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DNA-damage repair; the good, the bad,
and the ugly
Razqallah Hakem
1,2,
*
1
Department of Medical Biophysics, Ontario Cancer Institute/UHN,
University of Toronto, Toronto, Ontario, Canada and
2
Department
of Immunology, Ontario Cancer Institute/UHN, University of Toronto,
Toronto, Ontario, Canada
Organisms have developed several DNA-repair pathways
as well as DNA-damage checkpoints to cope with the
frequent challenge of endogenous and exogenous DNA
insults. In the absence or impairment of such repair or
checkpoint mechanisms, the genomic integrity of the
organism is often compromised. This review will focus
on the functional consequences of impaired DNA-repair
pathways. Although each pathway is addressed individu-
ally, it is essential to note that cross talk exists between
repair pathways, and that there are instances in which a
DNA-repair protein is involved in more than one pathway.
It is also important to integrate DNA-repair process with
DNA-damage checkpoints and cell survival, to gain a
better understanding of the consequences of compromised
DNA repair at both cellular and organismic levels.
Functional consequences associated with impaired DNA
repair include embryonic lethality, shortened life span,
rapid ageing, impaired growth, and a variety of syndromes,
including a pronounced manifestation of cancer.
The EMBO Journal (2008) 27, 589–605. doi:10.1038/
emboj.2008.15
Subject Categories: molecular biology of disease
Keywords: cancer; DNA repair; mouse models; syndromes
Introduction
Organisms have evolved to efficiently respond to DNA
insults that result from either endogenous sources (cellular
metabolic processes) or exogenous sources (environmental
factors). Endogenous sources of DNA damage include
hydrolysis, oxidation, alkylation, and mismatch of
DNA bases; sources for exogenous DNA damage include
ionizing radiation (IR), ultraviolet (UV) radiation, and
various chemicals agents. At the cellular level, damaged
DNA that is not properly repaired can lead to genomic
instability, apoptosis, or senescence, which can greatly
affect the organism’s development and ageing process.
More importantly, loss of genomic integrity predisposes the
organism to immunodeficiency, neurological disorders, and
cancer (O’Driscoll and Jeggo, 2006; Subba Rao, 2007; Thoms
et al, 2007). Therefore, it is essential for cells to efficiently
respond to DNA damage through coordinated and integrated
DNA-damage checkpoints and repair pathways.
DNA-damage checkpoints
The mechanisms of DNA-damage checkpoints are best
understood during their responses to double-strand breaks
(DSBs). Initiation of these checkpoints is dependent on the
transient recruitment of the MRE11/RAD50/NBS1 (MRN)
complex at DSB sites, followed by the recruitment/activation
of ataxia–telangiectasia mutated (ATM) a member of the
family of phosphoinositide-3-kinase-related kinases (PIKKs)
(Su, 2006). In addition, two other PIKKs, DNA-dependent
protein kinase (DNA–PK) and ATR (ATM and Rad3 related),
are also activated and involved in the response to DSBs.
However, the primary function of ATR is the initiation of
DNA-damage response to stalled replication forks (RFs)
(Su, 2006). ATM, ATR, and DNA–PK phosphorylate various
targets that contribute to the overall DNA damage response.
Therefore, within minutes of DSB formation, active ATM
phosphorylates different proteins that are essential for
DNA-damage response and repair. An example includes the
histone H2AX that, following its phosphorylation at the site
of DNA damage by ATM, DNA–PK, or ATR (gH2AX), recruits
other proteins and initiates the chromatin-remodelling
process that is essential for the repair of damaged DNA.
Other proteins recruited to sites of DSBs include
MDC1, 53BP1, and BRCA1, all of which are ATM substrates
and mediators in DNA-damage response. The MRN
complex-mediated resection of DSBs is followed by
single-stranded DNA coating with replication protein A
(RPA), which serves to recruit ATR and its binding partner
ATRIP, and subsequent ATR-dependent phosphorylation of
clapsin, Rad17, BRCA1, and others (Su, 2006).
ATM and ATR are essential for the G1/S, intra-S-phase, and
G2/M DNA-damage checkpoints, and are critical for the
maintenance of genomic integrity. Defects in either ATM or
ATR have been associated with human syndromes. AT M
mutations are associated with the human ataxia–telangiecta-
sia (AT), an autosomal recessive disorder characterized by
cerebellar ataxia, progressive mental retardation, impaired
immune functions, neurological problems, and malignancies
(O’Driscoll and Jeggo, 2006). At the cellular level, AT pheno-
types include chromosomal breakage and IR sensitivity.
Similarly, ATR mutations predispose individuals to Seckel
syndrome, a very rare autosomal recessive human disorder
Received: 31 October 2007; accepted: 16 January 2008
*Corresponding author. Department of Medical Biophysics, Ontario
Cancer Institute/UHN, University of Toronto, 610 University Avenue
PMH, Room 10-622, Toronto, Ontario, Canada M5G 2M9.
Tel.: þ1 416 946 2398/4501; ext: 5661; Fax: þ1 416 946 2984;
E-mail: rhakem@uhnres.utoronto.ca
The EMBO Journal (2008) 27, 589–605 |
&
2008 European Molecular Biology Organization |All Rights Reserved 0261-4189/08
www.embojournal.org
&2008 European Molecular Biology Organization The EMBO Journal VOL 27 |NO 4 |2008
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characterized by growth and mental retardation, as well as
microcephaly (O’Driscoll and Jeggo, 2006). Spontaneous and
IR-induced genomic instability and immunological defects
have also been observed in Seckel syndrome patients. In
contrast to ATM and ATR, no human syndrome has yet been
associated with defective DNA–PK. However, studies of
mouse models have linked mutations of DNA–PK to severe
immunodeficiency (see section Non-homologous end joining
repair pathway).
Activated ATM and ATR mediate the phosphorylation and
subsequent activation of Chk2 and Chk1, respectively; this
process is necessary in the induction of phosphorylation of
CDC25A, marking it for proteosomal degradation (Su, 2006).
The consequential loss of CDC25A results in G1/S arrest, due
to the inefficient loading of CDC45 at the origin of replication.
In addition, activated ATM, ATR, DNA–PK, Chk2, and Chk1
all aid in the phosphorylation and activation of p53, a key
player in DNA-damage checkpoints. Activated p53 transacti-
vates p21, which inhibits two G1/S-promoting cyclin-depen-
dent kinases (CDKs), CDK2 and CDK4. This leads to
sustained G1 arrest, which ultimately hampers the replication
of damaged DNA.
The intra-S-phase checkpoint serves to arrest DNA synth-
esis during S phase of cells with damaged DNA (Su, 2006). In
these cells, CDC25A phosphorylation, mediated by Chk2 or
Chk1, leads to its degradation and the subsequent inactiva-
tion of the S-phase cyclin E/CDK2 complex. Consequently,
these events prevent the loading of CDC45 at the origin of
replication and result in intra-S-phase arrest. It has been
reported that other proteins, including Nijmegen breakage
syndrome 1 (NBS1), BRCA1, SMC1, 53BP1, and MDC1, all
contribute to the intra-S-phase checkpoint.
The activation of the G2/M DNA-damage checkpoint pre-
vents mitotic entry of the damaged cells (Su, 2006). This
checkpoint is mediated by the dual-specificity phosphatase
CDC25C, as well as p53. In normal conditions, CDC25C
dephosphorylates CDC2, allowing the CDC2–cyclin B kinase
to facilitate entry into mitosis. However, phosphorylation of
CDC25C by Chk2 or Chk1, initiates its binding with 14-3-3,
which leads to its cytoplasmic sequestration away from its
substrate, thus preventing mitotic entry. p53 also contributes
to the G2/M checkpoint through its transactivation of p21 and
14-3-3. P21 effectively blocks the phosphorylation of CDC2,
initiating the onset of the G2/M cell-cycle arrest. 14-3-3
sequesters CDC25C in the cytoplasm and promotes the
activation of Wee1, a tyrosine kinase that negatively regulates
CDC2, thus blocking entry into mitosis.
The activation of DNA-damage checkpoints enforces the
growth arrest of damaged cells and allows the DNA-repair
mechanisms to mend the damaged DNA. Once repair
is completed, cells are able to exit the checkpoints and
resume their cell-cycle progression and functions. However,
unsuccessful DNA repair leads to p53-dependent apoptosis
(Chipuk and Green, 2006), in addition to senescence (Collado
et al, 2007).
Defects of DNA-damage checkpoints, similar to impaired
DNA-damage repair, promote genomic instability and predis-
pose individuals to immunodeficiency, neurological defects,
and cancer (Niida and Nakanishi, 2006). Although important
advances have been made in understanding the cellular
mechanisms behind the initiation and maintenance of check-
points, the mechanisms that control checkpoints exit, as well
as how the cell decides survival, death, or senescence,
require further investigation.
Defects associated with DNA-damage
repair pathways
Different DNA-repair pathways exist and perform major roles
at both cellular and organismic levels. These pathways
include (1) the direct reversal pathway, (2) the mismatch
repair (MMR) pathway, (3) the nucleotide excision repair
(NER) pathway, (4) the base excision repair (BER) pathway,
(5) the homologous recombination (HR) pathway, and (6) the
non-homologous end joining (NHEJ) pathway (Figure 1). The
mechanisms for these pathways will not be discussed in
detail in this review; instead we will focus on the functional
consequences associated with their defects.
Direct reversal of DNA damage
In contrast to other DNA-damage repair pathways, direct
reversal of DNA damage is not a multistep process and
does not involve multiple proteins (Sedgwick et al, 2007).
Furthermore, unlike excision repair, direct reversal of DNA
damage does not require the excision of the damaged bases.
An example of a DNA lesion that is repaired by direct reversal
is the O
6
-alkylguanine. Alkylating agents can transfer methyl
or ethyl groups to a guanine, thereby modifying the base and
interfering with its pairing with cytosine during DNA replica-
tion. The cytotoxic and mutagenic O
6
alkyl adduct in DNA is
repaired by direct reversal, which is mediated by the enzyme
Ada in Escherichia coli (E.coli) and the mammalian
O
6
-methylguanine-DNA methyltransferase (MGMT). MGMT,
also known as AGT, removes the DNA adducts by transferring
the alkyl group from the oxygen in the DNA to a cysteine
residue in its active site. This reaction leads to the reversal of
the base damage; however, the alkylation of MGMT leads to
its inactivation and subsequent ubiquitination and proteoso-
mal degradation. MGMT has attracted a great deal of atten-
tion, as certain anticancer chemotherapeutic drugs produce
O
6
-alkylguanine, further supporting its role in modulating the
therapeutic response of tumors to these drugs. Mouse models
for Mgmt inactivation have been generated (Tsuzuki et al,
1996b; Glassner et al, 1999). These mutants were viable and
MMR
Direct
reversal
NER
BER
HR
NHEJ
DNA damage repair pathways
CH3
Figure 1 DNA-repair pathways. Several DNA-repair pathways exist
and deal with various types of DNA insults. These pathways include
(1) the direct reversal pathway, (2) the MMR pathway, (3) the
NER pathway, (4) the BER pathway, (5) the HR pathway, and (6) the
NHEJ pathway.
DNA-damage repair
R Hakem
The EMBO Journal VOL 27 |NO 4 |2008 &2008 European Molecular Biology Organization590
showed no increase in spontaneous tumorigenesis (Table I).
However, Mgmt homozygous mice and cells were highly
sensitive to chemotherapeutic alkylating agents such as
methylnitrosourea. Mgmt homozygous mutant females, but
not males, developed larger numbers of dimethylnitrosa-
mine-induced liver and lung tumors compared with controls
(Iwakuma et al, 1997). Additionally, transgenic mice
over-expressing human MGMT or E. coli Ada have also
been generated. In response to alkylating carcinogens that
produce O
6
-alkylguanine in DNA, these transgenic mice
demonstrated a significantly reduced susceptibility to
developing cancers, including thymomas (Dumenco et al,
1993), liver tumors (Nakatsuru et al, 1993), and skin tumors
(Becker et al, 1997).
AlkB is another enzyme that mediates direct DNA damage
reversal in E. coli. This dioxygenase is involved in the repair
of alkylation damage, particularly 1-methyladenine (1meA)
and 3-methylcytosine (3meC). Two mammalian AlkB homo-
logues, ABH2 and ABH3, have been shown to possess DNA-
repair functions similar to the bacterial AlkB (Duncan et al,
2002; Sedgwick et al, 2007). Similar to AlkB, ABH2 and ABH3
have the ability to repair 1meA and 3meC residues. However,
whereas ABH2 prefers double-stranded DNA, ABH3 and AlkB
favour single-stranded DNA and RNA (Aas et al, 2003; Falnes
et al, 2004). Further insight into the function of the mamma-
lian ABH2 and ABH3 came from studies of mice carrying
targeted mutations of these genes. Mice deficient in Abh2,
Abh3, or both, were viable (Ringvoll et al, 2006). Abh2
/
,
but not Abh3
/
, mice showed age-dependent accumulation
of 1meA in their genomic DNA. As in AlkB mutants in E.coli,
mouse embryonic fibroblasts (MEFs) deficient in Abh2
were hypersensitive to methyl methane-sulfonate (MMS)
treatment. However, mice deficient in Abh2 or Abh3 did
not show increased spontaneous cancer development
(Table I). Further studies are required to assess the role
of these dioxygenases, and other AlkB homologues, in
alkylation damage-induced cancer.
These examples of direct DNA-damage reversal mediated
by MGMT/Ada or ABH/AlkB demonstrate the conserved role
of this mechanism in DNA repair. In addition, increased
tumorigenesis of Mgmt mutants, together with the resistance
of MGMT transgenic mice to alkylating carcinogens that
produce O
6
-alkylguanine, further demonstrate the important
role that direct reversal plays in cancer.
The MMR pathway
The MMR pathway plays an important role in both prokar-
yotes and eukaryotes in repairing mismatches, which are
small insertions and deletions that take place during
DNA replication (Figure 1; Jiricny, 2006). Failure of MMR
commonly results in microsatellite instability (MSI). Several
homologues of the bacterial MMR genes MutS and MutL have
been identified in yeast and mammals.
The importance of the MMR pathway became evident
upon identification of mutations in certain human MMR
genes in hereditary non-polyposis colorectal cancer
(HNPCC), a highly penetrant autosomal dominant cancer
syndrome (Figure 2; Vasen et al, 2007). HNPCC, also
known as Lynch syndrome, is characterized by early-onset
colorectal cancer, with elevated levels of MSI in the tumors.
Individuals with HNPCC have an approximate 80% lifetime
risk for colorectal cancer, and are also predisposed to the
development of endometrial, ovarian, gastric, and other types
of malignancies.
Approximately 70–80% of germline mutations identified in
HNPCC families are mutations in MLH1 or MSH2, whereas
mutations in MSH6 are found in approximately 10% of
HNPCC families (Peltomaki and Vasen, 1997). Germline
mutations in other human MMR genes, including PMS1,
PMS2,MLH3, and exonuclease 1 (EXO1), have also been
found in HNPCC families; however, they occur at a much
lower frequency (Vasen et al, 2007). In addition, inactivation
of MLH1 by mutations at the promoter or coding sequences,
or by promoter methylation, has been identified in sporadic
colorectal tumors (Kane et al, 1997; Veigl et al, 1998). Recent
Table I Examples of mouse models for direct reversal
Genotype Developmental
defects
Fertility defects Spontaneous
tumorigenesis
Induced tumorigenesis References
Mgmt/None None Not affected Increased DMNA-
induced lung and
liver cancer in females
(Tsuzuki et al, 1996a, b;
Iwakuma et al, 1997;
Glassner et al, 1999)
Abh2/
Abh3/
Abh2/Abh3/
None None Not affected Not tested (Ringvoll et al, 2006)
Defective repair
HR pathway NHEJ pathway
–SCID
–RS-SCID
BER pathway
–MAP
MMR pathway
–HNPCC
NER pathway
–XP
–CS
–TTD
–BS
–WS
–RTS
–ATLD
–NBS
Figure 2 Examples of human syndromes and disorders associated
with defective DNA-damage repair. Impaired MMR pathway leads to
the hereditary HNPCC. Mutations of certain human NER genes have
been associated with syndromes and disorders including the XP, CS,
and TTD. MAP, a rare disorder, has been shown associated with
mutations of the BER gene MUTYH. Various human syndromes and
disorders have been associated with defects of the HR pathway.
They include ATLD, NBS, BS, WS and RTS. Mutations of certain
human genes involved in NHEJ lead to the SCID or RS-SCID.
DNA-damage repair
R Hakem
&2008 European Molecular Biology Organization The EMBO Journal VOL 27 |NO 4 |2008 591
studies, although very limited, have identified rare patients
with homozygous germline mutations for MLH1,MSH2,
MSH6,orPMS2 (Felton et al, 2007). Typically, these indivi-
duals have a reduced life span and, in contrast to hetero-
zygous MMR individuals, tend to develop juvenile
haematological malignancies and brain cancer.
In yeast, Msh2 forms heterodimers with Msh3 and Msh6,
proteins that bind DNA mismatches and initiate the MMR
process. In Saccharomyces cerevisiae (S.cerevisiae), Msh2,
Msh3, and Msh6 mutants are viable (Marsischky et al, 1996).
Both Msh2 and Msh6 S.cerevisiae mutants show high fre-
quencies of base substitution, whereas only Msh2 mutants
exhibit high frameshift mutations. Msh3 mutations in
S.cerevisiae result in low rates of frameshift mutations.
However, on Msh6-mutant background, synergistic effects
of the dual mutations have been observed, including in-
creased MSI and mutability similar to Msh2 mutants.
Mutant mice for MutS and MutL MMR homologues have
also been generated using gene targeting (Table II). Mutant
mice for the MutS homologues include Msh2,Msh3,Msh4,
Msh5, and Msh6. Mice carrying homozygous mutations for
Msh4 or Msh5 did not exhibit cancer phenotypes; however,
males and females were infertile, consistent with the role of
MutS homologues in processing meiotic recombination inter-
mediates (de Vries et al, 1999; Edelmann et al, 1999). In
contrast, homozygous mutants for Msh2 (de Wind et al,
1995; Reitmair et al, 1995), Msh3 (Edelmann et al, 2000),
and Msh6 (Edelmann et al, 1997) have increased risk for
developing cancers such as lymphoma, gastrointestinal, and
skin cancer.
Mutant mice for MutL homologues include Pms1
/
,
Pms2
/
,Mlh1
/
, and Mlh3
/
mice (Table II). These
mutants are viable; however, males and females deficient in
Mlh1 (Baker et al, 1996; Edelmann et al, 1996) or Mlh3
(Lipkin et al, 2002) and males deficient in Pms2 (Baker
et al, 1995) are sterile, demonstrating a requirement for
these proteins during meiosis. In addition, mouse MutL
homologues are differentially required for cancer suppres-
sion. Pms1
/
mice do not show any increased risk for cancer
(Prolla et al, 1998), whereas Mlh1
/
(Prolla et al, 1998; Chen
et al, 2005) and Mlh3
/
mice (Chen et al, 2005) are predis-
posed to developing lymphomas and gastrointestinal tumors.
Similarly, Pms2-null mutants (Prolla et al, 1998; Chen et al,
2005) are prone for lymphoma development.
EXOI physically interacts with MSH2, MSH3, and MLH1,
and is involved in the excision of mismatched bases in DNA
(Tishkoff et al, 1997; Schmutte et al, 2001). Mutant mice for
ExoI have impaired MMR, accumulate MSI, and exhibit a
greater risk for developing lymphomas (Wei et al, 2003).
These mutants also have meiotic defects and are sterile,
demonstrating the requirement of ExoI in meiosis.
Double mutant mice carrying dual mutations of different
MMR genes have also been reported. For example, Msh3-
mutant mice develop cancer with low frequency and at a later
age, whereas Msh3
/
Msh6
/
mice (Edelmann et al, 2000)
die prematurely and develop tumors including lymphomas,
gastrointestinal, and skin tumors. This phenotypic outcome
is similar to that of Msh2
/
or Mlh1
/
mice that are the
most cancer-prone MMR mutants, as half of these mutants
die around 6 months of age. This cooperation between
mutations of Msh2 and Msh6 in mice is reminiscent of their
collaboration in the maintenance of genomic integrity of
S.cerevisiae.
Immunoglobulin (Ig) diversification, an essential process
for immunity, involves somatic hypermutation (SHM) of the
Ig genes, as well as VDJ recombination and class-switch
recombination (CSR), two processes mediated by NHEJ
(Maizels, 2005). Interestingly, studies of the various MMR-mutant
strains have implicated a role for this pathway in SHM and CSR.
Thus, Msh2,Msh6,andExoI, but not Msh3-mutant mice, have
reduced CSR and SHM (Rada et al, 1998; Wiesendanger et al,
2000; Bardwell et al, 2004; Li et al, 2004).
Table II Examples of mouse models for the MMR pathway
Genotype Developmental
defects
Fertility defects Spontaneous tumorigenesis References
Msh2/None None High frequency and early onset
of lymphomas, gastrointestinal,
and skin cancer
(de Wind et al, 1995;
Reitmair et al, 1995)
Msh3/None None Low frequency and late onset
of lymphomas, gastrointestinal,
and skin cancer
(Edelmann et al, 2000)
Msh4/None Infertile Not affected (Kneitz et al, 2000)
Msh5/None Infertile Not affected (de Vries et al, 1999;
Edelmann
et al, 1999)
Msh6/None Unaffected Lymphoma, gastrointestinal, and
skin cancer
(Edelmann et al, 1997)
Msh3/Msh6/None None Higher frequency of lymphomas,
gastrointestinal, and skin
tumours compared to single
mutants
(Edelmann et al, 2000)
Pms1/None None Not affected (Prolla et al, 1998)
Pms2/None Male infertility Lymphomas (Baker et al, 1995; Prolla et al,
1998; Chen et al, 2005)
Mlh1/None Infertile High frequency and early onset
of lymphomas and gastrointest-
inal tumours
(Baker et al, 1996; Edelmann
et al, 1996; Prolla et al, 1998;
Chen et al, 2005)
Mlh3/None Infertile Lymphomas and gastrointestinal
tumours
(Lipkin et al, 2002; Chen et al,
2005)
ExoI/None Infertile Lymphomas (Wei et al, 2003)
DNA-damage repair
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The EMBO Journal VOL 27 |NO 4 |2008 &2008 European Molecular Biology Organization592
Whereas most HNPCC human individuals carry heterozy-
gous germline mutations of MMR genes, which predisposes
them to cancer, mice heterozygous for MMR mutations do
not appear to have an increased risk for developing cancer.
This difference is not specific for MMR mutations, as
heterozygous mutations in certain genes involved in other
DNA-damage repair pathways are also able to predispose
humans, but not mice, to cancer. The reasons for these
differences remain unknown, although species differences
in the DNA-damage repair pathways, metabolism, or life
span could contribute to these observed human–mouse
discrepancies. Despite these differences, mouse models
have significantly improved our understanding of the MMR
and other repair mechanisms, and their roles in preserving
genomic integrity and suppressing cancer.
The NER pathway
The NER pathway is a multistep process that serves to repair
a variety of DNA damage, including DNA lesions caused by
UV radiation, mutagenic chemicals, or chemotherapeutic
drugs that form bulky DNA adducts (Figure 1; Leibeling
et al, 2006). Over 30 different proteins are involved in the
mammalian NER, whereas only three proteins (UvrA, UvrB,
and UvrC) are required by prokaryotes (Truglio et al, 2006).
Two NER sub-pathways that have been identified are as
follows: the global genome NER (GG-NER) that detects and
removes lesions throughout the genome, and the transcrip-
tion-coupled NER (TC-NER), which repairs actively tran-
scribed genes. NER begins with the recognition of the DNA
lesion, followed by incisions at sites flanking the DNA lesion,
and culminates in the removal of the oligonucleotide contain-
ing the DNA lesion. Ligation of a newly synthesized oligonu-
cleotide, complementary to the pre-existing strand, serves to
fill the gap, thus ending the NER process. GG-NER and
TC-NER involve several common proteins and proceed
through the same repair steps, except during recognition of
the DNA lesion. In GG-NER this recognition involves the
XPC–RAD23B and DDB1–DDB2/XPE proteins, whereas re-
cognition in TC-NER is mediated by cockayne syndrome
group A (CSA) (ERCC8) and CSB (ERCC6). NER has attracted
a great deal of attention due to its role in three rare human
syndromes characterized by increased cancer frequencies,
neurodegeneration and ageing (Figure 2). These syndromes
are xeroderma pigmentosum (XP), Cockayne syndrome (CS),
and trichothiodystrophy (TTD) (Thoms et al, 2007).
XP individuals show extremely severe skin sensitivity to
short intervals of sun exposure and most develop freckles at
an early age. In addition, XP individuals may exhibit eye
damage as they suffer chronic UV-induced conjunctivitis and
keratitis as a consequence of continual sun exposure. XP
individuals have greater than 1000-fold increased skin cancer
risk, which first appears at an average age of 10 years.
Approximately 20% of XP individuals also develop neurolo-
gical abnormalities. XP is caused by mutations of the NER
gene XPA,XPB (ERCC3), XPC,XPD (ERCC2), XPE (DDB2), or
XPF; XPG. Whereas XP individuals carrying mutations of XPC
or XPE (DDB2) are only deficient in GG-NER, the remaining
XP individuals are deficient in both GG-NER and TC-NER
(Thoms et al, 2007).
CS is a very rare human autosomal recessive inherited
genetic disease (Thoms et al, 2007). Similar to XP individuals,
CS individuals suffer excessive sun sensitivity, but without
increased predisposition for skin cancer. Common CS symp-
toms include growth retardation (dwarfism), progressive
cognitive impairment, and ophthalmologic disorders such
as cataracts or retinitis pigmentosa. CS individuals typically
die in the first or second decade of life. CS is caused by
mutations of either CSA or CSB, two proteins essential
for DNA-damage recognition and initiation of TC-NER.
Therefore, although CS individuals are deficient in TC-NER,
they remain proficient in GG-NER.
TTD is a rare human autosomal recessive disorder asso-
ciated with defective NER; the most severe cases are asso-
ciated with mutations in the XPB or XPD genes. Clinical
characteristics of TTD include brittle hair and nails, dwarf-
ism, and ataxia (Thoms et al, 2007). In addition, half of TTD
individuals exhibit sensitivity to sunlight; however, skin
cancer predisposition has not been linked to this syndrome.
Several mouse models for NER mutations have been
generated (Table III). Homozygous mutants for XP genes
are viable (Nakane et al, 1995; Sands et al, 1995; Harada
et al, 1999; Itoh et al, 2004; Tian et al, 2004a, b; Yoon et al,
2005), with the exception of the pre-implantation embryonic
lethality of Xpd mutants (de Boer et al, 1998b). Xpd
R722W
-
mutant mice carrying an amino-acid substitution that mimics
a human XPD allele associated with TTD have been gener-
ated (de Boer et al, 1998a). Similarly, Xpd
G602D
-mutant mice
carrying a substitution at amino acid 602 have been gener-
ated to mimic the human combined XP/CS (Andressoo et al,
2006). Both Xpd
R722W
and Xpd
G602D
mutants have been
proven viable and reproduced some of the characteristics of
individuals that carry these XPD mutations.
With the exception of Xpe (Ddb2) mutants (Itoh et al,
2004; Yoon et al, 2005), homozygous mutant mouse cells for
Xpa (Nakane et al, 1995), Xpc (Sands et al, 1995), Xpf (Tian
et al, 2004b), Xpg (Harada et al, 1999; Tian et al, 2004a),
Xpd
R722W
(de Boer et al, 1998a), or Xpd
G602D
(Andressoo
et al, 2006) were all UV sensitive, correlating to the results
obtained from human mutant cells. Increased predisposition
for UV-induced skin cancer was observed with Xpa,Xpc,
Xpd
R722W
,Xpd
G602D
, and Xpe (Ddb2)-mutant mice (Nakane
et al, 1995; Sands et al, 1995; de Boer et al, 1999; Itoh et al,
2004; Yoon et al, 2005; Andressoo et al, 2006). Thus, as seen
in humans, XP proteins play a major role in murine NER.
DDB1 and HR23B are two proteins involved with XPC in
the recognition of DNA damage and the initiation of GG-NER.
However, in contrast to Xpc
/
mice (Sands et al, 1995),
Ddb1 mutants die during early embryonic development
(Cang et al, 2006). Inactivation of Ddb1 in developing CNS
and lens resulted in massive p53-dependent apoptosis of
dividing cells and lethality just after birth (Cang et al,
2006). MEFs deficient in Ddb1 showed defective proliferation
and were UV-sensitive. A total of 90% of HR23B mutants
suffer intrauterine or neonatal death (Ng et al, 2002). The
surviving HR23B
/
mice were growth retarded and males
were sterile; however, their NER and UV sensitivity remained
normal. In contrast, mutants for HR23A, another homologue
of the S.cerevisiae NER gene Rad23, are viable and their cells
proficient in UV responses (Ng et al,2003).However,mutations
of both HR23A and HR23B lead to embryonic lethality and
increased UV sensitivity, showing a redundancy between these
two NER proteins (Ng et al, 2003). It remains to be shown
whether inactivation of Ddb1 or mHR23A/B would facilitate
spontaneous or UV-induced cancer in mouse models.
DNA-damage repair
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&2008 European Molecular Biology Organization The EMBO Journal VOL 27 |NO 4 |2008 593
Mice mutant for Csa (Ercc8)orCsb (Ercc6) have been
generated (van der Horst et al, 1997, 2002). These mutants
are viable, but their cells, although competent in GG-NER, are
nonetheless impaired in TC-NER and are UV-sensitive.
Surprisingly, in contrast to CS individuals, Csa- and Csb-
mutant mice are prone to UV-induced skin cancer. This
discrepancy is also evident in TTD mouse models, as in
contrast to TTD individuals, Xpd
R722W
mice are susceptible
to UV-induced skin cancer (de Boer et al, 1999).
As mentioned earlier, cross talk exists between DNA-
damage repair pathways and certain DNA-damage repair
proteins. For example, during NER, the endonuclease
ERCC1/XPF cleaves a DNA strand on the 50-side of the lesion,
allowing the repair to proceed. However, ERCC1/XPF is also
implicated in interstrand crosslink (ICL) repair and HR,
suggesting that the phenotypes observed in Ercc1 or Xpf
mutants are unlikely to result only from the impairment of
NER. Cells deficient in Ercc1 or Xpf show high sensitivity
to UV and to the ICL mitomycin C (MMC), and mice
with inactivation of Ercc1 or Xpf are runted, dying at
approximately 3–4 weeks of age (McWhir et al, 1993;
Weeda et al, 1997; Tian et al, 2004b).
Thus, NER is an important DNA-repair pathway and its
impairment is associated with growth defects, excessive UV
sensitivity, and in certain cases, increased skin cancer.
The BER pathway
The BER pathway deals with base damage, the most common
insult to cellular DNA (Figure 1; Wilson and Bohr, 2007). Two
sub-pathways, short-patch BER and long-patch BER, are
involved in BER. The short-patch BER sub-pathway typically
replaces a single nucleotide, whereas the long-patch
sub-pathway results in the incorporation of 2–13 nucleotides.
The two sub-pathways progress through different major
processes that initially involve the removal of the damaged
base by glycosylases such as Ogg1 and Mutyh (Myh). This is
followed by strand incision of the apurinic or apurimidinic
(AP) site by the endonuclease APE1. The newly generated
gap is filled by incorporation of nucleotide(s), mediated by
DNA polymerase-b(Polb) in the case of the short-patch BER
sub-pathway, and by Polband/or Poleor din the case of the
long-patch BER sub-pathway. Strand ligation is carried out by
the XRCC1/ligase III (LigIII) complex in the case of the short-
patch sub-pathway. For the long-patch BER sub-pathway,
other proteins are involved in the repair process before the
ligation takes place. Thus, FEN1, PARP1, and LigI participate
in the DNA synthesis–ligation step and displace the 2- to
13-base DNA flap. FEN1, a 50-flap endonuclease and
50–30exonuclease, excises the flap and the strand ligation is
carried out by LigI.
For a period of time, little evidence existed to support the
involvement of BER in human cancer or any other disorders.
However, recent studies have demonstrated the existence of a
human disorder linked to defective BER (Figure 2). This
autosomal recessive disorder, referred to as MUTYH-asso-
ciated polyposis (MAP), is associated with biallelic germline
mutations of the human MUTYH, and is characterized by
multiple colorectal adenomas and carcinomas (Cheadle and
Sampson, 2007). The glycosylase MUTYH, a bacterial mutY
homologue, functions in the BER of oxidative DNA damage
by excising adenines misincorporated opposite 8-oxoG.
Deficient repair of this damage results in G:C-T:A
mutations, typically found in the adenomatous polyposis
coli (APC) gene in MAP tumors.
In addition to Myh, several mammalian glycosylases
are involved in BER, including Nth1, Ogg1, Ung, and Aag.
Table III Examples of mouse models for the NER pathway
Genotype Developmental defects Fertility
defects
Induced tumorigenesis References
Xpd/Pre-implantation
embryonic lethality
NA NA (de Boer et al, 1998b)
Xpd
R722W/R722W
Growth retardation None UV- and DMBA-induced
skin cancer
(de Boer et al, 1998a, 1999)
Xpd
G602D/G602D
Growth retardation None Early onset of UV-induced
skin and/or eye tumours
(Andressoo et al, 2006)
Xpe/None None UV-induced skin cancer (Itoh et al, 2004;
Yoon et al, 2005)
Xpa/None None UV- and DMBA-induced
skin cancer
(Nakane et al, 1995)
Xpc/None None UV-induced skin cancer (Sands et al, 1995)
Xpg/Growth retardation and
premature death
NA NA (Harada et al, 1999)
Ddb1/Early embryonic lethality NA NA (Cang et al, 2006)
HR23A/Unaffected None NT (Ng et al, 2002)
HR23B/Intrauterine/neonatal
death. 10% viable but
growth retarded
Male infertility NT (Ng et al, 2002)
HR23A/;
HR23B/
Embryonic lethality NA NA (Ng et al, 2003)
Csa/None None UV-induced skin cancer (van der Horst et al, 1997)
Csb/None None UV-induced skin cancer (van der Horst et al, 2002)
Ercc1/Growth retardation and
death before weaning
NA NA (McWhir et al, 1993;
Weeda et al, 1997)
Xpf/Growth retardation and
death before weaning
NA NA (Tian et al, 2004b)
NA, not applicable; NT, not tested.
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The EMBO Journal VOL 27 |NO 4 |2008 &2008 European Molecular Biology Organization594
Murine homozygous mutants for the glycosylases Nth1,
Ogg1,Ung,Aag,andMutyh have been generated by gene
targeting, and surprisingly, these mutants were viable
(Table IV). Nth1
/
,Ogg1
/
, and Aag
/
mice showed no
overt abnormalities (Engelward et al, 1997; Klungland et al,
1999; Ocampo et al, 2002; Takao et al, 2002); however after a
long latency, Ung
/
and Mutyh
/
mice developed B-cell
lymphomas and intestinal tumors, respectively (Nilsen et al,
2003; Sakamoto et al, 2007). These findings suggest func-
tional redundancy of certain BER glycosylases. This is further
supported by the pronounced phenotypes of Ogg1
/
Mutyh
/
mice; these mice had shorter life span and elevated
risk for cancer, with 50% of double mutants developing lung
tumors, lymphomas, sarcomas, and others tumors by
15 months of age (Xie et al, 2004).
In contrast to glycosylases, targeted inactivation of en-
zymes that act downstream of the glycosylases in BER
resulted in embryonic or post-natal lethality. Thus, homo-
zygous mutants for Ape1 (Ludwig et al, 1998), LigI (Petrini
et al, 1995), LigIII (Puebla-Osorio et al, 2006), Xrcc1 (Tebbs
et al, 1999), or Flap endonuclease 1 (Fen1) (Kucherlapati et al,
2002) died during embryonic development, whereas homo-
zygous mutants for Polbdied immediately after birth
(Gu et al, 1994; Sugo et al, 2000). The death of mutants
such as Xrcc1
/
,LigIII
/
and Polb
/
was preceded by
elevated levels of apoptosis (Gu et al, 1994; Tebbs et al,
1999; Sugo et al, 2000; Puebla-Osorio et al, 2006).
Interestingly, inactivation of p53 rescued the apoptosis but
not the lethality of these mutants, suggesting the contribution
of other p53-independent mechanisms in the deaths of
these mutants.
Despite the presumed important role for the BER pathway
in maintaining genomic integrity, mutations in this pathway
have not significantly predisposed mutant mice for cancer.
This is in contrast to mutations of other excision repair
pathways, such as NER and MMR.
The HR repair pathway
DSB repair can be mediated by two major repair pathways
depending on the context of the DNA damage, HR or NHEJ
repair pathways (Figure 1; Kanaar et al, 2008). In bacteria
and yeast, DSBs are preferentially repaired by HR, whereas
more than 90% of DSB in mammalian cells are repaired
by NHEJ. Both pathways are well defined and their
impairment is associated with defects and pathologies,
including increased cell death, cell-cycle arrest, telomere
defects, genomic instability, meiotic defects, immuno-
deficiency, and cancer (Krogh and Symington, 2004; Sung
and Klein, 2006).
HR is a multistep process that requires several proteins and
operates at the S or G2 phase of the cell cycle. Although it
accounts only for the repair of B10% of DSBs in mammalian
cells, HR defects can have severe consequences, as demon-
strated by the human syndromes AT-like disorder (ATLD) and
the NBS (Figure 2; Thompson and Schild, 2002). The predis-
position to either syndrome has been linked to mutations in
the MRN complex, which is important for the resection of
DSBs (Thoms et al, 2007). However, it is important to note
MRN functions are not restricted to HR, as is also involved in
NHEJ, checkpoint activation, and telomere maintenance
(Niida and Nakanishi, 2006).
The very rare human ATLD is associated with hypo-
morphic mutations in the MRE11 gene (Taylor et al, 2004).
Patients with this disorder exhibit clinical features similar to
AT, including immunodeficiency and progressive neurolo-
gical degeneration (Thoms et al, 2007). However, in contrast
to AT, ATLD is not associated with ocular telangiectasia and
the course of the disease is considerably milder. Similar to AT
and NBS, cellular features of ATLD include defective DSB
repair and repair-related cell responses, hypersensitivity to
IR, as well as increased spontaneous and IR-induced genomic
instability.
The NBS is a rare human autosomal recessive disorder
caused by hypomorphic NBS1 (NIBRIN) mutations (Thoms
et al, 2007). This disorder is characterized by growth retarda-
tion, immunodeficiency, microcephaly, and cancer predispo-
sitions, particularly lymphomas. Cellular characteristics of
NBS include radiosensitivity, increased levels of spontaneous
and IR-induced chromosome breakage, and defective
cell-cycle checkpoints.
Table IV Examples of mouse models for the BER pathway
Genotype Developmental
defects
Fertility defects Spontaneous tumorigenesis References
Nth1/None None Not affected (Ocampo et al, 2002;
Ta k a o et al, 2002)
Ogg1/None None Not affected (Klungland et al, 1999)
Ung/None None Late onset of B-cell
lymphomas
(Nilsen et al, 2003)
Aag/None None Not affected (Engelward et al, 1997)
Mutyh/None None Late onset of intestinal
tumours
(Sakamoto et al, 2007)
Ogg1/
Mutyh/
None None Late onset of lung tumours,
lymphomas, sarcomas, and
others tumours
(Xie et al, 2004)
Ape1/Embryonic lethality NA NA (Ludwig et al, 1998)
LigI/Embryonic lethality NA NA (Petrini et al, 1995)
LigIII/Embryonic lethality NA NA (Puebla-Osorio et al, 2006)
Xrcc1/Embryonic lethality NA NA (Tebbs et al, 1999)
Fen1/Embryonic lethality NA NA (Kucherlapati et al, 2002)
Polb/Death immediately after birth NA NA (Gu et al, 1994; Sugo et al,
2000)
NA, not applicable.
DNA-damage repair
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&2008 European Molecular Biology Organization The EMBO Journal VOL 27 |NO 4 |2008 595
In contrast to both MRE11 and NBS1, no data have yet been
reported to support a role for RAD50 in human chromosomal
breakage or immunodeficiency syndromes.
The functions of the MRN complex have been studied in
various organisms. S.cerevisiae strains carrying null muta-
tions in components of the Mre11p–Rad50p–Xrs2p (MRX)
complex have been generated, where Xrs2p is the functional
homologue of mammalian NBS1. MRX mutants are viable,
hypersensitive to IR and MMS, and are defective in meiotic
recombination (Krogh and Symington, 2004). Mutations of
the MRN complex have also been assessed in DT40. This
chicken B-lymphocyte cell line is deficient in p53, but highly
competent for homologous recombination and gene target-
ing. Deficiency of Mre11 in DT40 is lethal and its conditional
inactivation leads to radiosensitivity, defective proliferation,
genomic instability, and decreased HR (Yamaguchi-Iwai et al,
1999). DT40 deficient in NBS1 also display increased IR
sensitivity, abnormal S-phase checkpoints, and decreased
HR (Tauchi et al, 2002).
In contrast to S.cerevisiae, null mutations of Mre11
(Xiao and Weaver, 1997), Nbs1 (Zhu et al, 2001), and
Rad50 (Luo et al, 1999) result in early mouse embryonic
lethality (Table V). Therefore, to circumvent embryonic leth-
ality, hypomorphic and tissue-specific mutants for the three
MRN components were generated. Mre11
ATLD1/ATLD1
-mutant
mice carrying an A to T substitution at amino acid 1894 of
Mre11, which results in a 75-amino-acid truncation, exhibited
reduced female fertility, defective ATM functions, and
increased genomic instability. However, no cancer pheno-
types were observed (Theunissen et al, 2003).
Rad50
s
(Rad50
k22M
) hypomorphic mutant mice have also
been generated (Bender et al, 2002). Approximately 40% of
Rad50
s/s
mutants die in utero; however, those that are viable
show progressive haematopoietic failure, short life span, and
increased predisposition to thymic lymphoma. In contrast to
rad50
s
in yeast, Rad50
s/s
did not impair meiotic progression
and the mutants were fertile; however, p53-mediated
apoptosis was increased in the testes. Inactivation of the
Table V Examples of mouse models for the HR pathway
Genotype Developmental defects Fertility
defects
Spontaneous
tumorigenesis
References
Mre11/Early embryonic
lethality
NA NA (Xiao and Weaver, 1997)
Mre11
ATLD1/ATLD1
None Reduced female
fertility
None (Theunissen et al, 2003)
Rad50/Early embryonic
lethality
NA NA (Luo et al, 1999)
Rad50
s/s
(Rad50
k22M/k22M
)
40% die in utero. The
one that survive show
haematopoietic failure
None Thymic lymphoma (Bender et al, 2002)
Nbs1/Early embryonic
lethality
NA Mild cancer predisposition
of Nbs1+/mice
(Zhu et al, 2001)
Nbs1
D23/D23
Growth retardation,
lymphoid defects
Female sterility Thymic lymphomas (Kang et al, 2002)
Nbs1
D45/D45
None None Twofold increase (Williams et al, 2002)
Rad52/None None Not affected (Rijkers et al, 1998)
Rad51/,Rad51B/
Rad51D/
Embryonic lethality NA NA (Lim and Hasty, 1996; Tsuzuki
et al, 1996a; Shu et al, 1999;
Pittman and Schimenti, 2000)
Xrcc2/Embryonic lethality NA NA (Deans et al, 2003; Orii et al,
2006; Adam et al, 2007)
Dmc1/None Mutants are
sterile
Not affected (Pittman et al, 1998; Yoshida
et al, 1998)
Rad54/,
Rad54B/, and
Rad54/;Rad54B/
None None Not affected (Essers et al, 1997; Bross et al,
2003; Wesoly et al, 2006)
Brca1/Embryonic lethality NA NA (Gowen et al, 1996; Hakem
et al, 1996; Liu et al, 1996;
Ludwig et al, 1997; Xu et al,
2001)
Brca1 mutation in
mammary epithelial
cells
NA Mammary tumorigenesis
that is enhanced on
Chk2-orp53-mutant
backgrounds
(Xu et al, 1999; McPherson
et al, 2004b)
Brca2/Early embryonic
lethality
NA NA (Ludwig et al, 1997; Suzuki
et al, 1997)
Brca2 mutation in
mammary epithelial
cells
Mammary tumorigenesis
on p53-mutant background
(Jonkers et al, 2001)
Blm/None None Predisposition to a wide
range of tumours
(Luo et al, 2000)
Mus81/None None T- and B-cell lymphomas (McPherson et al, 2004a)
Mus81/p53/Female embryonic
lethality
None Multiples tumours
including lymphomas
and sarcomas
(Pamidi et al, 2007)
NA, not applicable.
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Atm–Chk2–p53 pathway by the hypomorphic mutation
Mre11
ATLD1
or the Nbs1
DB
(Nbs1
D45
) mutation, was able to
rescue the depletion of haematopoeitic cells in the Rad50
s/s
mutants (Morales et al, 2005). Surprisingly, tumorigenesis,
senescence, and radiosensitivity associated with Atm muta-
tion were all partially suppressed by the Rad50
s
mutation
(Morales et al, 2005). Although the exact mechanism for this
rescue is not fully understood, it might involve compensatory
activation of other checkpoint pathways, such as the ATR
pathway.
Similar to Rad50 and Mre11 mutations, Nbs1-null muta-
tions resulted in early embryonic lethality (Zhu et al,2001;
Dumon-Jones et al, 2003). In contrast, Nbs1
D23
and
Nbs1
D45
hypomorphic mutants were viable (Kang et al,
2002; Williams et al, 2002). Cells from these mutants were
defective in the intra-S-phase and G2/M checkpoints.
Heterozygous mice carrying a null Nbs1 mutation (Dumon-
Jones et al, 2003), as well as homozygous mice carrying
Nbs1
D23
or Nbs1
D45
hypomorphic mutations (Kang et al,
2002; Williams et al, 2002), demonstrated a mild pre-
disposition for cancer. Interestingly, whereas p53 inactivation
shortened the tumor latency of Nbs1
D45
mutants, Atm
inactivation, or its impaired function on Mre11
ATLD1/ATLD1
-
mutant background, resulted in synthetic embryonic
lethality of Nbs1
D45
mutants (Williams et al, 2002;
Morales et al, 2005).
In addition to the previous models, a conditional mutant
strain for Nbs1 has also been generated (Frappart et al, 2005).
Neuronal inactivation of Nbs1 in these mice leads to chro-
mosomal breaks, microcephaly, growth retardation, cerebel-
lar defects, and ataxia, representing a combination of features
characteristic of NBS, AT, and ATLD. p53 inactivation in this
model also significantly rescued the neurological defects
associated with Nbs1 mutations (Frappart et al, 2005).
Thus, the MRN complex is essential for maintaining geno-
mic integrity, cell viability, and checkpoint activation.
Moreso, its requirement in various species demonstrates its
essential conserved functions.
Similar to Rad50, Rad52 is a member of the Rad52 epistasis
group of proteins originally identified by their requirement
for the repair of IR-induced DNA damage (Krogh and
Symington, 2004). Inactivation of rad52 in S.cerevisiae
results in increased radiation sensitivity and decreased
recombination (Symington, 2002). However, its inactivation
in DT40 reduced gene-targeting frequency, but did not lead to
increased IR sensitivity and viability and growth of the
mutant cells were not affected (Yamaguchi-Iwai et al,
1998). The effects of inactivation of mouse Rad52 were also
investigated. Null Rad52 mouse embryonic stem (ES) cells
obtained by gene targeting demonstrated a 30–40% decrease
in the frequency of HR compared with controls (Rijkers
et al, 1998). In contrast to rad52 mutants in S.cerevisiae,
Rad52-deficient ES cells were not hypersensitive to IR or
agents that induce DSBs. Rad52
/
mice, were viable, fertile,
and showed no overt abnormalities (Rijkers et al, 1998).
Rad52 inactivation was shown to extend the life span of
Atm
/
mice, partially rescue their T-cell development, and
suppress/delay their tumorigenesis (Treuner et al, 2004).
However, growth defects, infertility, and radiosensitivity of
Atm
/
mice were not rescued on by a Rad52-mutant
background. The reasons for the rescue of certain Atm
/
phenotypes by Rad52 inactivation are not clear, but
nevertheless this is reminiscent of the rescue mediated
by Rad50
s/s
mutation of tumorigenesis, senescence, and
radiosensitivity associated with Atm inactivation (Morales
et al, 2005).
The mammalian Rad51 family is also important for HR.
This family is composed of RAD51, disrupted meiotic cDNA 1
(Dmc1), and five RAD51 paralogues, RAD51B, RAD51C,
RAD51D, XRCC2, and XRCC3. RAD51, a homologue of the
bacterial DNA-dependent ATPase, RecA, is central for the
homology search and strand exchange during HR.
Mutants rad51 in S.cerevisiae are viable, IR sensitive, have
meiotic defects, and accumulate meiosis-specific double-
strand breaks (Shinohara et al, 1992). Rad51 deficiency in
DT40 leads to chromosomal breakage, G2/M arrest, and
cell death (Sonoda et al, 1998). Rad51-null mutation in
mice results in early embryonic lethality associated
with chromosomal loss, radiosensitivity, decreased cell
proliferation, and increased apoptosis (Lim and Hasty,
1996; Tsuzuki et al, 1996a).
Mutants for Rad51 paralogues were also generated. Thus,
for example, inactivation of Rad51 paralogues in DT40 did not
compromise cell survival (Takata et al, 2000, 2001). However,
decreased gene-targeting efficiency, increased spontaneous,
and induced cell death in response to IR or MMC, as well as
elevated chromosomal aberrations, were observed in these
mutants. Mutant mice for the Rad51 paralogue Rad51B (Shu
et al, 1999), Rad51D (Pittman and Schimenti, 2000), or Xrcc2
(Deans et al, 2003) were also generated. These mutations
resulted in lethality at different stages of embryonic develop-
ment and their effects on cell growth were variable.
Consistent with the role of Rad51 and its paralogues in HR,
their mutations result in accumulation of DNA damage
and activation of cellular checkpoints, including p53.
Consequently, the embryonic lethality of Rad51 (Lim and
Hasty, 1996), Rad51 B (Shu et al, 1999), and Rad51 D
(Smiraldo et al, 2005) mutants was delayed on p53-null
background. The role of p53 in the embryonic lethality
of Xrcc2
/
mutants remains controversial (Orii et al,
2006; Adam et al, 2007).
The meiosis specific the Rad51 paralogue Dmc1 is essential
for meiotic recombination in S.cerevisiae (Bishop et al,
1992). In mice, Dmc1 expression is restricted to the testis
and ovaries, and null Dmc1 mutants are sterile and exhibit
arrested gametogenesis in the first meiotic prophase (Pittman
et al, 1998; Yoshida et al, 1998). These results demonstrate
the conservation of the essential meiotic function of Dmc1.
Rad54 is another member of the Rad52 epistasis group
involved in HR. Similar to rad51 and rad52 mutants, S.
cerevisiae deficient in rad54 are highly sensitive to IR. DT40
cells mutant for Rad54 are viable, hypersensitive to IR,
exhibit slow growth, decreased rate of Ig gene conversion,
and show a drastic decrease in gene-targeting efficiency
(Bezzubova et al, 1997). Rad54 paralogues exist and include
rdh54 in S.cerevisiae and Rad54B in mammalian cells. In
contrast to rad54 mutants in S.cerevisiae,rdh54 mutants are
not sensitive to IR and show no defects in mitosis (Shinohara
et al, 1997). However, meiotic recombination was affected in
rdh54 mutants and was completely defective in S.cerevisiae
lacking the two Rad54 orthologues (Shinohara et al, 1997).
Homozygous mutant mice lacking Rad54 and/or its para-
logue Rad54B are viable and ES cells deficient in Rad54 or
Rad54B are hypersensitive to IR, MMS, and MMC (Essers
DNA-damage repair
R Hakem
&2008 European Molecular Biology Organization The EMBO Journal VOL 27 |NO 4 |2008 597
et al, 1997; Bross et al, 2003; Wesoly et al, 2006). Rad54
/
,
but not Rad54B
/
, ES cells have a 3- to 40-fold decrease in
HR as assessed by gene targeting. Dual inactivation of both
paralogues further impairs HR, as double mutant ES cells
show a further 10-fold reduction in their gene targeting
efficiency compared with Rad54-null ES cells. Although
Rad54 is important for HR, mice deficient in Rad54 and/or
Rad54B do not have a predisposition to cancer.
BRCA1 and BRCA2, the early-onset breast cancer-
susceptibility genes, have also been demonstrated to partake
in HR-mediated DSB repair. Germline mutations of BRCA1 or
BRCA2 predispose women to familial human breast and
ovarian cancer (Narod and Foulkes, 2004). Individuals with
germline mutations for BRCA1 or BRCA2 also have increased
risk for other cancers, including prostate cancer. Although the
BRCA1 gene has important functions in DNA-damage signal-
ling/repair it is also involved in other cellular processes,
including transcription, ubiquitination, oestrogen receptor
signalling, and chromatin remodelling. In response to DSBs,
BRCA1 is phosphorylated by ATM, ATR, and Chk2, under-
scoring its functional regulation by other molecules involved
in DNA-damage checkpoints. BRCA2 functions in the loading
of the HR protein RAD51 during filament formation. It
directly binds to RAD51 and its phosphorylation on Ser3291
inhibits this binding (Esashi et al, 2005). In addition to its role
in HR, recent studies have suggested a role for BRCA2 in the
stabilization of stalled RFs (Lomonosov et al, 2003).
Homozygous mutations of murine Brca1 resulted in em-
bryonic lethality. (Gowen et al, 1996; Hakem et al, 1996; Liu
et al, 1996; Ludwig et al, 1997; Xu et al, 2001). Premature
death of Brca1-mutant embryos is likely due to accumulation
of damaged DNA and activation of DNA-damage checkpoints,
including Chk2–p53. Inactivation of p53 delayed the embryo-
nic lethality of Brca1-null mutants (Hakem et al, 1997;
Ludwig et al, 1997) and completely rescued survival of
Brca1 hypomorphic mutants (Xu et al, 2001). Studies of
hypomorphic and conditional null Brca1 mutant females
demonstrated that similar to humans, Brca1 mutations
increase the risk for mammary cancer (Xu et al, 1999;
McPherson et al, 2004b). Interestingly, inactivation of p53,
or Chk2, drastically facilitates development of mammary
tumors on Brca1-mutant backgrounds (Xu et al, 1999;
McPherson et al, 2004b). A targeted Brca1-null mutation to
the T-cell lineage resulted in increased genomic instability,
apoptosis, cell-cycle arrest, and a drastic depletion of the
T-cell lineage (Mak et al, 2000). Interestingly, development
of Brca1-null T-cells was completely rescued in p53-or
Chk2-mutant backgrounds, and this rescue was at the
expense of increased genomic instability and increased risk
for tumorigenesis (McPherson et al, 2004b).
BRCA1 plays a major role in the DNA-damage response as
it interacts with the MRN complex and g-H2AX at the site of
DSB. Likewise, it is also required for the recruitment of other
proteins such as Rad51, BRCA2, and BARD1 to sites of DSBs
(Greenberg et al, 2006). HR, as measured by the efficiency of
gene targeting in ES cells, was drastically reduced in Brca1
hypomorphic mutants (Moynahan et al, 1999). Other defects,
including compromised telomeres integrity and male sterility,
were also associated with Brca1 mutations (Xu et al, 2001;
McPherson et al, 2006).
Similar to Brca1 and Rad51 mutants, null mutations for
Brca2 result in early mouse embryonic lethality and impaired
HR (Ludwig et al, 1997; Suzuki et al, 1997; Moynahan et al,
2001). Furthermore, loss of Brca2 in murine cells resulted in
increased genomic instability and activation of p53. A small
subset of Brca2 hypomorphic mutants survived embryonic
development (Connor et al, 1997; Friedman et al, 1998).
These mice were growth defective, sterile, and predisposed
for thymic lymphoma, and their cells were sensitive to DNA
damaging agents, including IR, MMS, and UV radiation.
Conditional mutant strains have also been generated using
the Cre/loxP system and similar to Brca1 mutation, dual
inactivation of Brca2 and p53 in mammary epithelial cells
resulted in increased frequency and decreased latency for
mammary tumors (Jonkers et al, 2001).
Beside the proteins described above, efficient HR requires
other proteins such as members of the family of RecQ
helicases (Hickson, 2003). The human family of RecQ heli-
cases includes BLM, REQ, WRN, RECQL4, and RECQL5, and
is important for unwinding DNA, repairing stalled RFs,
promoting HR, and maintaining overall genomic integrity.
Mutations of some of these helicases are associated with rare
human syndromes (Hickson, 2003). Thus, the Werner syn-
drome (WS) and the Rothmund Thomson syndrome (RTS)
are associated with mutations of human WRN and RECQL4
genes, respectively (Figure 2). WS features include premature
ageing, short stature, and cancer predisposition. Clinical
features of RTS include short stature, skin pigmentation
changes, skin atrophy, and increased cancer predisposition.
In addition mutations of the human BLM gene are asso-
ciated with the Bloom syndrome (BS), a rare autosomal
recessive disorder characterized by growth defects, immune
deficiency, reduced fertility, and predisposition to a large
spectrum of cancers. Cells from BS patients have elevated
sister-chromatid exchange (SCE) and genomic instability.
Three mouse models for Blm mutation have been generated,
but only one was viable (Chester et al, 1998; Luo et al, 2000;
Goss et al, 2002). Similar to BS patients, viable Blm mice are
prone to a wide range of tumors, and cells from these mutant
mice show elevated levels of SCE, a hallmark cellular phe-
notype of BS. In addition, Blm deficiency leads to increased
mitotic recombination and somatic loss of heterozygosity
(Luo et al, 2000).
While most steps essential for HR in eukaryotes have been
well characterized, the identification of the resolvase(s)
required for the resolution of Holliday junctions (HJs) during
mitotic and meiotic recombination turned out to be very
challenging. Resolution of HJs is critical for HR and is
mediated in E.coli by the resolvase RuvC (West, 1997);
however, the search for true HJ resolvase(s) in eukaryotes
is still ongoing.
Yeast Mus81 (MMS, UV-sensitive, clone 81) with its partner
Eme1 (Schizosaccharomyces pombe) or MMS4 (S.cerevisiae)
forms a structure-specific endonuclease important for DNA-
damage repair (Osman and Whitby, 2007). Yeast mus81,
eme1,ormms4 mutants show increased sensitivity to DNA-
damaging agents that interfere with normal progression of
RFs caused by agents such as UV radiation, MMS, and
camptothecin, but their sensitivity to IR is not affected
(Osman and Whitby, 2007) . These mutants also show
meiotic defects supporting a role for this endonuclease
in this process.
In vitro studies indicate that yeast and mammalian Mus81,
with their partner Eme1/Mms4, efficiently cleaves various
DNA-damage repair
R Hakem
The EMBO Journal VOL 27 |NO 4 |2008 &2008 European Molecular Biology Organization598
DNA structures that mimic RFs, D-loops, and nicked HJs, but
the cleavage activity of intact HJs was weak (Osman and
Whitby, 2007). Thus, at least in mammalian cells, Mus81 with
Eme1 or with Eme2, another eme1 homologue, forms a
heterodimeric 30-flap/RF endonucleases that process stalled
RFs and recombination intermediates, but does not possess
typical characteristics of an HJ resolvase (Abraham et al,
2003; Ciccia et al, 2003, 2007; Ogrunc and Sancar, 2003).
Gene-targeted inactivation of Mus81 or Eme1 in mouse and
human cells increased sensitivity to ICL, hydroxyurea, but
not UV or IR and enhanced spontaneous and MMC-induced
genomic instability (Abraham et al, 2003; McPherson et al,
2004a). Mus81 was shown to be important for generating
ICL-induced DSBs, as well as for mediating the restart of
stalled or blocked RFs (Hanada et al, 2006). Our studies of a
mouse model for Mus81-null mutation demonstrated that
Mus81 is a haploinsufficient tumor suppressor (McPherson
et al, 2004a). Heterozygous and homozygous Mus81 mutants
show cancer predisposition, particularly to T- and B-cell
lymphomas. The MMC hypersensitivity of Mus81-mutant
cells and mice is p53-dependent (Pamidi et al, 2007).
Interestingly, on a p53-null background, Mus81-mutant
mice have more genomic instability and develop sarcomas
and multiple different tumors with short latency and high
penetrance, demonstrating a role for p53 in suppressing
cancer associated with Mus81 mutation (Pamidi et al,
2007). Both Mus81- and Mus81p53-mutant mice are fertile,
thus failing to support a requirement for Mus81 in meiotic
recombination. A second Mus81-mutant strain (Mus81
D912
)
showed increased genomic instability but failed to show
increased tumorigenesis (Dendouga et al, 2005). A role in
cancer for Eme1 or Eme2 requires further investigations.
Recent studies have implicated the mammalian RAD51
paralogues RAD51C–XRCC3 in HJ resolution (Liu et al,
2004). Rad51C–XRCC3 binds HJs in vitro and cell extracts
from Rad51C- or XRCC3-deficient hamster cells, exhibit low
HJ resolvase activities. In addition, depletion of RAD51C from
HeLa cell extracts strongly impairs in vitro HJ branch migra-
tion and resolution. Similar to other Rad51 paralogues, null
mutation of mouse Rad51C results in embryonic lethality.
However, a viable mouse model carrying intronic integration
of a Neomycine selection cassette resulting in decreased
Rad51 expression has been recently reported (Kuznetsov
et al, 2007). About 40% of mutant males and 10% of mutant
females were infertile. Cell extracts from MEFs null for
Rad51C and p53 show reduced in vitro HJ resolvase activities
compared with controls. Thus, the current data support a role
for the mammalian RAD51C in HJ resolution; however,
further studies are still required to establish RAD51C as
a typical HJ resolvase and to demonstrate whether other
mammalian HJ resolvases also exist.
Thus, the human syndromes and early onset of breast
cancer, and the developmental defects, increased genomic
instability and tumorigenesis associated with impaired HR in
mouse models, all demonstrate the in vivo requirement for
HR-mediated DNA-damage repair.
The NHEJ repair pathway
NHEJ is the predominant pathway of DSBs repair in mam-
malian cells (Figure 1; Kanaar et al, 2008). This repair path-
way is active especially at the G1, but is error prone. NHEJ is
also essential for T-cell receptor-a/band Ig V(D)J recombina-
tion, and thus this repair pathway is required for the devel-
opment of the T and B-cell repertoires. The core protein
components of the mammalian NHEJ include the Ku subunits
(Ku70 and Ku80), DNA–PKcs, XRCC4, DNA ligase IV (LigIV),
Artemis, and the recently identified Cernunnos–XLF (also
known as NHEJ1).
DNA–PK is composed of the catalytic subunit DNA–PKcs
and the heterodimer Ku70/Ku80, important for DNA end
binding. DNA–PKc is a serine/threonine kinase that is
activated following its recruitment by Ku70/Ku80 to sites of
DSBs. Active DNA–PKcs autophosphorylate themselves as
Table VI Examples of mouse models for the NHEJ repair pathway
Genotype Developmental defects Fertility
defects
Spontaneous
tumorigenesis
References
DNA-PKcs/T and B-cell development arrested
at early progenitor stages
None Not affected (Gao et al, 1998a; Taccioli
et al, 1998; Kurimasa et al,
1999)
Ku70/Growth retardation and early
arrest of T and B-cell development
None Thymic lymphomas (Gu et al, 1997; Ouyang
et al, 1997)
Ku80/Growth retardation and early
arrest of T and B-cell development
None Not affected (Nussenzweig et al, 1997)
Ku80/p53/Growth retardation and block of
T and B-cell development
None Early onset of pro
B-cell lymphomas
(Difilippantonio et al,
2000)
Artemis/Developmental arrest at early
T and B cell progenitor stages
None None (Rooney et al, 2002;
Li et al, 2005)
Artemis/p53/Developmental arrest at early
T and B cell progenitor stages
None Pro-B cell
lymphomas
(Rooney et al, 2004)
LigIV
Y288C
Growth retardation and progres-
sive loss of haematopoeitic stem
cells
NA None (Nijnik et al, 2007)
LigIV/Late embryonic lethality NA NA (Barnes et al, 1998;
Frank et al, 1998)
LigIV/p53/Viable but growth retarded None Early onset of pro
B-cell lymphomas
(Frank et al, 2000)
Xrcc4/Late embryonic lethality NA NA (Gao et al, 1998b)
Xrcc4/p53/Viable but growth retarded None Early onset of pro
B-cell lymphomas
(Gao et al, 2000)
NA, not applicable.
DNA-damage repair
R Hakem
&2008 European Molecular Biology Organization The EMBO Journal VOL 27 |NO 4 |2008 599
well as several other targets, including the Ku subunits, p53,
H2AX, Artemis, XRCC4, and WRN (Collis et al, 2005).
The important role of DNA–PKcs in vivo became evident
following the identification of its mutation in the severe
combined immunodeficient ‘SCID’ mice (Bosma et al, 1983;
Blunt et al, 1995; Kirchgessner et al, 1995). SCID mice exhibit
impaired V(D)J recombination and have arrested T- and
B-cell development at early progenitor stages. The role of
DNA–PKc mutation in SCID phenotypes was confirmed in
DNA–PKc-null mice obtained by gene targeting (Table VI; Gao
et al, 1998a; Taccioli et al, 1998; Kurimasa et al, 1999). These
mutants are severely immunodeficient, have impaired V(D)J
coding joining but normal signal joining, and their
T- and B-cell development are blocked at early progenitor
stages. Moreso, MEFs deficient for DNA–PKcs are hypersen-
sitive to IR, further supporting its requirement for DSB repair.
Similar to DNA–PKcs, targeted inactivation of Ku70 or
Ku80 in mice results in SCID phenotypes associated with
early arrest of T- and B-cell development, although the T-cell
arrest in Ku70 mice is leaky (Nussenzweig et al, 1996; Gu
et al, 1997; Ouyang et al, 1997). However, in contrast to
DNA–PKcs mice, null mutants for Ku70 or Ku80 showed
growth retardation and their cells were impaired in both
V(D)J coding and recombination signal (RS) end joining.
Inactivation of either Ku70 or Ku80 resulted in elevated IR
sensitivity but did not affect mice fertility. Taken together,
these data demonstrate the essential role of DNA–PK in NHEJ
and V(D)J recombination.
Increased apoptosis and proliferative arrest of pro-B cells
in Ku80
/
mice were rescued by a p53-null background
(Difilippantonio et al, 2000). Genomic instability associated
with Ku80 mutation was significantly increased in the ab-
sence of p53, and the double mutants developed pro B-cell
lymphomas with 100% penetrance and died within 3 months
of age (Nussenzweig et al, 1997; Difilippantonio et al, 2000).
Similarly, the incidence of thymic lymphomas was increased
in Ku70-null mice (Gu et al, 1997) and inactivation of p53 on
DNA–Pk
scid
-mutant background resulted in the rapid onset of
lymphomas/leukaemia (Guidos et al, 1996).
The nuclease Artemis is a phosphorylation target for
DNA–PK and forms a complex with DNA–PKcs. This complex
is important for the hairpin-opening step of V(D)J recombi-
nation and for the 50and 30overhang terminal end processing
in NHEJ. ARTEMIS mutation causes the severe combined
immunodeficiency with sensitivity to ionizing radiation
(RS-SCID), a rare human disorder (Figure 2; O’Driscoll and
Jeggo, 2006). Similar to DNA–PKc mutants, mice deficient for
Artemis are viable, have normal size, and suffer severe
combined immunodeficiency associated with developmental
arrest at early T- and B-cell progenitor stages (Rooney et al,
2002; Li et al, 2005). Artemis deficiency impaired coding but
not RS joining. Artemis-mutant MEFs are radiosensitive and
exhibit increased chromosomal instability. This spontaneous
loss of genomic integrity is likely the basis for increased
tumorigenesis associated with dual inactivation of Artemis
and p53 (Rooney et al, 2004; Woo et al, 2007).
Following the terminal end-processing step of NHEJ,
LigIV/XRCC4 complex serves to perform the ligation and
final step of NHEJ. Hypomorphic mutations of human LigIV
are associated with the hereditary autosomal LigIV syndrome
(Figure 2; O’Driscoll and Jeggo, 2006). This syndrome is very
rare, as only eight patients have been identified so far. This
syndrome is characterized by SCID phenotype, growth
defects, microcephaly, radiosensitivity, and leukaemia.
Recently, Cernunnos–XLF, a novel NHEJ factor that interacts
with the XRCC4–DNA LigIV complex, has been identified
(Ahnesorg et al, 2006; Buck et al, 2006). Mutation of
Cernunnos–XLF was found to be associated with a
rare inherited human syndrome characterized by growth
retardation, microcephaly, severe immunodeficiency, and
radiosensitivity (Figure 2).
Null mutation of LigIV or Xrcc4 in mouse models results in
late embryonic lethality associated with extensive apoptosis
in the embryonic central nervous system (Barnes et al, 1998;
Frank et al, 1998; Gao et al, 1998b). V(D)J joining does not
occur, lymphopoiesis is blocked, and MEFs deficient for LigIV
or Xrcc4 are radiosensitive, growth defective and enter sene-
scence prematurely. p53 inactivation rescued the extensive
apoptosis in the central nervous system, the defective pro-
liferation/senescence and the embryonic lethality associated
with LigIV or Xrcc4 mutation (Frank et al, 2000; Gao et al,
2000). However, p53 inactivation did not rescue defective
V(D)J recombination or lymphocyte development of LigIV-or
Xrcc4-null mutants. In addition, on a p53-null background,
both LigIV- and Xrcc4-null mutants were growth-retarded
and developed pro-B lymphomas with short latency. More
recently, the hypomorphic mutation LigIV
Y288C
has been
reported to lead to growth retardation, progressive loss of
haematopoeitic stem cells, and immunodeficiency (Nijnik
et al, 2007). In addition, mice carrying a targeted Xrcc4
mutation to neuronal progenitors demonstrated increased
genomic instability and predisposition for medulloblastomas
(Yan et al, 2006). Although mice deficient for Cernunnos–XLF
have not yet been reported, ES cells null for this gene are
radiosensitive, show defective V(D)J coding and RS joining,
and accumulate spontaneous genomic instability (Zha et al,
2007). These data suggest that similar to other NHEJ
core components, inactivation of Cernunnos–XLF could
potentially lead to cancer development.
The radiosensitivity, genomic instability, immuno-
deficiency, growth retardation, embryonic development, and
cancer predisposition associated with defective NHEJ all
demonstrate the major role this DNA-damage repair pathway
plays in vivo.
Concluding remarks
Although there exist numerous proteins employed by several
DNA-repair pathways in response to DNA damage, loss or
partial inactivation of only one of these proteins can have
extremely devastating consequences. Conversely, there are
some mutated DNA-damage repair proteins that go unnoticed
due to a lack of functional consequences at both the cellular
and organism level. This is likely due to genetic redundancy
and compensatory mechanisms of repair. The onset of var-
ious syndromes, increased cancer predisposition, immuno-
deficiency, and neurological defects associated with impaired
DNA-damage repair, are all strong indications for the require-
ment of a tight regulation of these pathways. Our current
knowledge of the mechanisms that regulate DNA repair
has grown significantly over the past years. In addition to
improving diagnosis, this overwhelming knowledge of
the mechanism of DNA-damage repair and DNA-damage
DNA-damage repair
R Hakem
The EMBO Journal VOL 27 |NO 4 |2008 &2008 European Molecular Biology Organization600
checkpoint will likely contribute to a better design for both
drugs and therapies for diseases, such as cancer.
Our understanding of the DNA-repair mechanisms in hu-
mans, and how defects in these processes lead to human
syndromes and pathologies, has greatly benefited from stu-
dies in other organisms, including mice and yeast. Further
studies in these organisms are required to better assess the
effect of the amino-acid substitutions and point mutations of
DNA-repair genes that associate with human syndromes
and diseases. Characterization of the post-translational
modifications that control the function and stability of DNA-
repair proteins, such as phosphorylation and ubiquitination,
is essential for future manipulation of the repair machinery to
aid in better therapeutic responses.
Acknowledgements
We thank the members of the Hakem laboratory for helpful discus-
sions, and Amanda Fenton, Anne Hakem, Jacinth Abraham, and
Renato Cardoso for reviewing the paper. Research in the Hakem
laboratory was supported by the Canadian Institutes of Health
Research, the National Cancer Institute of Canada, the Cancer
Research Society, and the Leukemia & Lymphoma Society Of
Canada. We apologize to those whose work was not cited directly
owing to space limitations.
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