Homologous recombination and maintenance of genome integrity:
Cancer and aging through the prism of human RecQ helicases
Karen J. Ouyanga, Leslie L. Woob, Nathan A. Ellisa,c,*
aCommittee on Genetics, University of Chicago, Chicago, IL, United States
bDepartment of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, United States
cDepartment of Medicine, University of Chicago, Chicago, IL, United States
1. Maintenance of genome integrity
Genome integrity is the capacity of the cell to avoid mutations.
Tantamount to genome integrity is the accurate replication and
faithful transmission of the genetic information. Tightly associated
with the DNA replication and chromosome segregation mechan-
isms are biochemical pathways that contend with DNA damage.
Because cells are continuously exposed to DNA damage, generated
by either the products of normal cellular metabolism or the
environment, cells have evolved biochemical pathways that
respond to specific DNA lesions that are encountered (Friedberg
et al., 2004). Cells coordinate proficient DNA repair with a highly
regulated DNA damage-signaling network that controls the
progression of cells through the cell-cycle, referred to as
checkpoints (Hartwell, 1992).
Error-free repair of DNA damage preserves the genome
and allows continued normal function. If DNA damage is too
extensive, the cell may commit to apoptosis. Alternatively, the
cell may repair the damage but introduce one or more errors,
thereby surviving with acquired mutations that potentially
compromise normal cellular functions. Mutations in proteins
that are themselves important in the repair of DNA damage and
cell-cycle checkpoints are especially insidious for the cell.
Mutations in DNA repair and checkpoint functions increase the
rate at which mutations accumulate in a cell, a condition
referred to as genomic instability (Loeb, 2001). Genomic
instability is a common condition of cancer cells (Lengauer
et al., 1998). An increased rate of mutation increases the
probability that cancer-causing mutations will occur, and
failure to engage checkpoints allows cells to proliferate in
the presence of DNA damage. Genomic instability is also the
unifying characteristic of a group of hereditary disorders
that contain germline mutations in the DNA repair or the
checkpoint genes (Table 1). In these syndromes, mutations
accumulate at higher rates than normal given the same level of
exposure to the relevant mutagen. Although genetic defects in
each repair or checkpoint pathway are associated with clinically
distinct entities, as a group they are characterized by develop-
mental abnormalities, cancer predisposition, and accelerated
Mechanisms of Ageing and Development 129 (2008) 425–440
A R T I C L EI N F O
Available online 15 March 2008
Double-strand break repair
A B S T R A C T
Homologous recombination (HR) is a genetic mechanism in somatic cells that repairs DNA double-strand
breaks and restores productive DNA synthesis following disruption of replication forks. Although HR is
indispensable for maintaining genome integrity, it must be tightly regulated to avoid harmful outcomes.
HR-associated genomic instabilities arise in three human genetic disorders, Bloom syndrome (BS),
Werner syndrome (WS), and Rothmund–Thomson syndrome (RTS), which are caused by defects in three
individual proteins of the RecQ family of helicases, BLM, WRN, and RECQL4, respectively. Cells derived
from persons with these syndromes display varying types of genomic instability as evidenced by the
presence of different kinds of chromosomal abnormalities and different sensitivities to DNA damaging
agents. Personswith these syndromesexhibitavarietyofdevelopmental defectsand arepredisposedtoa
wide range of cancers. WS and RTS are further characterized by premature aging. Recent research has
shown many connections between all three proteins and the regulation of excess HR. Here, we illustrate
the elaborate networks of BLM, WRN, and RECQL4 in regulating HR, and the potential mechanistic
linkages to cancer and aging.
? 2008 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author at: Department of Medicine, MC 4080, University of
Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, United States.
Tel.: +1 773 702 7868; fax: +1 773 702 5790.
E-mail address: email@example.com (N.A. Ellis).
Contents lists available at ScienceDirect
Mechanisms of Ageing and Development
journal homepage: www.elsevier.com/locate/mechagedev
0047-6374/$ – see front matter ? 2008 Elsevier Ireland Ltd. All rights reserved.
2. Homologous recombination repair
A double-strand break (DSB) is the most toxic form of DNA
damage that a proliferating cell can sustain. In the absence of DSB
repair, the broken ends are inherently unstable because they are
unprotected and therefore subject to nucleolytic degradation,
resulting indeletion ofgenes both proximaland distal to the break.
If a break occurs on one of the two sister chromatids, the resulting
acentric fragment can be lost or attached to a non-homologous
chromosome, leading potentially to segmental trisomy. If a break
involves both sister chromatids, the ends distal to the break can
These rejoining events form an acentric fragment and a dicentric
chromosome, respectively. The dicentric chromosome can form an
anaphase bridge in metaphase; the stretched DNA is susceptible to
leading to additional bridge-breakage-fusion cycles (McClintock,
Exposure of cells to gamma irradiation generates high levels of
reactive oxygen species (ROS), which in turn generates large
numbers of strand breaks (both single and double) through
oxidation and cleavage of the sugar-phosphate backbone. Endo-
genous DSBs, however, are relatively rare in most cells. A major
source of DSBs originates from the process of DNA replication
(Haber, 1999). During S phase, the DNA is unraveled and exposed,
and the replication complex may clash with DNA lesions or
proteins that have engaged the DNA. For example, if the replicative
polymerase encounters a single-strand break or gap that was
introduced during repair of DNA damage, then the replication fork
will collapse and devolve to an intact duplex and a single DSB
(Fig. 1A). The replication fork must be re-established by a process
that involves homologous recombination (HR) (Kuzminov, 1999;
Michel et al., 2007).
With a replication-associated DSB, only a single broken end is
available (Cox, 2001). The broken end is first processed by a 50to 30
exonuclease to expose a single-stranded DNA (ssDNA) tail with a 30
end. The free ssDNA tail is rapidly complexed with ssDNA binding
protein (referred to as SSB in bacteria and RPA in eukaryotes).
Mediator proteins, such as RAD52 and the RAD55/57 complex,
actively substitute recombinase (RecA protein in bacteria and
RAD51 in eukaryotes) for ssDNA binding protein, and the
recombinase-ssDNA nucleoprotein filament invades the intact
duplex to produce a displacement loop (D-loop). The invading 30
end creates a primer for DNA synthesis, which restarts the
D-loop to generate a four-stranded recombination intermediate
replication fork. A prevailing view is that the Holliday junction is
resolved by cutting and rejoining, and half of the cutting/rejoining
events lead to a crossover that produces a sister-chromatid
exchange (SCE) (Wilson and Thompson, 2007).
Another potential source of SCEs is generated by base-damage
encountered by the replicative polymerase during S phase. When
DNA polymerase is stalled at base damage, DNA replication can
restart downstream of the stalled fork, producing a daughter-
strand gap (Fig. 1B). Hypothetically, recombinase is loaded onto
the ssDNA gap by mediators, and the recombinase-ssDNA filament
invades the intact DNA duplex. The available 30end can anneal
with the displaced DNA strand of the D-loop, which primes DNA
synthesis across the gap. By ligation with the 50end on the other
side of the gap, a double Holliday junction can form (Agarwal et al.,
2006; Cahill et al., 2006). The double Holliday junction can be
resolved with or without SCE formation, depending on the
molecular mechanism used for resolution (Fig. 1B depicts a case
in which SCE did not occur). The result of this HR process is lesion
bypass not DNA repair, which can be achieved at a later time.
broken ends must be processed to expose ssDNA tails. Mediators
displace SSB protein and load recombinase onto one of the tails.
Genetically determined genomic instability disorders in humansa
Disorder Gene mutatedDNA repair pathwayCancer riskb
Autosomal recessive disorders
Nijmegen breakage syndrome
FANCA, -B, -C, -D1, -D2,
-E, -F, -G, -I, -J, -L, -M, -N
XPA, -B, -C, -D, -E, -F, -G
CSA, -B, XPB, -D, -G
XPB, -D, TTDA, -B
LIG4, Artemis, XLF
Double-strand break signaling
UV and replication damage signaling
Double-strand break repair
Double-strand break repair
Regulation of recombination
Double-strand break repair?
Homologous recombination and
interstrand crosslink repair
Nucleotide excision repair (NER)
Non-homologous end joining
XPF-ERCC1 progeroid syndrome
Xeroderma pigmentosum variant
Ligase I deficiency
Spinocerebellar ataxia with neuropathy
Ataxia oculomotor apraxia
Single-strand break repair
Single-strand break repair
Autosomal dominant disorders
Hereditary breast cancer
MLH1, MHS2, -6
Homologous recombination repair
aInformation in table is from (Niedernhofer et al., 2006; Prigent et al., 1994; Rass et al., 2007; Taniguchi and D’Andrea, 2006; van Brabant et al., 2000a).
bA plus (+) indicates that cancer susceptibility is present, a minus (?) indicates it is absent, and a question mark (?) indicates that the presence of cancer susceptibility is
cWRN protein has also been implicated in homologous recombination and base excision repair (see text for details).
dXPF-ERCC1 (XFE) progeroid syndrome is associated in humans with a single identified mutation in the XPF gene (Niedernhofer et al., 2006). The XPF and ERCC1 complex
has been implicated in interstrand crosslink DNA repair, which may be defective in this syndrome.
K.J. Ouyang et al./Mechanisms of Ageing and Development 129 (2008) 425–440
Recombinase catalyzes invasion of the intact duplex and primes
DNA synthesis. After some amount of DNA synthesis, the extended
strand can be unwound from the intact duplex and annealed with
the free ssDNA tail from the other broken end. If necessary, flaps are
removed; gaps are filled by DNA polymerase, and the ends are
ligated together to finish repair of the DSB. This pathway of HR has
been called synthesis-dependent strand annealing (SDSA) (Adams
the broken and the intact chromosomes.
HR is an error-free pathway because it repairs DSBs using the
genetic information from an intact homologous DNA molecule,
which in higher eukaryotes is almost always the sister chromatid.
Excessive HR, however,can generate DNA damage. The genomes of
higher eukaryotes contain a great deal of tandem and dispersed
repeat sequence DNA, for example, centromeric DNA, telomeres,
repeat gene clusters, and of course ‘‘junk’’ DNA. Genetic exchange
between non-homologous copies of these repeat sequences
(illegitimate recombination) generates translocations, deletions,
or inversions (Myung et al., 2001). Illegitimate recombination
events are more likely to occur when the cell is replicating its DNA
because the DNA is exposed. Replication forks that have stalled
because, for example, the polymerase has encountered a damaged
base, can regress to form a structure that resembles a Holliday
junction (a so-called regressed replication fork). Up-regulated HR
could convert these structures into DSBs.
3. Anti-recombination and the RecQ family of DNA helicases
To oppose these potentially adverse effects of HR, the cell holds
recombination activity (Radman, 1989). Anti-recombination refers
tofactorsthatareinhibitorytothe HRreaction.Forexample, ssDNA
ssDNA when available, and can prevent recombinase from binding
Fig. 1. Homologous recombination (HR) in repair of lesions arising during DNA replication: the pro- and anti-recombinogenic activities of BLM. (A) Replication restart. A
single-ended double strand break (DSB) can arise during DNA replication when the replication fork encounters a single-strand DNA (ssDNA) break. The fork must be re-
the 30tail. Mediator proteins displace SSB and load recombinase. Recombinase catalyzes the invasion of the 30tail into the intact duplex to form a D-loop. BLM could
antagonize the formation of stable D-loops by preventing the formation of recombinase-nucleoprotein filaments or by unwinding the invading strand. (The triangle
represents the BLM protein, and the direction that thetriangle points indicates the direction of BLM’s helicase function.) If a stable D-loop is formed, the 30end of the invading
strandprovidesaprimer forDNApolymerase.BLMmayfacilitatetheearlystagesofthisDNAsynthesis.ResolutionoftheHollidayjunctionformedbehind thereplicationfork
(breaking and rejoining at the arrowheads) can lead to a sister-chromatid exchange. (B) Lesion bypass. When DNA polymerase encounters base-damage that blocks the
polymerase at the site of damage (depicted by an asterisk), DNA replication can restart downstream, leaving a daughter-strand gap. As in part (A), mediators substitute
recombinase for SSB on the ssDNA in the gap. After strand invasion, lesion bypass can be achieved. The displaced strand (in red) can serve as a template for DNA synthesis
using the 30end on the left side of the gap. A double Holliday junction is then formed, and this structure can be ‘‘dissolved’’ by the BLM/Topoisomerase 3 (Top3) complex
without crossing-over (see text for details). Alternatively, the Holliday junctions can be resolved by breaking and rejoining, which half the time will result in sister-chromatid
exchange (not shown). (C) DSB repair-synthesis-dependent strand annealing (SDSA). If the ssDNA in a daughter-strand gap is nicked (right arrow connecting part B with part
C), a DSB is formed that consists of two broken ends. As in part (A), strand resection and SSB loading prepare the DNA substrate. Recombinase substitutes SSB on only one of
strand behind the DNA polymerase, and the invading strand is captured by the other broken end, a function that can also be facilitated by BLM’s strand-annealing activity
(Bartos et al., 2006; Cheok et al., 2005). The resulting annealed substrate is polished by DNA synthesis, and possibly also flap removal, and the duplex is sealed by ligation to
repair the duplex.
K.J. Ouyang et al./Mechanisms of Ageing and Development 129 (2008) 425–440
a balance of pro- and anti-recombinogenic activities and the
the cell engages. In some situations, for example, when the sister
called non-homologous end-joining (NHEJ) pathway (Lieber et al.,
2006). NHEJ is the pathway that precursors to B and T cells use to
recombine the immunoglobulin and T-cell receptor loci. In this
prone because the ends are often processed by exonuclease or by
terminal nucleotide additions.
The specialized DNA helicases of the RecQ family (Fig. 2) are
as will be explained in greater detail below, on the one hand
inhibitionofHRthroughthe stabilizationofstalledreplication forks
and on the other hand prevention of stable recombinase-nucleo-
protein filaments, and facilitation of HR through the release of DNA
strands for SDSA and resolution of Holliday junctions.
Like other DNA helicases, most RecQ family members possess
DNA-dependent ATPase and ATP- and Mg2+-dependent DNA
unwinding activities (Karow et al., 1997; Neff et al., 1999; Shen
et al., 1998b; Suzuki et al., 1997). RecQ helicases bind ssDNA and
translocate unidirectionally in a 30to 50direction, disrupting the
hydrogen bonds that hold together the DNA duplex. Unlike most
other helicases, the RecQ helicases have a strong preference for
DNA substrates that resemble recombination intermediates,
including Holliday junctions (Karow et al., 2000), double Holliday
junctions (Wu and Hickson, 2003), D-loops (van Brabant et al.,
2000b), and G-quadruplex DNA (Huber et al., 2002; Sun et al.,
1998). G-quadruplex DNA is a tetrahelical DNA structure that
Gilbert, 1988; Williamson et al., 1989). DNA sequences in the
genome that can fold into G-quadruplex DNA are found in G-rich
regions, including telomeres, ribosomal DNA gene clusters, and
immunoglobulin switch regions.
resemble replication forks. In vitro, they can promote the
annealing of homologous single strands (Cheok et al., 2005; Garcia
et al., 2004; Sharma et al., 2005) and the regression of a replication
fork (Machwe et al., 2006a); they can also do the opposite—reverse
the regressed replication fork to reform the fork (Courcelle et al.,
2003; Ralf et al., 2006). It also has been shown that RecQ helicases
are capable of catalyzing the branch migration of Holliday
junctions (Karow et al., 2000). The observation of many
structure-specific substrate preferences of RecQ helicases has
led to a multitude of speculations about where RecQs function in
the universe of DNA transactions. However, cell and molecular
data from normal cells and from cells that carry mutations in RecQ
helicases have implicated specific DNA transactions that occur at
the interface of DNA replication and HR repair.
The human RecQ helicases have been a major source of interest
in the DNA repair field because of their links to human disease.
Humans possess five distinct RecQ helicases – RECQL1, BLM, WRN,
of these helicases, BLM, WRN, and RECQL4, result in the genomic
instability disorders Bloom syndrome (BS), Werner syndrome
(WS), and Rothmund–Thomson syndrome (RTS), respectively.
These three human disorders share the general characteristics of
genomicinstability and cancer predisposition syndromes, buteach
syndrome possesses unique clinical, cellular, genetic, and bio-
chemical features that point to non-overlapping roles in the
maintenance of genome integrity.
4. Bloom syndrome helicase and replication-fork functions
4.1. Consequences of mutations in the Bloom syndrome gene
BS is a rare autosomal recessive disorder caused by bi-allelic
loss-of-function mutations in the BLM gene. The disorder is
characterized by small but proportional size, a sun-sensitive facial
erythema, hypo- and hyperpigmented skin lesions, immune
deficiency, infertility in males and sub-fertility in females, a
paucity of subcutaneous fat and defects in sugar metabolism,
susceptibility to type 2 diabetes, and, most prominently, a
predisposition to all types of cancers (Diaz et al., 2006; German
et al., 2007). The cancer predisposition in BS is notable for its high
incidence, broad spectrum (including leukemias, lymphomas, and
carcinomas), early diagnosis relative to the same cancer in the
general population, and the development of multiple cancers in
single individuals (German, 1997).
has disclosed a striking chromosome instability. This instability
includes excesses of chromatid breaks and gaps, dicentric and ring
associations, and anaphase bridges (German et al., 1965; German
and Crippa, 1966). However, BS cells exhibit two unusual
Fig. 2. TheRecQfamily ofDNA helicases.Selected members fromvarious species areshown alignedby theconserved central helicase domain whichconsists ofseven helicase
motifs depicted ingreen. Also shownarethe conserved,C-terminaldomains presentin mostRecQ familymembers,includingtheRecQ C-terminalregion (RQC)in red andthe
helicaseand RNaseD C-terminal (HRDC)in blue,along withstretchesof acidicamino acids inpurple, thenuclear localizationsignals (NLS)inblack, andnonconserved regions
of the protein in white. The exonuclease (exo) domain of WRN is shown in yellow. aa, amino acid.
K.J. Ouyang et al./Mechanisms of Ageing and Development 129 (2008) 425–440
chromosomal abnormalities that are pathognomic of BS and
cytogenetic signs of dis-regulated HR. The first of these abnorm-
alities is the quadriradial, which is a symmetrical, four-armed
apparently have undergone somatic crossing-over (German, 1964).
Quadriradials appear very rarely in metaphases from normal
lymphocytes (<1 per 1000) but in BS they are relatively common
(1–2 per 100). The second is the SCE, which is an exchange event
prepared from normal persons, the average number of SCEs ranges
ranges from 50 to 150 per 46 chromosomes. At the molecular level,
the expected consequences of this excessive HR include increased
and re-arrangements – all of which are seen in BS cells – along with
1990; Langlois et al., 1989; Vijayalaxmi et al., 1983; Warren et al.,
1981). As one would expect of a cell with a cytogenetically manifest
defect in genome integrity, BS cells are hypersensitive to various
genotoxic agents, including ultraviolet light, mitomycin C, and
topoisomerase inhibitors, especially when these and other agents
are administered to cells synchronized in S phase (Davalos and
Campisi, 2003; Heddle et al., 1983; Ishizaki et al., 1981; Krepinsky
et al., 1979; Kurihara et al., 1987).
4.2. Balance between inaction and action – trafficking of BLM in the
BLM is a 1417 amino acid protein with a predicted molecular
weight of 159 kDa, possessing the biochemical activities described
above for RecQ helicases. Most BLM mutations in persons with BS
cause premature protein translation termination and are effec-
tively nulls (German et al., 2007). The mutation spectrum of BLM in
persons with BS also includes amino acid substitutions at
conserved residues of the helicase domain. These mutated BLM
proteins are expressed in BS cells but the helicase is inactive,
indicating that helicase activity is essential to BLM’s anti-
recombination functions (Neff et al., 1999).
BLM is cell-cycle regulated, exhibiting its lowest expression
levels inearly G1phase and highest inlate S and G2 phases (Bischof
et al., 2001; Dutertre et al., 2000; Sanz et al., 2000). In normal cells,
BLM is distributed throughout the nucleoplasm predominantly in
fine granules and concentrates focally in bodies known as the PML-
NBs, after the promyelocytic leukemia protein PML (Sanz et al.,
2000; Zhong et al., 1999). The PML-NBs range in number from 10 to
30 per nucleus and range in size from 0.2 to 1mm (Dellaire and
Bazett-Jones, 2004). They contain a wide variety of nuclear proteins
involved in such processes as apoptosis and cell-cycle regulation,
viral infection, gene regulation, cellular senescence, and DNA repair
(Dellaire et al., 2006). Besides BLM, the PML-NBs contain other DNA
repair proteins, including topoisomerase 3a, the tumor suppressor
PML-NBs are thought of as sites for storage of DNA repair and other
proteins, but there is also evidence that they participate in DNA
damage-sensor functions (Dellaire et al., 2006), providing access to
sites of DNA damage and regulating the available pools of active
protein (Matunis et al., 2006).
PML is a non-essential protein and the major structural
component of the PML-NBs. Proteins normally focally concen-
trated in the PML-NBs are diffusely distributed throughout the
nucleoplasm in cells that lack PML (Ishov et al., 1999). Pml null
mice have defects in DNA damage-mediated apoptosis and are
highly susceptible to the development of tumors after treatment
with carcinogens (Wang et al., 1998). Pml null cells also exhibit
twofold higher levels of SCEs compared to wild type cells (Zhong
et al., 1999). Coincidentally, experimentally derived BLM mutants
that are unable to localize to the PML-NBs also exhibit
approximately twofold higher than normal SCE levels, consistent
with the hypothesis that BLM function is regulated by its
localization in the PML-NBs (Hu et al., 2001).
The most important factor that regulates accumulation of
proteins in the PML-NBsis modificationby the small ubiquitin-like
modifier (SUMO). There are three known SUMOs (SUMO-1, SUMO-
2, and SUMO-3) in higher eukaryotes. Proteins are modified by
SUMOs through a system that is closely analogous to the ubiquitin
modification system (Johnson, 2004). The system consists of an E1
UBC9), and E3 ligases that specifically target particular substrates
for modification. SUMO modification consists in the formation of
an isopeptide bond between the carbonyl group of the C-terminal
glycine on the activated SUMO and the e amino group of a lysine on
the substrate protein. PML itself is modified by SUMO-1, and
expression in Pml null cells of a PML protein with mutations of its
three SUMO-acceptor sites results in failure to form PML-NBs
(Zhong et al., 2000). It has also been demonstrated that PML
contains a SUMO-binding motif, and experimentally introduced
mutations within these motifs also block PML-NB formation (Shen
et al., 2006). These observations have led to a model in which
dimeric, SUMO-modified PML proteins nucleate the formation of a
PML network via homotypic interactions between the SUMOs and
the SUMO-binding motifs of neighboring SUMO-modified PMLs
(Matunis et al., 2006). Many of the proteins that normally
accumulate in the PML-NBs are either themselves SUMO modified
or are able to bind SUMO through a SUMO-binding motif, allowing
their integration into the PML-NB network. Frequently, a PML-NB
proteincanbothbe modifiedbySUMO andbindSUMO,justasPML
does. BLM is modified by SUMO-1 and SUMO-2 (Eladad et al.,
2005), and it also contains a SUMO binding motif (M. Matunis,
personal communication). Expression of a BLM that contains an
experimentally derived deletion of amino acid sequences contain-
ing the SUMO binding motif results in failure of BLM to accumulate
in PML-NBs, indicating that localization of BLM in the PML-NBs is
also dependent on SUMO interactions (Hu et al., 2001).
Recent reports have emphasized the importance of SUMO
modification in DNA repair and damage signaling processes.
Proliferating cell nuclear antigen (PCNA) is a key component at the
replication fork that serves as a sliding clamp, ensuring processiv-
ity of replicative DNA polymerases. It also can recruit proteins to
the replication fork, stabilizing different protein-protein interac-
tions through ubiquitin and SUMO modifications (Hoege et al.,
2002; Stelter and Ulrich, 2003). In S. cerevisiae, PCNA recruits the
helicase Srs2 to stalled replication forks induced by hydroxyurea
(HU) treatment (Papouli et al., 2005; Pfander et al., 2005).
IncreasedSUMO modificationof PCNApromotes increased binding
of Srs2. Srs2 is an anti-recombination helicase that disrupts the
binding of Rad51 to ssDNA, preventing the stable formation of
nucleoprotein filaments and thereby blocking unwanted recom-
bination events (Branzei et al., 2006; Krejci et al., 2003).
Mammalian cells do not contain a homolog to yeast Srs2, yet
a way similar to Srs2 (Bugreev et al., 2007).
4.3. BLM and DNA damage during S phase
When DSBs occur in cells, repair proteins are recruited to and
accumulate at sites of active DNA repair to form cytologically
detectable foci, which we will refer to here as repair foci. Central to
this process of recruitment and accumulation of DNA repair
proteins is the histone H2AX, which is a member of the H2A
histone family (Fernandez-Capetillo et al., 2004). At amino acid
K.J. Ouyang et al./Mechanisms of Ageing and Development 129 (2008) 425–440
residue 139, H2AX contains an invariably conserved serine-
glutamine (SQ) motif that other histones do not contain (McManus
and Hendzel, 2005). Immediately upon induction of DNA breaks,
histone H2AX is phosphorylated at serine 139, designated gH2AX
(Paull et al., 2000). The phosphorylation of histone H2AX is
accomplished through the activation of the phosphoinositide-3-
kinase-related kinase (PI3KK) family members ATM, ATR, and
DNA-PK (Pilch et al., 2003). gH2AX marks a region of chromatin
extending over one million base pairs on both sides of the DNA
break (Rogakou et al., 1999), and it acts to recruit other repair
proteins to the site (Bassing and Alt, 2004). Mice deficient in H2AX
exhibit sensitivity to ionizing radiation, defective DSB repair, and
increased genomic instability (Celeste et al., 2002). H2AX deficient
cells also exhibit a twofold increase in SCEs, suggesting that H2AX
functions in pathway choice, anti-recombination, or both.
It has long been known that BS cells exhibit defects in DNA
DNA-chain growth (Hand and German, 1975). In vitro, BLM can
stimulate flap endonuclease activity on substrates resembling
maturing Okazaki fragments, and BLM could prevent association
of flap DNA with homologous DNA sequences on the sister
chromatid (Bartos et al., 2006). BS cells are hypersensitive to
replication inhibitors (Davies et al., 2004), and replication forks
inefficiently recover from HU-induced stalling, promoting the
accumulation of DSBs (Davies et al., 2007). In normal cells treated
with various DNA damaging agents during S phase (e.g., HU, DNA
crosslinking agents, and ultraviolet irradiation), within minutes of
drug treatment BLM leaves the PML-NBs and relocates to sites of
stalled or damaged replication forks, colocalizing with gH2AX-
marked repair foci (Davalos and Campisi, 2003; Sengupta et al.,
2003). BLM has functional and physical interactions with both
gH2AXandthe ATR and ATM kinases(Beamish etal.,2002; Davalos
and Campisi, 2003; Davies et al., 2004; Franchitto and Pichierri,
2002).Anearlyeventinthe S-phase specific damageresponse isthe
et al., 2004). Accumulation of BRCA1 protein and the MRN complex
accumulation of these proteins in gH2AX-marked foci occurs with
slower kinetics in BLM-deficient BS cells (Davalos and Campisi,
of p53 to the repair foci (Sengupta et al., 2003). If p53 is degraded in
BS cells, the kinetic defect in the accumulation of BRCA1 and the
MRN complex at repair foci is alleviated (Davalos and Campisi,
2003). Because BLM is one of the earliest components to associate
with S-phase repair foci and because it interacts with the major
signaling factors known to be involved in repair focus formation
(FANCD2, BRCA1, p53, and the MRN complex), many have
speculated that BLM’s function in pathway choice and anti-
recombination is mediated via these early events at stalled
replication forks. SUMO modification may regulate BLM’s anti-
tion of gH2AX-marked foci in cells that express a BLM that is
mutated attwo ofitsfour SUMO-acceptorsites(Eladad etal.,2005).
4.4. Is BLM for or against homologous recombination?
Topoisomerase 3 has also been associated with anti-recombi-
nation activities (Oakley and Hickson, 2002). Topoisomerase 3
preferentially binds to ssDNA and has a cleavage and re-joining
activity (Champoux, 2001). Unlike topoisomerase 1 or 2, topoi-
somerase 3 is inefficient at supercoiling orrelief from supercoiling;
instead, it is efficient at decatenation of DNA molecules (DiGate
and Marians, 1988). Yeast cells that lack the topoisomerase 3 gene
(top3 mutants) exhibit excessive recombination, increased chro-
mosome mis-segregation, and increased cell death (Bailis et al.,
1992; Goodwin et al., 1999; Maftahi et al., 1999; Wallis et al.,
1989). Mutants in the yeast RecQ genes (SGS1 in budding yeast and
Rqh1+ in fission yeast) suppress the top3 hyper-recombination and
cell death phenotypes. The topoisomerase 3 genes and RecQ
helicases of different yeasts physically interact, and their inter-
action is mediated by a region in the N-terminal portion of the
RecQ helicase (Bennett et al., 2000; Fricke et al., 2001; Gangloff
et al., 1994; Onodera et al., 2002; Ui et al., 2001). The genetic and
physical interactions are well conserved in evolution, because BLM
interacts with mammalian topoisomerase 3a (Johnson et al., 2000;
Wu et al., 2000), and expression in BS cells of a BLM protein that is
incapable of interaction with topoisomerase 3a only partially
rescues their high-SCE phenotype (Hu et al., 2001).
In vitro, the BLM/topoisomerase 3a complex has the ability to
resolve DNA substrates that resemble double Holliday junctions –
an activity that neither protein has on its own (Wu and Hickson,
2003). Unlike a breaking-and-rejoining resolution of double
Holliday junctions, which would result in an equal distribution
of crossover and non-crossover products, the BLM/topoisomerase
3a complex acts by pushing the two Holliday junctions together
(convergent branch migration), generating non-crossover pro-
ducts only (Plank et al., 2006). Topoisomerase 3a permits the
branch migration to occur by relieving the build-up of superhelical
A third member of BLM/topoisomerase 3a complex BLAP75/RMI1
binds to both BLM and topoisomerase 3a and recruits the complex
to the Holliday junction (Raynard et al., 2006; Wu et al., 2006). A
plausible interpretation of the genetic and biochemical data is that
the BLM/topoisomerase/BLAP75 complex specifically ‘‘dissolves’’
the Holliday junctions that arise during DSB repair. In the absence
of BLM, as in BS cells, resolution of Holliday junctions would occur
by breakage and rejoining instead of dissolution, leading to
crossover events and increased SCEs (see Fig. 1B).
It seems unlikely, however, that dissolution of double Holliday
junctions can explain all the defects present in BS cells. The
formation of Holliday junctions in the HR pathway is a late event,
whereas BLM’s appearance at and stabilization of stalled replica-
tion forks implicate BLM as an early responder to replication-fork
stress. BLM interacts directly with the RAD51 recombinase, and
BLM and RAD51 colocalize after replication arrest (Bischof et al.,
2001; Wu et al., 2001). Consistent with its function in HR repair,
RAD51 localizes to sites of ssDNA after the induction of DSBs
can displace RAD51 recombinase from a recombinase-ssDNA
filament (see Fig. 1A) (Bugreev et al., 2007), an anti-recombination
activity that is similar to the activity of Srs2 (Branzei et al., 2006;
loops and help unwind DNA in front of the free 30end in advance of
the polymerase. This process might favor the SDSA branch of HR as
suggested by Adams et al. (2003) (see Fig. 1C). The biochemical
evidence for BLM’s involvement in each stage of the HR pathway
along with the cellular evidence linking BLM to stabilization of
stalled replication forks leads to a more complex view of BLM, in
which BLM possesses both anti-recombinogenic and pro-recom-
binogenic functions (Bugreev et al., 2007).
BLM protein in the cell may associate with various different
protein complexes when responding to DNA damage. BLM has
include the ssDNA binding protein RPA (p70, p32, and p14), the
Fanconi anemia (FA) gene product FANCA, the mismatch repair
protein MLH1 (Langland et al., 2001; Pedrazzi et al., 2001), and the
topoisomerase 3a and BLAP75 factors (as a conglomerate, these
complexes have been called BRAFT) (Yin et al., 2005). Although the
K.J. Ouyang et al./Mechanisms of Ageing and Development 129 (2008) 425–440
role of MLH1 in BLM-related repair has remained elusive, we
speculate that it assists in preventing illegitimate recombination,
by assuring that the invading strand is homologous to the duplex
invaded (Myung et al., 2001).
FA is a rare autosomal recessive disorder that features
chromosomal instability, sensitivity to DNA crosslinking agents,
and cancer predisposition. A DNA interstrand crosslink (ICL) will
(Thompson, 2005). A hypothetical crosslink repair pathway is as
follows: (1) intra-strand excision on either side of the crosslink to
generate a gap; (2) synthesis across the damaged base with a
specialized, error-prone polymerase that can incorporate nucleo-
tides independent of the normal base-pairing rules; and finally, (3)
HR to re-establish the replication fork. One of the main roles of the
G, -L, and -M proteins) is to ubiquitin modify the FANCD2 gene
product, which stabilizes stalled replication forks and recruits other
repair components to the site of damage, including BRAFT and the
2002). The intersection between mismatch repair and the FA
pathway gene products with BLM and HR is intriguing and requires
further functional studies in order to clarify their joint roles.
5. Werner syndrome helicase and telomere functions
5.1. Consequences of mutations in the Werner syndrome gene
WS is a rare autosomal recessive disorder caused by bi-allelic
loss-of-function mutations in the WRN gene (Yu et al., 1996). The
disorder is characterized by the premature development of
features that resemble aging, with the first signs appearing shortly
after adolescence (Epstein et al., 1966; Goto, 1997; Martin, 1978).
These features include the premature development of alopecia and
graying hair, artiosclerosis, atherosclerosis, osteoporosis, hypogo-
nadism, cataracts, and type II diabetes. Persons with WS are
smaller on average than nonWS persons. In addition, persons with
WS have an increased cancer predisposition, leading primarily to
rare cancers that are mesenchymal in origin (Goto et al., 1996).
as ‘‘variegated translocation mosaicism’’ (Melaragno et al., 1995;
Salk et al., 1981). Fibroblast cultures are frequently pseudodiploid
rearrangements. Lymphocytes also exhibit the propensity to
translocations (Grigorova et al., 2000; Scappaticci et al., 1990). As
detected using the spectral karyotyping technique, fragile sites
constitute preferred sites for chromosomal translocations (Melcher
et al., 2000). At the molecular level, WS cells accumulate mutations
at a higher rate, consisting predominantly of large spontaneous
deletions (Fukuchi et al., 1989; Poot et al., 2001). Cells are
hypersensitive to a number of DNA damaging agents, including 4-
nitroquinolone (4NQO) (Ogburn et al., 1997; Poot et al., 2002),
platinum, 8-methoxypsoralen(Poot etal.,2001, 2002),andthe base
analog O6-methylguanine (Blank et al., 2004).
Fibroblast cultures from persons with WS enter premature
replicative senescence (Epstein et al., 1966; Faragher et al., 1993;
Schulz et al., 1996; Tahara et al., 1997), implicating WRN in
telomere function. Telomeres are the ends of chromosomes, and
they provide two major functions. One is to ensure the complete
replication of chromosome ends and the other is to protect the end
from various enzymatic attacks to avoid the loss of genetic
information (McClintock, 1938; Sandell and Zakian, 1993). Human
telomeres consist of a repetitive sequence TTAGGG at the end of
the chromosome ranginginlength from 5 to 20 kb (Nurnberg et al.,
1993). The chromosome terminates with a 100–200 base 30ssDNA
overhang. The ssDNA overhang is annealed into the telomere DNA
to form a stable D-loop. As a result, a looped out structure forms at
the end of the telomere that is referred to as a t-loop (see Fig. 3)
(Griffith et al., 1999; Murti and Prescott, 1999). The free,
unannealed telomere overhang may also form G-quadruplex
DNA (Williamson et al., 1989). Telomere defects in WS cells
suggest that WRN operates at telomere DNA.
5.2. WRN exonuclease – important clue and major mystery
The WS gene WRN encodes a 1432 amino acid protein with a
predicted molecular weight of 162 kDa, containing the conserved
RecQ helicase regions described above (Yu et al., 1996). The
catalytic activities of the WRN helicase are in general the same as
those of BLM and the other RecQ helicases. However, unlike BLM,
WRN possesses a 30
exonuclease activity. The WRN
exonuclease can efficiently degrade a recessed 30end on dsDNA
or an RNA-DNA heteroduplex, whereas under most conditions it
shows little or no exonuclease activity on blunt-ended DNA, DNA
with a protruding 30tail, or ssDNA (Huang et al., 1998; Kamath-
Loeb et al., 2000; Shen et al., 1998a). WRN is also effective at the
removal of a single mismatched nucleotide at a recessed 30end but
not two mismatched nucleotides, and it is capable of initiating
exonucleolytic degradation from a gap or a nick (Huang et al.,
2000; Kamath-Loeb et al., 1998).
Fifty distinct WRN mutations have been reported (Huang et al.,
2006). Almost all of the mutations result in premature termination
making these mutations effectively nulls (Goto et al., 1997;
Matsumoto et al., 1997; Meisslitzer et al., 1997; Moser et al.,
1999; Oshima et al., 1996; Yu et al., 1997). Recently, two novel
missense mutations in the exonuclease domain were detected in a
single individual, both homozygous – namely p.Lys125Asn and
p.Lys135Glu (Huang et al., 2006). Structural analysis predicted that
the mutations would be on the surface of the protein and were
unlikely to affect exonucleaseactivity.Expression of doubly mutant
protein by transfection was nearly undetectable by Western blot
double mutations compromised protein stability. Thus, in contrast
to the finding of stably expressed, missense mutants of BLM, there
have been no reports of persons with WS having mutations that
eliminate only the helicase activity or only the exonuclease activity
(Huang et al., 2006). Therefore, in order to develop WS, both the
helicase and exonuclease activities of WRN must be lost.
How the helicase and exonuclease activities act in concert is a
question central to understanding WRN functions. Investigators
have compared the joint activities with each separate activity by
addition of non-hydrolyzable ATPgS, which inhibits the helicase
activity, and employment of a mutant WRN that contains an
amino acid substitution that specifically abrogates the exonu-
clease activity. On substrates that mimic replication forks and t-
loops, the WRN exonuclease can degrade the leading strand and
the annealed telomere overhang to promote helicase-dependent
fork regression and t-loop dissolution, respectively (Machwe
et al., 2007; Opresko et al., 2004). These activities can be
suppressed by the addition of proteins with which WRN interacts
(Opresko et al., 2002, 2004, 2005), prompting the question
whether the exonuclease is active on these substrates in the cell.
Recent work has even challenged the substrate specificities of the
exonuclease, because it was found WRN can degrade longer
ssDNA substrates (>40 nt) in a helicase-dependent manner
(Machwe et al., 2006b). As will be seen in the following section,
cellular studies that probe WRN-specific functions and WRN’s
cellular substrates help to make some sense of the biochemical
K.J. Ouyang et al./Mechanisms of Ageing and Development 129 (2008) 425–440
5.3. WRN and the replication of telomeres
Telomerase isanenzymethatfunctions inextendingtelomeres,
and expression of this enzyme in normal as well as in WS
fibroblasts allows these fibroblast cultures to proliferate indefi-
nitely, avoiding replicative senescence (Bodnar et al., 1998; Choi
et al., 2001; Vaziri and Benchimol, 1998; Wyllie et al., 2000). WS
fibroblasts with suppressed pRB and p53 DNA damage-response
E6 and E7 oncoproteins, rapidly accumulate chromosome fusions
(Crabbe et al., 2007). Fusions are detected in metaphase spreads as
anaphase bridges. Expression of both WRN and telomerase in the
WS fibroblasts can rescue the excess chromosome-fusion pheno-
type. By fluorescence in situ hybridization (FISH), the majority of
the anaphasebridges intheWS cellslackdetectabletelomere DNA,
suggesting that they were the consequence of total or partial
telomere loss. Substitution of a dominant-negative telomerase
allele that is incapable of telomere elongation failed to rescue the
excess chromosome-fusion phenotype, suggesting that mainte-
nance of stable telomere length is required for the rescue (Crabbe
et al., 2007).
Telomeres are protected by a complex of telomere-specific DNA
binding proteins, referred to as shelterin, that stabilize telomere
components: the duplex telomere-binding proteins TRF1 and
TRF2, the ssDNA telomere-binding protein POT1, and adaptor
proteins RAP1, TIN2, and TPP1. Overexpression of TRF2 in cells
leads to telomere shortening, supporting the hypothesis that TRF2
negatively regulates telomere length (Karlseder et al., 2002). On
the other hand, expression of a dominant-negative TRF2 mutant,
which fails to bind DNA, induces loss of the 30telomere overhang,
indicating that TRF2 protects the 30overhang (Karlseder et al.,
1999, 2002). Overexpression of TRF1 also leads to telomere
shortening, and expression of a dominant-negative TRF1 mutant in
telomerase-positive cells results in elongation of telomere length
(vanSteensel andde Lange, 1997). However, TRF1proteinlevels do
not affect telomere length in the absence of telomerase, indicating
(Smogorzewska and de Lange, 2004). Similarly, knockdown of
endogenous POT1 expression or overexpression of a dominant-
negative POT1 that cannot bind telomeres results in elongation of
telomeres (Veldman et al., 2004; Yang et al., 2005). POT1 can
Fig. 3. The hypothetical roles of the WRN helicase at telomeres. The t-loop structure that is formed at telomere ends is stabilized by the shelterin complex, which protects the
ends of chromosomes from being recognized as DNA damage. During replication, the G-rich telomere lagging strand DNA may fold into a G-quadruplex structure. The WRN
helicase aids in resolving these structures to allow lagging-strand synthesis to progress efficiently. (The triangle represents the WRN protein, and the direction that the
triangle points indicates the direction of WRN’s helicase function; the cleft on the other side of the triangle represents the exonuclease.) After the completion of telomere
replication, 30overhangs at the ends of each sister telomere are generated by resection 50to 30via a nuclease. After resection, the G-rich ssDNA overhangs can again form G-
quadruplex structures, which WRN could help resolve. These ssDNA ends then can form D-loops with other telomeres by invading duplex telomere DNA in trans. WRN helps
prevent these nascent recombination events between different telomeres through the coordinated activities of its helicase and exonuclease. Binding of shelterin proteins to
telomere DNA induces a change in the topology of the telomere, stimulating strand invasion of the 30overhang into the telomere in cis. Binding of TRF2 to telomere DNA has
been shown to change its topology by generating positive supercoiling, supporting the model that TRF2 enhances strand invasion (Amiard et al., 2007). The D-loop is
stabilized by further assembly of the shelterin complex, completing the t-loop and protecting the telomere.
K.J. Ouyang et al./Mechanisms of Ageing and Development 129 (2008) 425–440
regulate telomere length by competing with telomerase in binding
to 30telomere overhangs (Kelleher et al., 2005; Lei et al., 2005). In
summary, in the presence of telomerase, the coordinate actions of
TRF1, TRF2, and POT1 regulate telomere length (de Lange, 2005). In
the absence of telomerase, telomeres shorten by an average of 200
nucleotides with each cell division.
WRN physically interacts and colocalizes with TRF2 specifically
in S-phase (Crabbe et al., 2004; Opresko et al., 2002). Biochemical
assays using synthetic telomere D-loops demonstrate that TRF1
and TRF2 can limit the extent of WRN exonuclease activity on a
telomere D-loop, such that release of a longer invading 30overhang
is favored (Opresko et al., 2004). Addition of RPA or POT1
stimulates the helicase activity of WRN, and it also decreases
the extent of exonuclease digestion of the 30overhang (Opresko
facilitate leading-strand synthesis through telomere DNA. A more
intriguing possibility is that WRN prevents interchromosomal
interactions between telomeres (see Fig. 3). WS cells accumulate
abnormal recombination intermediates that impede cell growth
(Prince et al., 2001), and this impairment can be resolved through
the expression of a bacterial enzyme (RusA) that resolves Holliday
junctions (Saintigny et al., 2002). The biochemical evidence so far
indicates that abolishing the 30to 50exonuclease activity of WRN
does not affect unwinding of Holliday junctions formed between
the telomeres of two different chromosomes, unless the telomere
proteins are not bound to these structures, which could be the case
during replication of the telomeres during S phase. The proper
function of the exonuclease activity at telomeres is still elusive.
strand synthesis, and it may fold into G-quadruplex structures that
telomere DNA can be distinguished by chromosome orientation
FISH (CO-FISH), using incorporation of bromodeoxycytidine (lead-
ing strand) and bromodeoxyuridine (lagging strand), which can be
degraded by sequential UV and exonuclease III treatments (Crabbe
sequences allows differential imaging of newly replicated sister
telomeres. Inhibition of WRN by over-expression of a dominant-
sister telomeres, almost exclusively affecting the sister telomere
produced by lagging-strand synthesis (Crabbe et al., 2004). WRN’s
interaction with the flap endonuclease FEN-1, which helps process
and join Okazaki fragments that are produced in lagging-strand
synthesis,could assist inmaturation oftelomereOkazakifragments
(Brosh et al., 2002; Sharma et al., 2004). Altogether, these data
suggest that WRN assists in the replication of the G-rich lagging
(see Fig. 3).
WRN physically interacts with the DNA polymerase POLd, and it
stimulates POLd’s polymerization (Szekely et al., 2000). A synthetic
d(CGG)7structure can form G-quadruplex and hairpin structures in
vitro. Polymerases POLa, POLe, and POLd all stall upon contact with
successfully replicate through the d(CGG)7structure (Kamath-Loeb
et al., 2001). The stimulation of POLd activity only occurs in the
absence of PCNA, which suggests that WRN is not required for
processive DNA replication but instead may play a role at stalled
replication forks. The helicase activity of WRN is essential for this
function because a helicase-deficient WRN mutant fails to relieve
the G-quadruplex-induced stalling, and alleviation of POLd pausing
requires that unwinding of the G-quadruplex precedes or occurs
simultaneously with DNA synthesis. Abnormal DNA structures that
replicate late in S phase (including telomeres) may be particularly
prone to replication stalling in the absence of WRN. The under-
replicated DNA is subsequently prone to breakage.
Wrn null mice have been successfully produced, but surpris-
ingly do not develop any obvious age-related symptoms (Lebel and
Leder, 1998; Lombard et al., 2000). Two possible explanations for
this difference may be that mice possess much longer telomeres
than humans and telomerase activity is detectable in mouse
somatic cells. These biological differences would make telomere-
based replicative senescence a less likely event during the normal
aging process of a mouse. To test whether shortening telomere
lengths can increase the impact of mouse Wrn deficiency,
telomerase deficient (mTerc?/?), Wrn?/?double mutant mice were
generated. Strikingly, at later generations, mTerc?/?, Wrn?/?mice
display many of the clinical features persons with WS display,
including the premature development of wound-healing defects,
osteoporosis and skeletal fractures, hypogonadism, cataract
formation, type II diabetes, an increase in mesenchymal cancers,
and shortened life span (Chang et al., 2004; Du et al., 2004). Control
mTerc?/?, Wrn+/+mice expressing normal Wrn protein and earlier
generation double mutant mice with longer telomeres did not
Wrn coupled with short, dysfunctional telomeres is necessary for
the onset of phenotypes in the mouse model. While telomerase-
null mice also exhibit a variety of aging phenotypes and genomic
instabilities, they do not display the osteoporosis, cataract
formation, type II diabetes, and mesenchymal tumors that are
so characteristic of persons with WS (Chang et al., 2004; Du et al.,
are found at telomeres, including the Ku heterodimer, DNA-PK, the
MRN complex, and ATM (Riha et al., 2006). This finding seems
paradoxical because the last thing the cell would want to do is to
join two telomere ends together. Indeed, mouse cells that are
mutant for Trf2 generate chains of multiply joined chromosomes
fused end-to-end (Celli and de Lange, 2005), but Trf2?/?, Ku70?/?
double mutants exhibit a 10-fold decrease in chromosome fusions,
indicatingthattheabnormalend joiningismediated byNHEJ(Celli
et al., 2006). In co-immunoprecipitation and co-localization
studies, WRN interacts with Ku (Karmakar et al., 2002; Li and
Comai, 2000; Orren et al., 2001) and the MRN complex via
interaction with NBS1 (Cheng et al., 2004). In support of a role for
WRN in preventing interactions between telomeres, fibroblasts
from mTerc?/?, Wrn?/?mice exhibit elevated SCEs at telomeres (T-
SCEs). Expression of normal mouse and human WRN gene but not a
helicase-deficient mutant rescues the elevated T-SCE phenotype,
mutant exhibits increased T-SCEs, but the Trf2?/?, Ku70?/?double
mutant manifests a striking increase, indicating that under
unstable conditions Ku can suppress SCEs at telomeres (Celli
et al., 2006).
5.4. A connection between WRN and base excision repair in
Base excision repair (BER) is the main pathway in repairing
damaged bases, including oxidized, alkylated, methylated, and
deaminated bases. WS cells are hypersensitive to genotoxic agents
that produce ROS, such as 4-NQO, hydrogen peroxide, and g-
irradiation. WRN knockdown in human fibroblasts results in an
accumulation of DNA damage following oxidative stress and causes
increased sensitivity to alkylating agents, including MMS (Harrigan
for WRN in repair of oxidative DNA damage (Blank et al., 2004).
WRN interacts with various components in the BER pathway.
helicase activity is stimulated, and it can stimulate the strand
K.J. Ouyang et al./Mechanisms of Ageing and Development 129 (2008) 425–440
displacement and DNA synthesisactivities of POLb (Harrigan et al.,
2003). POLb has no 30to 50exonuclease activity to serve a
proofreading function, which theoretically WRN could provide.
Studies show that WRN helicase activity is inhibited in the
presence of AP-endonuclease 1, whereas in the presence of POLb,
this inhibition is relieved (Ahn et al., 2004).
There is also evidence that WRN activity in BER is regulated
through an interaction with PARP-1 (Li et al., 2004; von Kobbe
et al., 2004). PARP-1 is an abundant, chromatin-bound nuclear
enzyme that binds single and double-strand breaks in addition to
hairpins, loops, cruciforms, and X-junctions (Lonskaya et al., 2005;
Potaman et al., 2005). When PARP-1binds to strand breaks in vitro,
it is activated, catalyzing the transfer of ADP-ribose from the
cofactor NAD+ onto itself and other acceptor proteins and forming
chains of ADP-ribose up to 200 moieties long (Schreiber et al.,
2006). Ribosylation by PARP-1 is inhibited by a variety of agents,
including 3-aminobenzamide (3-AB). Sensitivity of cells to DNA
alkylating agents can be potentiated by inhibition of PARP-1
activity by 3-AB, but the same effect is not observed with UV
treatment (Morgan and Cleaver, 1982), indicating that strand
incision events generated during BER involve PARP-1 but not
incision events generated during nucleotide excision repair. In the
BER pathway, 3-AB treatment inhibits a step after strand incision
but prior to DNA ligation. When PARP-1 is poly-ribosylated, it no
longer binds strand breaks (D’Amours et al., 1999; Lindahl et al.,
1995), leading to the hypothesis that PARP-1 binds strand breaks
and prevents ligation until excess ribosylation clears PARP-1 from
the site. The unribosylated form of PARP-1 inhibits the WRN
helicase and exonuclease activities, whereas the ribosylated form
of PARP-1 relieves the inhibition of WRN catalytic activities. WS
cells exhibitdeficiencies in the ribosylationof target proteinsother
than PARP-1 itself after exposure to H2O2and MMS (von Kobbe
et al., 2004). Possibly PARP-1 blocks the strand break so that WRN
cannot gain access to it. Theoretically, PARP-1/WRN interaction
could up-regulate ribosylation of key NHEJ proteins, which would
reduce their affinity for strand breaks.
There is extensive evidence of a connection between on the one
hand PARP-1 and the BER pathway and on the other hand HR.
Cleaver, 1982; Natarajan et al., 1981; Otsuka et al., 1983; Shiraishi
the ligase III-associated factor XRCC1 leads to elevated SCEs
(Caldecott et al., 1992). These data suggest that un-repaired ssDNA
breaks stimulateHR. The stimulusofHRverylikely occursvia a DSB
break (see Fig. 1). In support of this hypothesis, Parp1 null cells
accumulate persistent RAD51 foci, and RAD51 foci persist longer
after HU treatment compared to normal cells (Schultz et al., 2003).
comparedtonormal,reactivation ofDNA replication isdelayed,and
colony survival is reduced (Yang et al., 2004).
Cells that carry mutations in the hereditary breast cancer genes
(BRCA1 and BRCA2), which have important functions in promoting
repair of DSBs by HR, are hypersensitive to PARP-1 inhibitors
because they accumulate persistent, unrepaired DNA damage
(Bryant et al., 2005; Farmer et al., 2005). Again, hypothetically,
inhibition of PARP-1 leads to persistent, unrepaired strand breaks
through inhibitionof ligation. Replicationcomplexes convertthese
persistent breaks into DSBs, which require the HR pathway for
replicationrestart. BRCA-deficientcells aredefectiveinHR,and the
cell succumbs to a vast DNA damage insult. As noted above, ICLs
are also repaired by HR. Both WRN- and BRCA1-deficient cells are
hypersensitive to agents that cause ICLs (Bohr et al., 2001; Yun
et al., 2005). WRN physically interacts with BRCA1. Interaction
specifically stimulates both the helicase and exonuclease activities
of WRN on forked and Holliday junction substrates. In comet
assays, cells with WRN and BRCA1 knockdowns exhibited less
efficient repair of ICLs, and the double knockdown was no different
from either single knockdown, suggesting WRN and BRCA1
participate in a common pathway for repair of ICLs (Cheng
et al., 2006). These data suggest that BRCA1 and WRN may act
cooperatively in the repair of ICLs by HR.
6. The RECQL4 helicase in DNA repair
RTS is an autosomal recessive disorder characterized by skin
(poikiloderma) and skeletal abnormalities, signs of premature
aging, consisting of premature graying and/or loss of hair and
cataracts (Kitao et al., 1999b; Larizza et al., 2006), and cancer
predisposition, especially to osteosarcomas (Wang et al., 2001,
2003). Cells derived from persons with RTS exhibit a mosaicism for
specific chromosome abnormalities, most commonly trisomy 8 or
isochromosome 8q (Der Kaloustian et al., 1990; Lindor et al., 1996;
Miozzo et al., 1998). Mutations in the RECQL4 gene are associated
with approximately 60% of all persons diagnosed with RTS (Kitao
et al., 1999a), strongly suggesting that RTS, as it is presently
defined, is a heterogeneous disorder that is caused by mutations in
more than one gene. RTS cells have been reported to be
hypersensitive to g-irradiation and H2O2 (Vennos and James,
1995; Werner et al., 2006), but the overall conclusion from these
studies is still controversial (Grant et al., 2000; Mann et al., 2005).
Recql4?/?mice exhibit high frequencies of premature centromere
separation and increased aneuploidy, supporting the picture of
genomic instability and suggesting that the absence of RECQL4
may adversely affect sister chromatid cohesion (Mann et al., 2005).
The RECQL4 gene encodes a 1208 amino acid protein with a
predicted molecular weight of 133 kDa (Kitao et al., 1998). The
protein bears no known homology to other proteins, aside from its
central RecQ helicase region (Fig. 2). A subset of mutations in the
RECQL4 gene has been associated with two additional recessive
disorders: RAPADILINO (RAdial hypoplasia, Patella hypoplasia and
limb malformation, Nose slender and nOrmal intelligence)
(Siitonen et al., 2003) and Baller–Gerold syndrome (BGS), which
is characterized by radial hypoplasia and craniosynostosis (Mega-
rbane et al., 2000; Van Maldergem et al., 2005). Although the three
syndromes share clinical features (e.g., radial ray abnormalities
and short stature), there are also syndrome-specific features. For
example, cataracts are seen only in RTS, joint dislocation and
patellar hypoplasia only in RAPADILINO, and craniosynostosis only
in BGS. Most of the RECQL4 mutations identified in RTS are
nonsense or frameshift mutations. These mutations destabilize the
mature mRNA through the process of nonsense-mediated mRNA
decay (Kitao et al., 1999a), and the overall, predicted outcome is
the expression of low levels of a truncated protein that lacks either
a portionof orthe entireRecQ helicase region. Incontrast,the most
common mutation in the RECQL4 gene resulting in RAPADILINO
syndrome is an in-frame deletion that leaves the helicase region
largely intact (Siitonen et al., 2003), suggesting that amino acid
sequences outside of the helicase region mediate important
RECQL4 functions. Only two mutations in the RECQL4 gene have
been detected in BGS – an R1021W missense mutation and a 2886
deletion T frameshift mutation in exon 9 (Van Maldergem et al.,
2005). The frameshift mutation is very likely a complete loss-of-
function mutation, whereas the missense mutation may affect
RECQL4 function partially. Further characterization at the mole-
K.J. Ouyang et al./Mechanisms of Ageing and Development 129 (2008) 425–440
cular level of the phenotypic consequences of specific RECQL4
mutations along with their effects on RECQL4 expression and
function is needed in order to determine how defects in RECQL4
result in these overlapping genetic disorders.
6.2. RECQL4 is not a DNA helicase
The biochemical properties of RECQL4 have been investigated
so far in a single study. Although RECQL4 exhibited ssDNA binding,
DNA-dependent ATPase, and ssDNA annealing activities, it was
unable to unwind a blunt-ended DNA molecule or a duplex
substrate with a protruding 50or 30ssDNA tail (Macris et al., 2005).
This conclusion seems unexpected because RECQL4 contains the
well-conserved, signature RecQ helicase motifs that define the
family (Kitao et al., 1998); however, it lacks important conserved
motifs C-terminal to the helicase region (see Fig. 2), and these
motifs are necessary for the helicase activities of BLM and E. coli
RecQ (Wu et al., 2005). Because, as previously mentioned, all RecQ
helicases tested to date prefer substrates that resemble recombi-
nation intermediates, the ability of RECQL4 to dissociate a forked
DNA duplex, a synthetic D-loop, a four-way X-junction, and G-
quadruplex DNA was also tested, and RECQL4 failed to unwind
these substrates (Macris et al., 2005). The authors concluded that
RECQL4 has no detectable DNA unwinding activity. If RECQL4
cannot unwind DNA, then it is reasonable to question whether it
has the ability to translocate on ssDNA. The presence of RECQL4’s
DNA-dependent ATPase and ssDNA annealing activities suggests,
however, that RECQL4 could have a function in HR. The ssDNA
annealing activity could bring together strands of DNA that are
released by one of the other helicases during an SDSA event or it
could promote ssDNA annealing in NHEJ, thereby influencing
pathway choice during DSB repair.
6.3. RECQL4 and DNA repair – conflicting views?
A role for RECQL4 in the initiation of DNA replication has been
suggestedby studiesintheX. laevisDNAreplicationsystem.TheN-
terminal region of the RECQL4 homolog in X. laevis displays
homology to the proteins Sld2 in S. cerevisiae and DRC1 in S. pombe,
which are components necessary in initiation of DNA replication
(Sangrithi et al., 2005). Immuno-depletion of xRTS, the X. laevis
RECQL4 gene, from egg extracts results in reduced amounts of DNA
synthesis in vitro and inhibition of the recruitment of RPA to the
pre-replication complex(Sangrithi et al., 2005). Addition of normal
human RECQL4 to the immuno-depleted extracts corrects these
defects. The N-terminal, non-helicase region of xRTS interacts with
the Cut5 protein, a homolog of Dpb11 in budding yeast, which is
important in loading DNA polymerase onto chromatin (Matsuno
et al., 2006). These data are consistent with a critical role for xRTS
in initiation of DNA replication; however, the relationship of these
data to human disease is unclear. RTS mutations in humans appear
to be nulls, and the lack of an essential replication initiation factor
is incompatible with life. Because the N-terminal region of xRTS,
which contains the homology to Sld2 and DRC1, is not homologous
to the mammalian RECQL4, it is possible that the role of xRTS in
initiation of DNA replication was not conserved in evolution.
In the Xenopus system, RECQL4 also associates with chromatin
that has been digested with restriction enzymes, an assay that
mimics DSB repair (Kumata et al., 2007). The binding of RECQL4 to
broken chromatin depends on RPA and ATM kinase activity, and by
chromatin-IP analysis RecQL4 binds in physically proximity to
where Ku binds DSBs. In HeLa cells, RECQL4 has been found in a
complex with the RAD51 recombinase, and after treatment with
etoposide, a fraction of RECQL4 colocalizes with RAD51 foci
(Petkovic et al., 2005). RECQL4 does not interact with Rad51 in the
Xenopus system. These observations place RECQL4 at DSBs after
damage recognition by the MRN complex and activation of ATM
and during DSB repair, suggesting the RECQL4 could be involved in
A portion of RECQL4 is also localized to the PML-NBs (Petkovic
et al., 2005), and RECQL4 can also be found in the nucleolus (Woo
et al., 2006). RECQL4 interacts with PARP-1, and RECQL40s nuclear
cells with hydrogen peroxide or streptonigrin, agents that induce
oxidative stress, the amount of RECQL4 in the nucleolus increases –
an effect that is not observed when cells are treated with g-
a role for PARP-1 in trafficking, RECQL4’s move from the
nucleoplasm to the nucleolus can be inhibited by pretreatment of
cellswiththe PARP-1inhibitor 3-AB. Coincidentally,Parp1null cells
peroxide and gamma irradiation. A defect in responding to damage
caused by ROS might help explain the development of premature
aging in RTS, as hydrogen peroxide-exposed RTS cells exhibit
decreased cell proliferation and a reduction in DNA synthesis
(Werner et al., 2006). Because PARP-1 is involved in a competing
end-joining pathway of DSB repair (Audebert et al., 2004; Wang
et al., 2006), it is also possible that RECQL4 acts in this alternative
pathway.With the amount of information available onRECQL4, it is
not possible at present to construct a strong model for its functions,
but it safe to say that RECQL4 is involved in some aspect of DNA
repair, most probably the repair of DSBs.
7. Concluding remarks
The mammalian RecQ helicases are components in the cellular
machinery that maintain the integrity of the genome. Although
as a group they appear to operate in pathways that process and
repair DSBs, influencing the balance of action between the various
BLM establishes it as a negative regulator of HR, and cellular
studies strongly suggest that BLM stabilizes DNA polymerases that
have stalled due to encounter with DNA damage. Knockdown
studies of the RECQL1 and RECQL5 genes (Hu et al., 2007; LeRoy
et al., 2005; Sharma et al., 2007) indicate that these RecQ helicases
play similar roles, because recombinationlevels and DNA breakage
increase in their absence as well, but more data is needed to
BLM is lethal to a mouse, but neither the absence of RECQL1 nor
RECQL5 is lethal. Mutations in RECQL1 and RECQL5 have not been
associated with genetic disorders in humans, consistent with
opposing interpretations that such mutations either are lethal or
have no clinically obvious human disease phenotype. In contrast,
the absence of WRN has no immediate impact on either a mouse or
a human; however, the process of aging is accelerated. The
connection to aging is supported by WRN’s action at telomeres,
which are genomic sites that can form G-quadruplex DNA. WRN
facilitates DNA replication as these sites and quite possibly
prevents abnormal recombination. Stabilization or facilitation of
DNA replication under stress and preventing aberrant recombina-
tion events provides as general a theme for RecQ helicases as can
be provided based on the current, sometimes confusing and
overlapping evidence. While many different molecular models and
many different proposed cellular substrates jockey for our
attention, much of what we know about these helicases is
dependent on the context in which the information was collected,
so that different models may be correct in different situations.
What could really prove useful then are cell physiological
experiments that rule out some of the models and situations.
K.J. Ouyang et al./Mechanisms of Ageing and Development 129 (2008) 425–440
Although a lot has been learned through the study of the RecQ
helicases in prokaryotes, yeasts, and lower eukaryotes, including
flies and worms, we inevitably need to test the ideas arising in
these fields in mice and in human cells. Besides increasing our
knowledge of fundamental genetic processes that maintain
genome integrity, the connections between RecQ helicases and
impact on ourefforts towards the betterment of humanhealth.Itis
not unreasonable to think that novel cancer chemotherapeutic
treatments could emerge from the study of these fundamental
mechanisms, while the exploration of RecQ helicases in DNA
replication and telomere functions continues to provide new
perspectives on cancer and aging.
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