Molecular Cell

Review

Regulation of DNA Damage Responses

by Ubiquitin and SUMO
Stephen P. Jackson1,* and Daniel Durocher2,3,*
1The Gurdon Institute and the Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, UK
2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada
3Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada

*Correspondence: [email protected] (S.P.J.), [email protected] (D.D.)

http://dx.doi.org/10.1016/j.molcel.2013.01.017

Ubiquitylation and sumoylation, the covalent attachment of the polypeptides ubiquitin and SUMO, respec-
tively, to target proteins, are pervasive mechanisms for controlling cellular functions. Here, we summarize
the key steps and enzymes involved in ubiquitin and SUMO conjugation and provide an overview of how
they are crucial for maintaining genome stability. Specifically, we review research that has revealed how ubiq-
uitylation and sumoylation regulate and coordinate various pathways of DNA damage recognition, signaling,
and repair at the biochemical, cellular, and whole-organism levels. In addition to providing key insights into
the control and importance of DNA repair and associated processes, such work has established paradigms
for regulatory control that are likely to extend to other cellular processes and that may provide opportunities
for better understanding and treatment of human disease.

Principles of Ubiquitylation and Sumoylation used to form chains, while SUMO1 can be conjugated as a
Although initially discovered as a mechanism targeting proteins chain-terminator. Consistent with different ubiquitin and SUMO
for destruction by the proteasome, ubiquitylation—the covalent chains having different structures and physical properties, they
attachment of the 76 amino acid residue protein ubiquitin to have distinct functions. For example, while Lys48-, Lys29-, and
other proteins—is now also known to regulate protein activity, Lys11-linked ubiquitin chains promote target-protein degrada-
localization, and interactions (Bergink and Jentsch, 2009; tion by the proteasome, Lys63 chains generally regulate
Komander and Rape, 2012). In addition to ubiquitin, there are protein-protein interactions. There is also evidence for ubiquitin
several ubiquitin-like proteins (UBLs) that are structurally related chains with mixtures of linkages (Komander and Rape, 2012),
to ubiquitin. Ubiquitin and most UBLs are attached via their as well as chains containing both ubiquitin and SUMO (Praefcke
C-terminal glycine residues to target proteins by enzymatic et al., 2012).
reactions mediated by E1, E2, and E3 ligases (Figure 1). The Like other posttranslational modifications, sumoylation and
most widely characterized UBL is the 100 residue protein ubiquitylation are reversible. While SUMO-protein isopeptide
SUMO (small ubiquitin-related modifier). Eukaryotes usually bonds are cleaved by a small family of peptidases (SENP1–
contain a single type of ubiquitin that is encoded by multiple SENP3 and SENP5–SENP7), there are 100 deubiquitylating
genes. By contrast, vertebrate cells possess two types of enzymes (also known as deubiquitylases or DUBs). DUBs are
SUMO: SUMO-1 and the highly related proteins SUMO-2 and grouped into five families: ubiquitin C-terminal hydrolases
SUMO-3 (SUMO2/3) that appear to be functionally redundant. (UCHs), ubiquitin-specific proteases (USPs) and ovarian tumor
Simpler organisms such as Saccharomyces cerevisiae and proteases (OTUs), the Josephins, and the Jab1/MPN/Mov34
Schizosaccharomyces pombe, however, contain a single SUMO family (JAMM/MPN+). The first four families are Cys proteases,
(Smt3 and Pmt3, respectively). In mammals, ubiquitylation whereas the latter comprises Zn2+-dependent metalloproteases
involves two E1s, over 35 E2s, and over 600 E3s, while sumoy- (Nijman et al., 2005). In addition to opposing ubiquitin/SUMO
lation is mediated by a single heterodimeric E1, one E2 (UBC9/ ligase activities, certain DUBs and SENPs process ubiquitin
UBE2I), and approximately ten E3s. and SUMO precursors, and some DUBs are intrinsic compo-
Ubiquitin and SUMO are usually attached to substrates via nents of the proteasome.
isopeptide linkages between their C termini and the εNH2 group
of Lys residues on target proteins. In some cases, the target DNA Repair and the DNA Damage Response
protein has a single ubiquitin or SUMO attached, while in others, Genome integrity is continuously undermined by exogenous and
several can be individually attached to multiple Lys residues on endogenously generated DNA-damaging chemicals, ionizing
the target. Furthermore, because ubiquitin and some SUMOs radiation (IR) and ultraviolet (UV) radiation, and by errors in
possess modifiable lysine residues, conjugation cycles can often DNA replication. To mitigate this, cells possess highly effective
be repeated to produce polymeric chains (Bergink and Jentsch, mechanisms—collectively called the DNA damage response
2009). In the case of ubiquitin, seven Lys residues can be used (DDR)—to detect, signal, and repair DNA lesions. These pro-
(Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) along cesses have profound impacts on normal cell and organism
with the amino group of the N-terminal Met. SUMO2/3 but not physiology, with their deregulation or loss causing genome
SUMO1 bear internal sumoylatable Lys residues that can be instability syndromes that are associated with cancer, stem

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Review

Figure 1. The Ubiquitin and UBL Conjugation Cycle

Ubiquitin and SUMO are produced as precursor polypeptides that are first
processed to reveal a C-terminal diglycine motif (1). An E1 enzyme then uses
ATP to convert this motif into a high-energy-bond containing adenylated
derivative, which is short-lived, rapidly reacting with a Cys in the E1 to form
a E1Ub or E1SUMO thioester intermediate (2). The ubiquitin or SUMO Figure 2. The roles of Ubiquitin and SUMO in Postreplication Repair
moieties are then coupled, via a transesterification reaction, onto a Cys residue (A) PCNA monoubiquitylation on Lys164 occurs when replication forks
in the E2 catalytic site to form E2Ub/SUMO intermediates (3). In most cases, encounter lesions (star). The single-stranded DNA is recognized by the Rad18
an E3 ligase serves as a substrate adaptor, linking the charged E2 and the E3 ligase, which interacts with Rad6, an E2, to ubiquitylate PCNA. PCNA
substrate (4). Ubiquitylation or sumoylation can be reversed by a DUB (for ubiquitylation recruits Y family polymerases such as Polh to bypass the lesion
ubiquitin) or a SENP (for SUMO) (5) or the modification cycle can be repeated in the process termed translesion synthesis (TLS). In vertebrates the USP1-
to produce chains of various topologies (6). The (*) indicates that other types of UAF1 DUB opposes PCNA monoubiquitylation.
enzymes can remove UBL modifications. (B) A second DNA damage tolerance pathway termed template switching
occurs when PCNA is polyubiquitylated by the E3 Rad5 and the Ubc13/Mms2
cell exhaustion, developmental defects, infertility, immune defi- dimeric E2. Template switching employs homologous recombination.
(C) In yeast, PCNA sumoylation on its Lys164 residue recruits Srs2, which acts
ciency, neurodegenerative disease, and premature aging (Jack- as an antirecombinase by disrupting Rad51 nucleoprotein filaments.
son and Bartek, 2009).
Different DNA lesions are repaired by distinct systems. Thus,
DNA double-strand breaks (DSBs) are repaired by nonhomolo- Ubiquitylation and Postreplication Repair
gous end joining (NHEJ), alternative NHEJ, or homologous The first association between DNA repair and ubiquitylation
recombination (HR), UV-induced DNA lesions, and other bulky arose when yeast Rad6, which functions in postreplication repair
DNA adducts are repaired by nucleotide excision repair (NER), (PRR), was shown to be a ubiquitin E2 (Jentsch et al., 1987). PRR
simpler base lesions are repaired by base-excision repair is a DNA-damage-tolerance pathway that allows replication past
(BER) whose components and reactions overlap with those bulky DNA lesions and is orchestrated by ubiquitylation and
of single-strand break repair, and DNA base mismatches are sumoylation of the DNA polymerase processivity factor PCNA
corrected by mismatch repair (MMR), while the Fanconi anemia (Figure 2). Two subpathways comprise eukaryotic PRR: trans-
(FA) pathway repairs DNA crosslinks. Lesions are first recog- lesion synthesis (TLS), and a template-switch mechanism asso-
nized by proteins that trigger and coordinate the recruitment ciated with HR (Ulrich, 2011). An early step in yeast PRR is PCNA
and activities of additional DNA repair components. DNA monoubiquitylation by the RING-type E3, Rad18, in conjunction
damage induction elicits cascades of posttranslational modi- with the E2, Rad6 (Hoege et al., 2002; Stelter and Ulrich, 2003).
fications, including phosphorylation, ubiquitylation, and sumoy- PCNA is primarily monoubiquitylated on Lys164, with this modi-
lation that orchestrate the aforementioned processes as well fication being recognized by specialized TLS polymerases such
as additional aspects of the DDR, such as regulating deoxyribo- as Polh, Pol i, Pol k, and Polz via their ubiquitin-binding domains
nucleotide supply and triggering of cell-cycle delays (cell-cycle (UBDs) of the UBM and UBZ families in conjunction with motifs
checkpoints). These events are largely initiated by the apical such as PIP boxes that recognize other features of PCNA
DDR kinases ATM and ATR, whose importance in coordinating (Lehmann, 2011). Unlike canonical high-fidelity polymerases,
the DDR is illustrated by their mutation in Ataxia-telangiectasia the catalytic sites of TLS polymerases can synthesize over and
(A-T) and Seckel syndrome, respectively (Durocher and Jack- past DNA lesions but usually at the cost of fidelity, thus making
son, 2001; Kerzendorfer and O’Driscoll, 2009). In this review, them error prone. Yeast PCNA can also be polyubiquitylated
we focus on the rapidly emerging functions of ubiquitin and on Lys164 by Rad5 (Hoege et al., 2002), an E3 ligase that coop-
SUMO in controlling various aspects of the DDR, with a particular erates with a dimeric E2 Ubc13-Mms2. Rad5-Ubc13-Mms2
emphasis on responses to DSBs, which are the most cytotoxic yields Lys63-linked ubiquitin chains on PCNA that promote the
of all DNA lesions. template-switching pathway that involves the newly synthesized

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sister chromatid and the HR machinery. While it is still unclear extraction, and degradation of RNAP II from chromatin (Figure 3).
how PCNA ubiquitylation promotes template switching, the TC-NER is dependent on CSB (ERCC6), a SWI/SNF family
mammalian ZRANB3 translocase was recently identified as an protein that associates with RNAP II (Gaillard and Aguilera,
effector of PCNA polyubiquitylation (Zeman and Cimprich, 2013). In addition to possessing chromatin-remodeling activity,
2012). CSB recruits CSA (ERCC8) to sites of DNA damage, the latter
While differences in PRR may exist between yeast and man, forming an E3 with DDB1 and CUL4. The action of CSB and
the pathway has been generally evolutionarily conserved, with CSA may be to license the TC-NER process, which includes
mammals having counterparts of Rad6 (human HR6A/UBE2A RNAP II backtracking and subsequent recruitment of the core
and HR6B/UBE2B), Rad18 (RAD18), and Rad5 (SHPRH and NER machinery (Gaillard and Aguilera, 2013). The identity
HLTF). Knockout or depletion of RAD18 in a variety of species of the key ubiquitylation event that initiates TC-NER is still
results in defective PRR, PCNA monoubiquitylation, and accu- unknown, but RNAP II and CSB ubiquitylation are possibilities.
mulation of TLS polymerases such as Polh at sites of replication In that regard, CSB possess a functionally important UBD, sug-
fork blockage (Lee and Myung, 2008). Furthermore, small inter- gesting that CSB recognizes this key ubiquitylation event (Anin-
fering RNA depletion studies suggest that human HLTF and dya et al., 2010). Furthermore, the DUB USP7 is recruited to
SHPRH contribute to PCNA polyubiquitylation in response to stalled polymerases by UVSSA, the product of a gene mutated
fork-causing lesions (Motegi et al., 2008) and in the suppression in a CS-like UV-sensitivity syndrome (Cleaver, 2012). UVSSA-
of mutagenesis (Lin et al., 2011), phenotypes consistent with USP7 interacts with RNAP II and delays the CSA-dependent
functions for these E3 ligases in error-free PRR. degradation of CSB by the proteasome.
In addition to being ubiquitylated, budding yeast PCNA is The degradation of RNAP II by the proteasome can be seen as
sumoylated on Lys164 (and to a lesser degree on Lys127) by a last-resort measure and provides a unique case study for the
the E3 Siz1 and the E2 Ubc9 (Pfander et al., 2005; Stelter and role of ubiquitin chain editing. In yeast, these processes involve
Ulrich, 2003). PCNA sumoylation prevents unscheduled recom- the Rsp5 E3 (NEDD4 in mammals), which catalyzes Lys63-linked
bination during DNA replication by recruiting Srs2, a UvrD-type ubiquitin chain formation on RNAP II (Anindya et al., 2007; Wilson
helicase that can strip the key HR protein Rad51 from chromatin et al., 2013). These chains are trimmed down by Ubp2, a DUB,
(Krejci et al., 2003; Pfander et al., 2005; Veaute et al., 2003). Srs2 resulting in monoubiquitylated RNAP II. Lys48-linked ubiquitin
harbors PCNA-binding PIP and SUMO-interaction motif (SIM) chains are then built from monoubiquitylated RNAP II by an
regions that simultaneously engage sumoylated PCNA (Arm- Elongin/Cullin 3 complex, which can then promote RNAP II
strong et al., 2012). While PCNA sumoylation is difficult to detect degradation after its extraction from chromatin with the Cdc48
in human cells, an Srs2-like protein, PARI, has recently been segregase, the yeast VCP/p97 homolog (Harreman et al.,
described (Moldovan et al., 2012). 2009; Verma et al., 2011; Wilson et al., 2013).

Control of NER by Ubiquitylation Ubiquitin-Based DSB Signaling by RNF8 and RNF168

NER repairs bulky DNA base adducts and ultraviolet light- An important paradigm for ubiquitin and SUMO acting in intra-
induced lesions. Inherited defects in NER factors yield patho- cellular signaling is provided by the orchestrated recruitment of
logies that include xeroderma pigmentosum (XP), Cockayne proteins such 53BP1 and the tumor suppressor BRCA1 onto
syndrome (CS), and trichothiodystrophy (TTD), which are char- chromatin surrounding DSB sites (Lukas et al., 2011) (Figure 4).
acterized by sun hypersensitivity, skin cancer predisposition These events are initiated by phosphorylation of the histone
(in the case of XP), cognitive impairments, premature aging, or variant H2AX (yielding gH2AX), which is recognized by MDC1
developmental defects (Hoeijmakers, 2009). NER comprises (Stucki et al., 2005). MDC1 is then phosphorylated by the DSB-
two main pathways: global genome repair (GG-NER) that oper- responsive kinase ATM, with these phospho-sites being bound
ates on all nuclear DNA, and transcription-coupled repair (TC- by the FHA domain of the RING-E3 ligase RNF8 (Huen et al.,
NER) that specifically targets the template strand of transcribed 2007; Kolas et al., 2007; Mailand et al., 2007). RNF8 then medi-
genes. During human GG-NER, DNA lesions can be detected ates ubiquitylation of proteins at DSB sites in a manner promoted
independently by two complexes, DDB1-DDB2/XPE and XPC- via interactions with the large HECT-type ligase HERC2 (Bekker-
RAD23 (Scrima et al., 2011) (Figure 3). However, DDB1-DDB2 Jensen et al., 2010). The RING-E3 ligase RNF168 is then re-
plays a unique role in NER owing to the observation that cruited by its UBDs, recognizing RNF8 ubiquitylation products
DDB1-DDB2 is required for the effective recruitment of XPC to and products of its own activity. The UBDs of RNF168 are not
chromatin (Fitch et al., 2003). Mechanistically, DDB2-DDB1 equivalent and are integrated in functional modules containing
forms an E3 ligase in association with CUL4A/B that mediates targeting motifs, the LRMs that are also present on RAD18 and
monoubiquitylation of histones and polyubiquitylation of DDB2 RAP80 (Panier et al., 2012). The primary outcome of RNF8/
and XPC (Scrima et al., 2011). While autoubiquitylated DDB2 is RNF168-dependent ubiquitylation is recruitment and/or reten-
targeted for degradation, XPC is not because it is protected tion of DSB repair and signaling factors on chromatin surround-
from proteasome action by RAD23, a proteasome-interacting ing the DNA lesion, which include 53BP1, RAD18, BRCA1, the
protein (El-Mahdy et al., 2006; Sugasawa, 2006; and references RAP80 complex (also known as BRCA1-A), HERC2, BMI1,
therein). RIF1, RNF169, NPM1, FAAP20, and NIPBL (Lukas et al., 2011).
When RNA polymerase II (RNAP II) stalls upon encountering At the functional level, RNF8/RNF168-dependent ubiquitylation
a DNA lesion, two independent cascades can be triggered. promotes NHEJ during immunoglobulin class switching and
The first event is TC-NER, and the second is the ubiquitylation, dysfunctional telomere fusion (Kracker and Durandy, 2011;

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Figure 3. The Role of Ubiquitin in
Nucleotide Excision Repair
(A) GG-NER promotes the repair of bulky lesions
on genomic DNA. The lesions can be recognized
by the DDB1-DDB2 and XPC-RAD23 complexes.
DDB1-DDB2 forms an E3 with CUL4 and RBX1
(not shown), which leads to DDB2 autoubiquity-
lation, resulting in its degradation, and XPC
polyubiquitylation. XPC is stabilized through its
interaction with RAD23, which contains a ubiq-
uitin-like domain that interacts with the protea-
some. This allows the subsequent steps of NER,
such as the recruitment of XPA.
(B) TC-NER repairs bulky lesions occurring on the
transcribed DNA strand. RNA polymerase II acts
as a lesion sensor. Two independent pathways
can occur when RNA polymerase stalls after a
lesion encounter. First (going right), TC-NER can
be activated, with the CSB and CSA proteins
playing a critical role in TC-NER. CSB associates
with RNA polymerase and recruits CSA, which
forms a CUL4-based E3 with DDB1. The exact
nature of the critical substrate of the E3 associated
with CSA is not known, but CSB is ubiquitylated
and its degradation is delayed by USP7, which is
brought to the stalled polymerase by UVSSA.
Second (going down), the RNA polymerase can
be ubiquitylated, extracted from chromatin, and
degraded by the proteasome as a last resort. This
pathway, in yeast, is initiated by Rad26-Def1 (not
shown), which leads to the Rsp5-dependent
ubiquitylation of RNA polymerase. Rsp5 can
promote Lys63-ubiquitin chains but the DUB
Ubp2 trims these down. Monoubiquitylated RNA
polymerase is then a substrate of an Elongin-Cullin
3 complex. The Lys48-linked ubiquitin chains on
RNA polymerase enable Cdc48 to extract the
stalled polymerase from chromatin, which then
leads to its degradation.

mine how TRIP12 and UBR5 affect

RNF168 levels and whether any physio-
logical conditions affect this regulation.
In this regard, TRIP12 contains a WWE
domain, which in other proteins binds
to poly(ADP) ribose, suggesting that
RNF168 levels might be controlled by
poly(ADP) ribosylation. Herpes simplex
virus (HSV) infection also regulates the
RNF8 pathway, with the HSV ICP0
Peuscher and Jacobs, 2011; Rai et al., 2011). In addition to protein—which is necessary for the transition between the viral
NHEJ, the RNF8 pathway can also promote HR. These repair latent and lytic phases—being an E3 that targets RNF8 and
defects likely contribute to the clinical phenotypes observed in RNF168 for degradation (Chaurushiya et al., 2012; Lilley et al.,
individuals with inactivating mutations in RNF168, which are 2010). Specifically, ICP0 targets RNF8 for degradation through
afflicted with an immunodeficiency and cellular radiosensitivity a ‘‘reverse-degron’’ mechanism mediated by a phospho-depen-
syndrome, RIDDLE, that is related to A-T (Stewart et al., 2009). dent interaction between ICP0 and the RNF8 FHA domain
Significantly, the RNF8 pathway is turned off during mitosis, (Chaurushiya et al., 2012). This RNF8 and RNF168 degradation
perhaps because chromatin ubiquitylation is incompatible with not only obfuscates the DDR initiated by the linear HSV DNA
mitotic progression (Giunta et al., 2010). In another striking but also relieves RNF8/168-dependent transcriptional repres-
example of regulation, it recently emerged that RNF168 is sion of the viral genome.
a limiting factor in the RNF8 pathway, with the E3 ubiquitin While early work pointed to H2A-type histones being primary
ligases TRIP12 and UBR5 collaborating to regulate RNF168 RNF8/168 targets, mutational studies suggested that RNF8/
levels, thereby preventing excessive histone ubiquitylation at RNF168 target sites were distinct from the canonical H2AK119
DSB sites (Gudjonsson et al., 2012). It will be interesting to deter- ubiquitylation sites (Huen et al., 2007). Indeed, RNF168 was

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potential crosstalk between H2A K13/K15 and H2B K120 ubiqui-

tylation might at least in part explain RNF8/RNF168-dependent
transcriptional silencing triggered near DSBs (Shanbhag et al.,
2010).
Histone ubiquitylation stimulated by DNA lesions is not limited
to H2A K13/K15 and H2B K120. Indeed, the E3 BMI1-RING1B
accumulates at DSB sites, where it is proposed to locally
increase H2A/H2AX K119 monoubiquitylation, which may partic-
ipate in DNA-damage-induced transcriptional silencing (Gieni
et al., 2011; Shanbhag et al., 2010). Furthermore, recent proteo-
mic work found that all core histones along with histones H1,
H2AZ, H2AX, and macro-H2A are ubiquitylated at multiple sites
(Kim et al., 2011; Wagner et al., 2011). It will clearly be of interest
to determine whether any of these modifications affect or are
affected by responses to DNA lesions.

Regulating DSB Responses by 53BP1 and BRCA1

53BP1 and BRCA1 are the two main effectors of the RNF8
pathway, yet they have diametrically opposed functions:
53BP1 opposes DNA end resection, an activity that promotes
DSB repair by NHEJ (Noon and Goodarzi, 2011), while BRCA1
promotes HR and is somehow linked to initiation of end resection
(Li and Greenberg, 2012). BRCA1 and 53BP1 are thus essentially
engaged in a tug of war that determines commitment to NHEJ
or HR. This functional antagonism has profound implications
for our understanding of BRCA1 function as a tumor suppressor,
since inactivation of 53BP1 suppresses the lethality, tumorigen-
Figure 4. The Role of Ubiquitin in the Chromatin-Based Response
to DNA Double-Strand Breaks esis, and sensitivity to most genotoxins associated with BRCA1
(A) DSBs trigger the ATM-dependent phosphorylation of H2AX, which is read loss of function (Bouwman et al., 2010; Bunting et al., 2010). A
by MDC1. ATM can also phosphorylate MDC1 to promote the recruitment of provocative implication of this work is that the function of
RNF8, an E3 ligase. RNF8 ubiquitylates an unknown factor on chromatin (X).
BRCA1 might act as a competitor or inhibitor of 53BP1, particu-
This ubiquitylation is then read by the N-terminal ubiquitin binding domains of
RNF168. RNF168 ubiquitylates H2A, which provides a second recruitment larly in S/G2 phases, when HR is upregulated (Chapman et al.,
signal for RNF168. In addition to RNF8/168, DSBs stimulate the recruitment of 2012).
the BMI1-RING1B, which ubiquitylates H2A on the C terminus. Also shown are The mechanism for 53BP1 recruitment to DSB sites has
DUBs that antagonize this ubiquitylation event.
(B) OTUB1 antagonizes RNF168-dependent ubiquitylation by binding to E2s. been puzzling because it does not possess recognizable ubiqui-
RNF8 and RNF168 also stimulate the formation of Lys63-linked ubiquitin tin-binding regions. Instead, it contains a tandem Tudor domain
chains on chromatin. that binds mono- or dimethylated histone H4 Lys20 (H4K20me1/
(C) The RNF168-dependent ubiquitylation of chromatin leads to the recruit-
ment of multiple effectors that include 53BP1, which promotes NHEJ, and
2) and a tandem BRCT domain involved in protein-protein inter-
BRCA1, which promotes HR. actions (Botuyan et al., 2006). Notably, the Tudor but not the
BRCT region of 53BP1 is needed for its accumulation at
DSB sites (Pryde et al., 2005). Recent work showed that the
recently shown to target Lys13/15 within the histone H2A N proteins L3MBTL1 and JMJD2A bind to H4K20me1/2 but are
terminus (Gatti et al., 2012; Mattiroli et al., 2012). It will be impor- removed from chromatin upon DNA damage induction (Acs
tant to determine whether and how H2A Lys13/15 ubiquitylation et al., 2011; Mallette et al., 2012). L3MBTL1 removal requires
may promote recruitment of proteins to chromatin flanking the segregase VCP/p97, which is targeted to DSB sites through
DSB sites and how this may functionally cooperate with other K48-linked ubiquitin and its cofactor NPL4 (Acs et al., 2011),
DSB-responsive histone modifications. In this regard, while whereas the RNF8 pathway promotes JMJD2A degradation
H2A Lys13, and Lys15 are located far from H2AK119 on the (Mallette et al., 2012). A role of VCP/p97 in 53BP1 retention at
nucleosome core particle, they are close to H2BK120, a residue DSB sites was also identified by Meerang et al. (2011), although
ubiquitylated by the RNF20 and RNF40 E3 ligases in collabora- the conclusions reached regarding mechanism differed. While
tion with the RAD6 E2 (Zhu et al., 2005). RNF20 and RNF40 are a model for ubiquitylation simply acting to remove JMJD2A/
also recruited to DSB sites via ATM-dependent mechanisms, L3MBTL1 is attractive, it is difficult to reconcile it with the consti-
to induce H2B monoubiquitylation (Moyal et al., 2011; Nakamura tutive, Tudor-dependent association of 53BP1 with chromatin
et al., 2011). These observations may help explain why Rnf8- (Bothmer et al., 2011) and with H2A K13/K15 ubiquitylation being
deficient mouse embryo fibroblasts display reduced steady- critical for 53BP1 DSB recruitment (Mattiroli et al., 2012). The
state H2B K120 ubiquitylation (Wu et al., 2009). Given the role mechanism(s) for 53BP1 recruitment at DSB sites will likely
of mammalian H2B ubiquitylation in regulating transcription remain unresolved until the biochemical reconstitution of its
and chromatin compaction (Weake and Workman, 2008), the RNF168-dependent chromatin binding is achieved.

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D’Andrea, 2012). There are currently 15 known genes whose

biallelic mutations yield FA (FANCA to FANCP), with their prod-
ucts functioning in the three prime events of the FA pathway
that occur in S phase: ICL recognition and excision, translesion
synthesis, and HR-mediated repair (Knipscheer et al., 2009).
The FA pathway is activated when replication forks encountering
ICLs trigger chromatin association of the FA core complex
(Figure 5). The FANCL subunit of this complex is a RING-type
E3 ligase that functions with the E2, UBE2T, to monoubiquitylate
both subunits of the heterodimeric FANCD2-FANCI complex,
which then provides a platform for coordinating repair processes
(Joo et al., 2011). Specifically, in addition to ubiquitylation
potentially helping anchor FANCD2-FANCI on chromatin, ubiqui-
tylated FANCD2 is recognized by the UBZ4 UBD present in the
structure-specific nuclease FAN1, although an interaction with
the nuclease scaffold SLX4 (FANCP) has also been proposed
Figure 5. The Role of Ubiquitin in the FA Pathway (Sengerová et al., 2011). This promotes recruitment of these
ICLs are potent fork-blocking lesions. DNA replication fork stalling, along with
factors to sites of DNA damage, with the ensuing nucleolytic
FANCM and associated proteins (not shown), stimulates the recruitment of the
multi-subunit FA core complex, which is an E3 ligase. The catalytic subunit of incisions triggering further stages of repair that include TLS
this E3 is FANCL and its E2 is UBE2T. The FA core complex monoubiquitylates and HR events discussed elsewhere in this review. Additional
FANCD2-FANCI. FANCD2-FANCI ubiquitylation is read by FAN1 and perhaps connections between the FA pathway and ubiquitin are high-
SLX4, which both contain a UBZ4-type ubiquitin binding domain. The USP1-
UAF1 DUB reverses FANCD2-FANCI ubiquitylation and is essential for the FA lighted by work demonstrating that the FA core-complex-associ-
pathway. The recruitment of nucleases enables incision, unhooking of the ated factor, FAAP20, contains a UBD that binds RNF8-mediated
lesion, TLS, and HR. ubiquitylations and cooperates with RNF8 to promote recruit-
ment of the FA core complex and FANCD2 to ICLs (Yan et al.,
2012).
BRCA1 forms a dimeric RING-type E3 with BARD1 and, in
addition to its role in HR, has also been linked to transcription- Deubiquitylases Acting in the DDR
coupled DNA repair, DNA crosslink repair, and checkpoint A well-characterized DUB is USP1, the prime FANCD2 deubiqui-
control. While BRCA1 recruitment at DSB sites clearly relies on tylase, whose inactivation recapitulates many FA phenotypes,
RNF8- and RNF168-dependent formation of ubiquitin conju- implying that both ubiquitylation and deubiquitylation must
gates (Lukas et al., 2011), exactly how BRCA1 recognizes such be appropriately controlled for the FA pathway to operate
ubiquitin conjugates is still under intense investigation. Long- effectively (Kim and D’Andrea, 2012). In this regard, the USP1-
term maintenance of BRCA1 at DSB sites depends on its activating factor, UAF1/WDR48, possesses two tandem
interaction with the RAP80 complex—and the RAP80 ubiquitin- SUMO-like domains that mediate interactions with a SIM-related
binding UIM modules—but RAP80 is dispensable for the initial sequence in FANCI, thus targeting USP1 to FANCD2/FANCI
accumulation of BRCA1 at DSB sites (Hu et al., 2011; Yin (Yang et al., 2011) (Figure 5). The DUBs USP3, USP16,
et al., 2012b). There is also vigorous debate about whether BRCC36, POH1, and OTUB1 are associated with negative
BRCA1 E3 ligase activity is crucial for DSB repair and tumor regulation of the RNF8 pathway, with USP3 and USP16 being
suppression, with the balance of opinion tilting toward it not first linked to this pathway through their ability to oppose H2A
being essential for DSB repair by HR (Li and Greenberg, 2012; ubiquitylation (Weake and Workman, 2008) and by USP3 over-
Shakya et al., 2011; Zhu et al., 2011). Indeed, a mutation in the expression blocking RNF168 accumulation at DSB sites (Doil
RING domain (Brca1I26A) that only affects its interaction with et al., 2009). USP16, on the other hand, opposes RNF8/
E2 conjugating enzymes results in cells that are proficient in RNF168-mediated, DSB-induced transcriptional silencing
repairing a targeted DSB by HR, along with mice that are pheno- (Shanbhag et al., 2010). BRCC36 (BRCC3)—a JAMM/MPN(+)
typically normal with wild-type tumor latency (Li and Greenberg, isopeptidase that displays strong selectivity for K63-linked ubiq-
2012; Shakya et al., 2011). However, a similar mutation in uitin chains and that is a component of the BRCA1-RAP80
chicken DT40 cells caused hypersensitivity to DNA damage complex—accumulates at DSB sites downstream of RNF8/
caused by the topoisomerase I poison camptothecin (Sato RNF168 (Cooper et al., 2009; Dong et al., 2003; Shao et al.,
et al., 2012). Additional work is clearly needed to define the 2009; Sobhian et al., 2007; Wang and Elledge, 2007). In addition,
specific function(s) for BRCA1 E3 ligase activity. a second JAMM/MPN(+) protein with specificity against K63-
linked ubiquitin chains, the proteasome-associated POH1/
Ubiquitin Control of the Fanconi Anemia Pathway PSMD14 DUB, has been linked to negative regulation of the
Fanconi anemia (FA) is a rare recessive genetic disorder charac- RNF8 pathway (Butler et al., 2012). Collectively, these findings
terized by developmental abnormalities, cancer predisposition, suggest that proteasome-associated POH1 and BRCA1/
progressive bone-marrow failure, and defective repair of DNA RAP80-associated BRCC36 may collaborate to restrict K63-
interstrand crosslinks (ICLs) that prevent transcription and linked, RNF8/RNF168-dependent polyubiquitylation at DSB
DNA replication (Garner and Smogorzewska, 2011; Kim and sites. Notably, the OTU family DUB, OTUB1, is a negative

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Figure 6. Examples of the Role of SUMO in
Multiple DNA Damage Response Pathways
(A) Thymine-DNA glycosylase (TDG), which acts
during BER, is inhibited by the product of its
reaction. TDG sumoylation produces a conforma-
tional change that enables it to leave DNA.
(B) Sumoylation acts at multiple steps during the
chromatin-based DSB response. See the main
text for details.
(C) Sumoylation can also stimulate ubiquitylation
through the action of STUbLs, E3 ligases that are
recruited to substrates by recognizing sumoyla-
tion. In the shown example, RNF4 promotes the
turnover of MDC1 from chromatin during the DSB
response.

playing crucial roles in these events.

Due to the breadth of such research, we
are only able to review it here in a cursory
manner (for a more comprehensive
review, see Lane and Levine, 2010). In
brief, over ten ubiquitin E3s have been
linked to p53 regulation, with most
research having been focused on the
RING E3, Mdm2/HDM2 (in mouse and
human, respectively) (Brooks and Gu,
2011). Mdm2 functions as a homodimer,
or as a heterodimer with the related E3
Mdmx/HDMX, to bind to and ubiquitylate
p53, thus promoting proteasome-medi-
ated p53 degradation and helping keep
p53 levels low under normal conditions.
In response to DNA damage, however,
various modifications on p53, Mdm2,
and their regulators, including those cata-
regulator of RNF168-dependent ubiquitylation, but, surprisingly, lyzed by and promoted by the checkpoint kinases ATM, ATR,
this is independent of the OTUB1 isopeptidase activity (Nakada CHK1, and CHK2, inhibit p53 ubiquitylation and degradation
et al., 2010). Instead, OTUB1 binds and inhibits the RNF168- and promote p53 transcriptional activity, allowing it and its
associated E2s (UBC13/UBE2N and E2s of the UBE2D/UBE2E cofactors to induce target genes. These genes in turn trigger
families) through assembly of a pseudocleavage product in its various responses, including cell-cycle delays, induction of
catalytic site (Juang et al., 2012; Wiener et al., 2012). DUBs DNA repair proteins, and, in some cases permanent cell-cycle
undoubtedly affect many other DDR processes, as highlighted arrest or apoptosis. Furthermore, recent work has highlighted
by USP1-UAF1 also regulating PCNA ubiquitylation and TLS how various DUBs, including USP7/HAUSP, USP10, USP29,
(Huang et al., 2006). Other notable DUBs are USP47, which and USP42, play important roles in modulating p53 levels and
affects BER by modulating DNA polymerase b levels (Parsons activity (Hock et al., 2011).
et al., 2011), the tumor suppressor BAP1, which regulates H2A
K119ub (Scheuermann et al., 2010), and USP7, which regulates A Paradigm for Sumoylation in BER
the stability of multiple DDR proteins, including p53 and ERCC6 Sumoylation was first firmly linked to DNA repair by studies on
(Fraile et al., 2012). Given their likely tractability to small- BER (Figure 6), a pathway that is initiated by various base lesions
molecule drug development, it will be interesting to assess being recognized by DNA glycosidases, proteins that enzymati-
the potential of various DUBs as targets for pharmacological cally remove the lesions, yielding abasic sites that are then pro-
intervention. cessed and repaired. Thymine-DNA glycosylase (TDG) removes
thymine or uracil from G:T or G:U mismatches that arise through
Ubiquitylation Controls p53 in Response to DNA Damage deamination of 5-methylcytosine or cytosine, respectively.
A prime executer of DNA damage signaling in vertebrates is the Biochemical studies established that, like certain other DNA
transcription factor p53, mutations in which are associated with glycosylases, TDG strongly binds to the abasic site it generates,
various human cancers. Extensive work by many laboratories with such product inhibition impairing further steps in BER.
has established that p53 levels and function are regulated in Notably, SUMO1 conjugation in the TGD C-terminal region
response to DNA damage, with ubiquitylation and sumoylation triggers interactions between this and SIMs elsewhere within

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the protein, leading to a conformational change in the TDG N it markedly reduced SUMO1 and SUMO2/3 accrual. This is
terminus that reduces product binding, thereby enhancing its probably because RNF8/168 mediate the recruitment of factors
enzymatic turnover (Baba et al., 2005; Steinacher and Schär, such as BRCA1 and 53BP1 that are then sumoylated, although
2005). Such studies suggested a molecular handover model direct effects of RNF8/RNF168 on PIAS1/PIAS4 activities are
wherein unconjugated TDG binds and mediates base hydrolysis, also possible. Furthermore, although PIAS1/4 are not required
with subsequent SUMO binding and sumoylation of TDG for RNF8 recruitment to DNA damage sites, PIAS4 but not
promoting its dissociation. This promotes the handover of the PIAS1 depletion impaired RNF168 recruitment. Notably,
abasic BER intermediate to the APE1 endonuclease that RNF168 and HERC2 are sumoylated in a DNA-damage- and
mediates the next step of the repair process. PIAS4-dependent manner, with PIAS4 depletion reducing
RNF168 levels, impairing HERC2 binding to RNF8, and abro-
SUMO and Ubiquitin Crosstalk in DSB Repair gating IR-induced HERC2 accrual on chromatin (Danielsen
A well-characterized, direct link between sumoylation and DSB et al., 2012). BRCA1 is sumoylated by PIAS1 and/or PIAS4
responses came through studies on S. cerevisiae Rad52, a key once localized at DSB sites, which enhances BRCA1 ubiquitin
HR factor. Rad52 is sumoylated in a manner requiring the ligase activity (Morris et al., 2009), possibly via sumoylation
Mre11 complex, Ubc9, and the E3 Siz2 that is related to helping BRCA1 to productively associate with E2 enzymes
mammalian PIAS proteins (see below). In addition to this sumoy- and/or SIM-containing target proteins. Additional functions of
lation protecting Rad52 from degradation, it promotes Rad52 PIAS1 and PIAS4 are likely mediated by PIAS1/4-dependent
exclusion from nucleoli, thus preventing inappropriate recombi- sumoylation of factors such as 53BP1 (Galanty et al., 2009).
nation between repetitive ribosomal DNA sequences (Torres- Other DDR functions for SUMO E3 ligases are highlighted by
Rosell et al., 2007). Sumoylation also controls yeast HR in other the interaction between PIAS1 and SNM1A promoting ICL
ways. Cohesin is a multiprotein complex that encircles sister repair (Ishiai et al., 2004) and by sumoylation of tyrosyl DNA
chromatids, thereby promoting equal chromosome distribution phosphodiesterase (TDP1) enhancing repair of single-strand
during mitosis and postreplicative sister-chromatid recombina- DNA breaks (Hudson et al., 2012). In addition, while the
tion. In S. cerevisiae, in addition to cohesion generation during SUMO protease SENP6 interacts with RPA70 in S phase,
S phase, it is further enforced locally and globally upon DSB keeping RPA70 hyposumoylated, replication stress dissociates
induction in G2/M. The cohesin subunit Mcd1 is sumoylated the complex, allowing accumulation of SUMO2/3-modified
upon DSB formation in a manner that promotes DNA-damage- RPA70 that then promotes HR by recruiting RAD51 to damaged
induced cohesion and that largely relies on the SUMO E3 DNA (Dou et al., 2010). BLM, the RecQ family DNA helicase
Mms21 (Nse2), which is part of the cohesin-related Smc5- defective in human Bloom’s syndrome, is also sumoylated,
Smc6 complex (McAleenan et al., 2012). Related mechanisms with mutations blocking its sumoylation causing higher DNA
might operate in vertebrates because human Smc5-Smc6 is damage production during S phase, DNA damage hypersensi-
recruited to DNA damage sites, where it promotes sister chro- tivity, and impaired RAD51 localization to sites of replication
matid HR, at least in part by MMS21/NSE2’s sumoylation of stalling (Ouyang et al., 2009).
the human Mcd1 ortholog SCC1 and counteracting the negative Additional connections between ubiquitylation and sumoyla-
cohesin regulator WAPL (Wu et al., 2012). The demonstration of tion are highlighted by work on SUMO-targeted ubiquitin ligases
DNA-damage-induced sumoylation of various yeast HR, NHEJ, (STUbLs), which include human RNF4, S. pombe Rfp1/2-Slx8,
BER, NER, MMR, and checkpoint components highlights how and S. cerevisiae Slx5-Slx8 (Prudden et al., 2007). STUbLs
sumoylation is likely to impact various components of many contain SIMs that bind sumoylations or SUMO-like domains
DDR processes (Cremona et al., 2012). Indeed, recent work in on target proteins and then ubiquitylate such proteins, often
S. cerevisiae by (Psakhye and Jentsch, 2012) has established leading to their proteasomal degradation. Although work has
that while sumoylation of various HR factors is collectively crucial shown that S. cerevisiae Slx5-Slx4 and S. pombe Rfp1/2-Slx8
for effective DSB repair, loss of any one individual sumoylation regulate HR, the mechanisms for this are not yet clear. By
has little effect. It seems likely that this principle of ‘‘protein group contrast, recent work revealed that RNF4 inactivation in human
modification’’ will also apply to sumoylation in additional DNA or chicken cells caused defective DSB repair by both HR and
repair pathways. NHEJ (Galanty et al., 2012; Luo et al., 2012; Yin et al., 2012a).
Links between sumoylation and DSB responses in mamma- Moreover, RNF4 is recruited to DSB sites via interactions
lian cells came through the findings that SUMO1, SUMO2/3, between its N-terminal SIMs and sumoylated DSB-response
UBC9, and the PIAS and MMS21 SUMO E3s accumulate at proteins such as 53BP1, MDC1, and RPA, where it mediates
sites of DSBs or replication stalling (Galanty et al., 2009; Morris the accrual of ubiquitin adducts (Figure 6). While there are likely
et al., 2009) (Figure 6). Moreover, PIAS4 inactivation markedly multiple, functional substrates for RNF4, major ones appear
impaired RNF168 accrual and K63-ubiquitin accumulation to be MDC1 and RPA, with RNF4 promoting their rates of turn-
together with 53BP1 and BRCA1 recruitment at DSB sites, over at DSB sites. For example, decreased RPA turnover
while PIAS1 depletion only prevented RAP80 and BRCA1 caused by RNF4 depletion or mutation of RPA sumoylation sites
accumulation. Accordingly, PIAS1 or PIAS4 depletion markedly leads to RPA being ineffectively replaced by RAD51, thereby
impaired DSB repair by HR and NHEJ and caused hypersensi- impairing HR (Galanty et al., 2012). The fact that RNF4 pro-
tivity toward DSB-generating agents (Galanty et al., 2009; motes proteasome recruitment to DSB sites, together with pro-
Morris et al., 2009). Significantly, while RNF8 or RNF168 deple- teasome subunit depletion phenocopying many aspects of
tion did not prevent PIAS1/4 recruitment to DNA damage sites, RNF4 depletion, suggest that RNF4-mediated, SUMO-targeted

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ubiquitylation of DDR factors leads to their recognition by the provide better diagnostics and therapeutics for additional
proteasome, thus triggering their localized turnover and coordi- age-related diseases associated with DNA damage and/or
nating progression through multiple stages of the DNA repair DDR dysfunction.
process. In this regard, the S. pombe VCP/p97 counterpart,
Cdc48, binds to SUMO conjugates via a SIM in its cofactor Perspectives
Ufd1 (Nie et al., 2012). Because VCP also binds to ubiquitin, The past few years have witnessed a rapid increase in our under-
DNA repair might be promoted by VCP through binding STUbL standing of how ubiquitin and SUMO conjugation affect cellular
targets that are comodified by both ubiquitin and SUMO. Given DNA damage responses. This growth has been propelled by
that S. cerevisiae Ufd1 also binds SUMO and because human enhancements in our general understanding of the ubiquitin
VCP/p97 functions at DSB sites (see the preceding sections), and SUMO systems, by the advent of new techniques and
connections between STUbL targets, VCP/p97 complexes, approaches, and by the large number of researchers now oper-
and the proteasome are likely to exist throughout the eukaryotic ating in this exciting area of research. In addition to identifying
lineage. additional ubiquitin and SUMO system components that affect
DNA repair and the associated events, another potentially
Therapeutic Applications exciting area for future study will be to assess whether and
DNA-damaging chemotherapies and radiotherapies are widely how other ubiquitin-like modifier proteins connect to the DDR.
used and often-effective cancer treatments, with their efficacy The major—and in our view the most exciting—challenge
reflecting the induction of lethal loads of DNA damage in cancer for future work, however, will be to explain precisely how
cells. Unfortunately, cancer cell mechanisms frequently lead to ubiquitylation, sumoylation, and related events control DDR
tolerance or repair of therapy-induced DNA lesions, incomplete processes. As mentioned previously, in addition to being some-
eradication, and subsequent recurrence, while effects of DNA times coupled as monomers, alone or in combination, ubiquitin
damage on normal tissues limit dosing and are associated and SUMO can also be conjugated through different linkages
with toxicities. The success of DNA-damaging agents as anti- into a vast potential array of differing chain topologies with
cancer therapeutics seems to reflect cancer cells often prolifer- differing functional effects on target proteins. Emerging evidence
ating more than most normal cells, through them experiencing points to such biological specificity often being achieved via
higher DNA damage loads than normal cells and through modified proteins being bound by ‘‘reader’’ factors that combine
them often being impaired in certain DDR components, making ubiquitin/ubiquitin-like binding motifs and targeting sequences,
them particularly reliant on the ones they retain (Helleday et al., thus allowing recognition of a particular ubiquitin/SUMO chain
2008; Jackson and Bartek, 2009). In addition to providing or linkage in the context of a specific protein substrate. The
opportunities for better tailoring of DNA-damaging therapies potential scope for biochemical and functional diversity in a
to particular tumors, knowledge of DDR differences between ‘‘ubiquitin/SUMO/UBL code’’ is clearly enormous, and it is
cancers and normal cells also provides opportunities for killing even larger when one considers that such modifications may
cancers selectively via the concept of synthetic lethality, as first also be recognized in conjunction with other protein modifi-
demonstrated by killing of HR-defective, BRCA1/2-deficient cations such as phosphorylation and poly(ADP) ribosylation.
tumors by PARP1/2 inhibitors (Bryant et al., 2005; Farmer Other areas where we see the DDR contributing to our
et al., 2005). Our growing knowledge of how ubiquitylation knowledge of ubiquitylation or sumoylation are in the area of
and sumoylation impact DDR processes might therefore yield VCP/p97 function and ubiquitin chain editing. Given its strength
new opportunities for cancer management. For example, the and breadth, it seems likely that the DDR research community
fact that inherited defects in ubiquitylation result in DDR defi- will continue to discover new principles and paradigms for
ciencies raises the prospect that such defects might arise ubiquitylation and sumoylation, many of which should also
somatically during cancer evolution, thereby providing new apply to the wider array of cellular activities controlled by these
diagnostic and perhaps therapeutic opportunities. Furthermore, two related posttranslational modifications.
the array of proteins of the ubiquitin and SUMO systems—many
of which are inherently druggable enzymes—provides a vast
ACKNOWLEDGMENTS
and presently largely untapped resource for new therapeutic
targets. Notably, proteasome inhibition by bortezomib (Velcade)
We apologize to those whose important findings we could not mention as
can sensitize cells to radiotherapy and DNA-damaging chemo- primary literature and/or cite due to space constraints. We thank Kate Dry,
therapies (Motegi et al., 2009), and recent work has indicated Haico van Attikum, Stephane Richard, Roger Greenberg, Matthew Weitzman,
that it causes a HR-impaired state in multiple myeloma cells, Ralph Scully, and Yaron Galanty for communicating results prior to publica-
tion, advice, and/or valuable discussions. Research in the S.P.J. laboratory
thus causing hypersensitivity to PARP inhibitors and high- is funded by CRUK program grant C6/A11224, the European Research
lighting how combined use of such agents might have clinical Council, and the European Community Seventh Framework Programme grant
potential (Neri et al., 2011). Development of agents targeting agreement number HEALTH-F2-2010-259893 (DDResponse). Core infrastruc-
ture funding was provided by CRUK (C6946/A14492) and the Wellcome Trust
more DDR-specific components of the ubiquitin and SUMO (WT092096). S.P.J. receives his salary from the University of Cambridge,
systems might also have anticancer properties, and, being supplemented by CRUK. Research in the D.D. laboratory on ubiquitin and
more focused on certain DDR events, these might have fewer the DNA damage response is funded by grant MOP84297 from the CIHR.
D.D. is a Canada Research Chair (Tier 1) in the Molecular Genetics of the
toxicities than those caused by more general inhibitors of the
DNA damage response and the Thomas Kierans Chair in Mechanisms of
ubiquitin-proteasome system. It is also possible that linkages Cancer Development. S.P.J. has no conflicts of interest but discloses that
of ubiquitin and SUMO with DNA repair can be exploited to he is a founder and shareholder of Mission Therapeutics Ltd.

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