
M. Dixit (Genentech) (Newton et al., 2008). Antibodies to MDC1, RNF8, and
53BP1 were gifts from Drs. Stephen Jackson, Junjie Chen, and Thanos Hala-
zonetis, respectively. Commercially available antibodies are specified in the
Supplemental Experimental Procedures. Conditions for immunoprecipitation,
immunoblotting, and immunofluorescence were described (Bekker-Jensen
et al., 2006; Mailand et al., 2006). Preparation of chromatin-enriched fractions
and conditions for in vivo ubiquitylation were described (Mailand et al., 2007).
Strep-tagged RNF168 protein complexes were purified on Strep-Taction
Sepharose (IBA BioTAGnology) according to the manufacturer’s instructions.
Confocal images were acquired on LSM-510 (Carl Zeiss Microimaging Inc.)
mounted on an upright Zeiss-Axioimager, equipped with an oil-immersion
objective (Plan-Apochromat 403/1.3). Image analysis procedures, real-time
recruitment assays, and quantitative photobleaching techniques (covering
both screening and experimental parts of this study) are described in detail
in the Supplemental Experimental Procedures.
The Ensembl accession number for the RNF168 sequence reported in this
paper is ENSG00000163961.
The Supplemental Data include Supplemental Experimental Procedures and
11 figures and can be found with this article online at http://www.cell.com/
We thank Junjie Chen, Stephen Jackson, Thanos Halazonetis, Ze’ev Ronai,
Kim Newton, and Vishva M. Dixit for reagents; Mark R. Payne for help with
statistical analysis; and Holger Erfle and Beate Neumann for assistance with
the siRNA screen. This work was supported by the Danish Cancer Society,
Danish National Research Foundation, European Commission Integrated
Projects ‘‘DNA Repair’’ and ‘‘GENICA,’’ Lundbeck Foundation (R13-A1287),
Danish Research Council, and the John and Birthe Meyer Foundation. J.E.
and R.P. acknowledgesupport from the GermanFederal Ministryof Education
and Research (BMBF) grant within the National Genome Research Network
(NGFN-1, SMP-RNAi, 01GR0403) and the European Commission Integrated
Project Mitocheck (FP6-503464).
Received: July 3, 2008
Revised: October 17, 2008
Accepted: December 17, 2008
Published: February 5, 2009
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