Pathways and functions of the Werner syndrome protein
Jae Wan Lee, Jeanine Harrigan, Patricia L. Opresko, Vilhelm A. Bohr*
Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA
Available online 26 October 2004
Mutations in human WRN (also known as RECQ3) gene give rise to a rare autosomal recessive genetic disorder, Werner syndrome (WS).
WS is a premature aging disease characterized by predisposition to cancer and early onset of symptoms related to normal aging including
osteoporosis, ocular cataracts, graying and loss of hair, diabetes mellitus, arteriosclerosis, and atherosclerosis. This review focuses on the
functional role of Werner protein (WRN) in guarding the genetic stability of cells, particularly by playing an integral role in the base excision
repair, and at the telomere ends. Furthermore, in-depth biochemical investigations have significantly advanced our understanding of WRN
protein regardingits binding partners and the site of protein–protein interaction. The mapping analysis of protein interaction sites in WRN for
most of its binding partners have revealed a common site of protein–protein interaction in the RecQ conserved (RQC) region of WRN.
Published by Elsevier Ireland Ltd.
Keywords: Base excision repair; Telomere; WRN; RecQ conserved; RQC; Werner syndrome
One approach to the study of aging is to better understand
the biological mechanisms that are deficient in human
disorders of premature aging. These diseases resemble the
normal aging process to a large extent, and since they are
genetic disorderswithmutationsofsinglegenes,genetic and
biochemical tools can be employed in the study of disease
mechanism. This category of diseases is also termed
segmental progeria to emphasize that not all of the features
of the diseases resemble the normal aging process. Werner
syndrome (WS) has been considered a hallmark disease in
this category and is perhaps the one that most closely
resembles normal aging. In support of this notion, we
recently undertook an extensive gene expression analysis
using cDNA microarray with RNA isolated from human
primary fibroblasts. We found that among the over 6000
genes analyzed, therewere significant changes in expression
pattern going from young to old individuals, which closely
resembled the changes between young and WS patients.
There was a 92% concordance, suggesting that expression
array patterns in WS closely resemble those of normal aging
(Kyng et al., 2003).
In recent years, we have studied WS at the cellular level
and at the level of the defective protein, Werner protein
(WRN). We have characterized its biochemical function and
the molecular pathways in which it functions. One of these
pathways is the repair of oxidative DNA damage, the base
excision repair (BER) system. Another is a role in the
processing at telomere ends, and these two pathways will be
discussed in the following. In our analysis of protein
interactions, we have found that the protein predominantly
interacts through a conserved region, RQC, and we are
exploring the function of this region.This approach will also
be discussed in the Section 4.
2. Proposed biological roles for WRN in base excision
The accumulation of oxidative DNA damage resulting
from endogenous or exogenous agents has been implicated
in the aging process (Beckman and Ames, 1998). Oxidative
DNA lesions are repaired by the BER pathway. BER
involves the excision of a damaged base by a DNA
Mechanisms of Ageing and Development 126 (2005) 79–86
* Corresponding author.
E-mail address: email@example.com (V.A. Bohr).
0047-6374/$ – see front matter. Published by Elsevier Ireland Ltd.
glycosylase, incision of the resulting abasic site, removal of
the remaining 30or 50abasic residue, gap filling, and
predominant gap-filling enzyme during short-patch (one
nucleotide) BER, while polb, pold, and pole have been
implicated in long-patch (greater than one nucleotide) BER
(Wilson et al., 2003) (Fig. 1). The importance of BER in the
prevention of mutation accumulation and the attainment of
the full chronological life span is supported by studies in
yeast in which loss of multiple DNA glycosylases or
apurinic/apyrimidinic (AP) endonucleases severely shor-
tened life span (Maclean et al., 2003). Thus, human
premature aging disorders, such as WS that are hallmarked
by genomic instability, may be deficient in the repair of
oxidative DNA damage.
Reactive oxygen species (ROS) attack DNA bases and
sugars and are the major sources of endogenous single-
strand breaks (SSBs) (Caldecott, 2003). WS cells display
elevated sensitivity to agents which produce ROS and
generate SSBs, including 4-nitroquinoline 1-oxide, ionizing
radiation, and the topoisomerase inhibitor camptothecin.
WRN?/?chicken DT40 cells are also hypersensitive to the
et al., 2002). Recently, Blank et al. (2004) demonstrated that
WRN knockdown cells display elevated sensitivity to
methyl-lexitropsin, a site-specific alkylating agent. They
also found that the absence or inhibtion of O6-methylgua-
nine-DNA methyltransferase rendered WRN knockdown
cells sensitive to the methylating agent temozolomide
(Blank et al., 2004). Human diploid WS fibroblasts
accumulate more oxidative DNA lesions following treat-
ment with hydrogen peroxide compared to normal
fibroblasts (von Kobbe et al., in press). Cells from WS
(HMU) glycosylase activity (Ganguly and Duker, 1992)
suggesting that the lack of WRN may result in the inefficient
removal of HMU from DNA, and HMU accumulation may
contribute to the aging process.
WRN interacts physically and functionally with many
proteins involved in BER including pold, polb, proliferating
cell nuclear antigen (PCNA), replication protein A (RPA),
flap endonuclease 1 (FEN-1), and poly(ADP-ribose)
polymerase 1 (PARP-1). WRN stimulates pold to synthesize
past hairpin and tetraplex structures of trinucleotide repeat
sequences (Kamath-Loeb et al., 2001), which may be
important for the repair of genomic sequences which are
susceptible to alternate structure formation including
telomeric DNA. WRN interacts physically with polb and
WRN helicase activity stimulates polb strand displacement
DNA synthesis (Harrigan et al., 2003). Weak strand
displacement synthesis of polb results in the expansion of
CAG/CTG triplet repeats (Hartenstine et al., 2002). Weak
strand displacement during DNA repair atstrand breaks may
enable short tracts of repeat sequences to be converted into
longer, more mutable stretches. Since polymerase errors
during DNA repair synthesis events likely play a role in
human aging and disease, it is interesting to speculate that
WRN may increase the fidelity of polymerases by
unwinding alternate structures and/or by stimulation of
strand displacement synthesis.
J.W. Lee et al./Mechanisms of Ageing and Development 126 (2005) 79–86 80
Fig. 1. Implications of WRN in BER pathway. Pathways of base excision repair (BER). There is a short-patch, exchange of one nucleotide, and a long patch
pathway (several new nucleotides are inserted). WRN has functional interactions with a number of the proteins involved in long-patch BER as shown in the
figure. Cellular data also support the notion that WRN is involved in the BER process.
The helicase activity of WRN may be important for
facilitating long-patch BER by unwinding BER intermedi-
ates which contain 50termini that are refractory to the
deoxyribose phosphate lyase activity of polb. In addition,
polb initiated repair of 2-deoxyribonolactone, an oxidized
abasic site residue, results in the formation of a covalent
protein–DNA cross-link (DeMott et al., 2002). These cross-
linked intermediates are likely repaired by long-patch BER
to stimulate cleavage of these protein–DNA substrates.
FEN-1 flap cleavage (Brosh et al., 2001) which may be
important in the repair of polb–DNA cross-links.
The WRN exonuclease removes 30mismatches (Kamath-
function as an autonomous proofreader for polymerases, like
Hubscher, 2002). In addition, the WRN exonuclease arrests
at certain oxidative DNA lesions (Machwe et al., 2000)
WRN is the only member of the human RecQ family to
possess both helicase and exonuclease activities, working in
cooperation with proofreading-deficient polymerases, like
the same polypeptide.
We recently identified a physical interaction between
WRN and PARP-1, and found that WS cells are deficient in
the PARP-1 poly(ADP-ribosyl)ation pathway after exposure
to hydrogen peroxide and MMS (von Kobbe et al., 2003). In
addition, unmodified PARP-1 inhibits WRN helicase and
exonuclease activity (von Kobbe et al., 2004). However,
activation and auto-poly(ADP-ribosyl)ation of PARP-1 by
DNA strand breaks relieves the inhibition of WRN catalytic
activities (von Kobbe et al., 2004) suggesting coordination
between PARP-1 and WRN during repair events.
Although PARP-1 and WRN are not essential components
of BER, their presence may ensure correct and efficient DNA
lead to various pathologies associated with aging. In support,
PARP-1?/?/WRNDhel/Dhelmice have an increased frequency
of cancer and fibroblasts derived from those animals display
elevated levels of chromosomal breaks, fragments, and
rearrangements (Lebel et al., 2003). Recently, it was
demonstrated that incomplete BER intermediates which
cannot be properly repaired by the BER pathway can be
shuttled in the homologous recombination pathway (Sobol
et al., 2003). Therefore, the hyper-recombination phenotype
of WS cells may reflect the products of tolerated, but
inappropriate, resolution of SSBs.
3. Role of WRN in telomere maintenance
Many of the WS cellular phenotypes, including genetic
instability and a decline in proliferative competence, are
consistent with defects in telomere metabolism. Telomeres
protect the ends of linear chromosomes and consist of
tandem repeats of the hexameric sequence (TTAGGG) in
mammalian cells. The progressive telomere erosion that
occurs in normal somatic cells during cell division
eventually triggers telomere-associated replicative senes-
an enzyme that lengthens telomeres (reviewed in Campisi et
al., 2001). Several independent studies have found that the
expression of exogenous telomerase in WS fibroblasts
extends the cellular life span, suggesting that premature
senescence in WS cells may be related to telomere
dysfunction (Wyllie et al., 2000; Hisama et al., 2000;
Ouellette et al., 2000; Choi et al., 2001). Classical WS
phenotypes are recapitulated in late generation mice doubly
knocked out for the Wrn and Terc genes, butnot in the single
knock outs, indicating that telomere shortening and
dysfunction contributes to the WS pathology (Chang et
al., 2004). However, the decrease in replicative capacity of
WS cells may not be explained simply by acceleration of
telomeric loss. Although WS fibroblasts showed accelerated
rates of telomere shortening, at senescence mean telomere
lengths were longer than in senescent controls (Schulz et al.,
1996), and erosion rates in WS E6-expressing clones were
decreased compared to the WS bulk population (Schulz et
al., 1996; Baird et al., 2004). Furthermore, the expression of
a WRN helicase-dead mutant in a tumor cell line lead to
increased chromosome fusions and stochastic telomere loss
(Bai and Murnane, 2003). Several studies have shown that
alterations in telomere structure or state, rather than
telomere length, may trigger telomere uncapping and
replicative senescence (Karlseder et al., 1999, 2002; Rubio
et al., 2002). The probability of telomere uncapping is
related to telomere length and the status of telomere-
associated proteins, as well as perhaps the WRN protein.
Therefore, defects in telomere structure and/or integritymay
contribute to telomere dysfunction and premature senes-
cence in WS cells.
Biochemical and cellular evidence suggest that WRN
may function in dissociating alternate or secondary
structures at telomere, to allow for replication, repair and
telomerase activity at the telomere end. Mammalian
telomeres end in a 30single-strand G-rich tail that is
proposed to invade the duplex telomeric DNA and form a
large t-loop that is stabilized by an intra-telomeric D-loop
(Griffith et al., 1999). Telomeric DNA also forms G-
tetraplex structures in vitro that have the potential to block
processes at the telomeres invivo (Fry and Loeb, 1999). The
telomere repeat binding factors TRF1 and TRF2 are
involved in formation and stabilization of the telomere
structure, and function in telomere length regulation and
protection (reviewed in De Lange, 2002). Both TRF1 and
TRF2 regulate telomere length, and defects in TRF2 induce
telomere end fusions and either growth arrest or p53-
mediated apoptosis (Karlseder et al., 1999, 2002; van
Steensel et al., 1998). TRF1 and TRF2 interact with a
J.W. Lee et al./Mechanisms of Ageing and Development 126 (2005) 79–86 81
numerous proteins required for telomere maintenance
including enzymes involved in double-strand break repair,
namely the Ku heterodimer (Hsu et al., 2000; Song et al.,
2000; d’Adda et al., 2001) as well as the Mre11 Rad50 Nbs1
complex (Zhu et al., 2000). We and others have found that
the WRN and Bloom syndrome (BLM) proteins physically
interact with TRF2 (Machwe et al., 2004; Opresko et al.,
2002; Stavropoulos et al., 2002; Lillard-Wetherell et al.,
2004). TRF2 also modulates the activity of the BLM and
WRN proteins. We observed that TRF2 stimulates the
helicase activity of WRN and BLM on telomeric and non-
telomeric substrates in vitro, which has recently been
confirmed (Opresko et al., 2002; Lillard-Wetherell et al.,
2004). Furthermore, WRN and BLM helicases unwind
telomeric D-loops and G-tetraplex structures (van Brabant
et al., 2000; Orren et al., 2002; Mohaghegh et al., 2001; Sun
et al., 1998). The WRN helicase and exonuclease cooperate
to release the invading strand of a telomeric D-loop in a
manner that is regulated by TRF2 and TRF1 (Machwe et al.,
2004;Opresko etal.,2004).Thesestudiessuggest thatWRN
may function in a protein complex at telomeres, possibly to
resolve alternate structures.
Evidence in yeast indicates that RecQ helicases function
in recombination repair of telomeric ends. Several inde-
pendent reports showed that the RecQ homolog in S.
cerevisiae, Sgs1p, participates in a telomerase-independent
pathway for telomere lengthening, termed alternative
lengthening of telomeres (ALT) (Huang et al., 2001;
Johnson et al., 2001; Cohen and Sinclair, 2001). Expression
of the mouse Wrn homolog could partially substitute for
Sgs1 in a Rad50- and Rad52-dependent ALT pathway in
likely involves recombination since some recombination
enzymes are required (reviewed in Henson et al., 2002). A
similar pathway has been identified in telomerase-negative
immortalized mammalian cells (Yeager et al., 1999). These
cells display long heterogeneous telomeres and distinct
nuclear foci referred to as ALT-associated PML (AA-PML)
bodies, which contain Rad52, Rad51, RPA, TRF1, TRF2,
telomeric DNA (Yeager et al., 1999), Ku (d’Adda et al.,
J.W. Lee et al./Mechanisms of Ageing and Development 126 (2005) 79–86 82
Summary of WRN functional protein interactions
Protein-binding partner Interacting region of WRN (amino acid)The nature of functional
BLM51–120 and 949–1092 Inhibits exonuclease
activity of WRN
activity of FEN-1
of other proteins by
PARP-1 but does not
activity of WRN
activity of WRN
activity of EXO-1
Inhibits or stimulates
helicase activity of WRN
dependent on the structure
of the DNA-substrate
activities of p53
Stimulates TopoI DNA
activity of WRN
Relieves pol d from
replication stall at hairpin
and G’2 bimolecular
tetraplex structures of d(CGG)n sequence
Inhibits serum withdrawal-induced
nucleolar localization of WRN
von Kobbe et al. (2002)
FEN-1 949–1092 Brosh et al. (2001)
PARP-1 949–1092von Kobbe et al. (2003)
p97/VCP949–1092Indig et al. (2004) and
Partridge et al. (2003)
Opresko et al. (2002)TRF2 949–1092
Harrigan et al. (2003)
Ku heterodimers940–1432Cooper et al. (2000)
EXO-1940–1432 Sharma et al. (2003)
RAD52982–1432 Baynton et al. (2003)
p53 1014–1432Blander et al. (1999)
Topoisomerase I1–51, 168–246, and 949–1432Laine et al. (2003) and
Lebel et al. (1999)
Brosh et al. (1999)RPA 949–1094 (J. Lee and V. Bohr, unpublished)
949–1401Szekely et al. (2000)
Protein kinase A 859–1066Nguyen et al. (2002)
2001), NBS1 (Wu et al., 2000), and BRCA1 (Wu et al.,
2003). Both WRN and BLM also localize in AA-PML
bodies (Opresko et al., 2004; Johnson et al., 2001;
Yankiwski et al., 2000). These foci most likely represent
sites of activeALT, rather than storage depots, since they co-
localize with BrdU incorporation and are enriched during
S- and G2-phases of the cell cycles (Wu et al., 2003). Using
fluorescent tagged protein we found evidence for WRN
localization to replicating telomeres in live S-phase ALT
cells, and confirmed WRN association directly with
telomeric DNA using qFISH and chromatin immunopreci-
pitation (Opresko et al., 2004). The molecular mechanism of
ALT is poorly understood, but current studies support a
model in which the 30telomeric tail of one telomere invades
telomeric duplex of another chromosome (Dunham et al.,
2000). The resulting inter-telomeric D-loop requires
resolution, for which WRN is a likely candidate. ALT
may derive from a normal pathway important for repairing
broken or damaged telomere ends to prevent untimely or
premature replicative senescence, in telomerase deficient
cells. The precise role of RecQ helicases at the telomeres
remains to be determined, however, current data supports
roles in resolving alternate or recombinant intermediate
structures at the telomeres.
4. The implication of winged helix-turn-helix in the
RQC region of Werner
In human, five RecQ helicases have been discovered
including RECQ1 (Puranam and Blackshear, 1994; Seki
et al., 1994), RECQ2 (Ellis et al., 1995), RECQ3 (Yu et al.,
1996), RECQ4 (Kitao et al., 1998), and RECQ5 (Kitao
et al., 1998). Although not complete, the functions of RecQ
helicases based on cellular and biochemical evidence sug-
gest their close association with DNA replication, DNA
repair, recombination, and telomere maintenance. Clini-
cally, defects in some of thesegenes have been linked to rare
genetic disorders such as Bloom syndrome (RECQ2), WS
(RECQ3), and Rothmund-Thompson syndrome (RECQ4),
which emphasizes their integral role for the normal cellular
function and survival.
One of the prominent characteristics that most members
of RecQ DNA helicases share is a highly conserved region
called RecQ conserved (RQC) (Opresko et al., 2003). In
WRN, the RQC region is located around amino acid (aa)
residues 872–1045. Numerous mapping results support that
the C-terminal part of this RQC region, namely aa residues
949–1092, appears to be included in most of the interaction
sites between WRN and its binding partner. Table 1
summarizes the interface of WRN that mediates protein–
protein interactions that are not only physical, but also
functional, and thus likely to be biological relevant.
Although there are 17 previously reported WRN interac-
tions, only 14 of the 17 have been determined to produce
functional changes in either WRN or its binding partner.
Surprisingly, all of the 14 WRN interactions that are
functionally significant require aa 949–1092 or at least part
of this region for WRN’s interaction with other proteins.
This suggests for a possible unique structure in this region
specific for protein–protein interactions of WRN.
WRN was first demonstrated to bind human flap
endonuclease through aa region of 949–1092 which
stimulated endonucleolytic activities of FEN-1 (Brosh et
al., 2001). Another important functional interaction between
WRN and PARP-1 was shown to be mediated by aa 949–
1092, where the binding of WRN inhibited normal
ribosylation of other cellular proteins by PARP-1 but not
for its auto-ribosylation (von Kobbe et al., 2003). Similarly,
physical interaction of WRN with TTAGGG repeat binding
factor 2 (TRF2) was determined to involve aa 949–1092 of
WRN and this interaction stimulated WRN helicase activity
but not its exonuclease activity (Opresko et al., 2002).
Moreover, WRN has been shown to interact with BLM
through its N-terminal aa 51–120 and the region of aa 949–
1092 (von Kobbe et al., 2002). Also, human exonuclease 1
(EXO-1) interacted with WRN through aa 940–1432, and its
50–30exo- and flap endo-nuclease activities were stimulated
by this interaction (Sharma et al., 2003). Also, the regions of
WRN that interact with other proteins p53, RAD52, and Ku
heterodimers (Ku76/80) have been mapped to the regions of
aa 1014–1432, aa 982–1432 and aa 940–1432, respectively,
all of which contain at least some part of the RQC region
(Baynton et al., 2003; Cooper et al., 2000; Blander et al.,
Much needed clue about the structural information of
WRN or its fragments, especially the aa 949–1092, was
difficult to obtain due to the inability to crystallize them for
X-ray crystallography. However, the catalytic core of E. coli
RecQ helicase structurewas recently solved in 2003 through
X-ray crystallography revealing detailed structure for a
turn-helix (wHTH) motif within its structure (Bernstein et
al., 2003). Surprisingly, the sequence of aa 949–1092 of
WRN aligned with significant homology against the wHTH
motif of E. coli RecQ as shown in Fig. 2.
Since the first observation of a wHTH structural motif
binding DNA in 1993, over a 100 genes that contain this
motif have been reported (Clark et al., 1993; Kaufmann and
Knochel, 1996). Topologically, the wHTH motif is a tight
alpha/beta complex consisting of two wings, three alpha
helices and two beta sheets (Gajiwala and Burley, 2000).
The roles of the wHTH motif in mammalian proteins
have been demonstrated to be important for protein–DNA
interaction as well as protein–protein interaction. In general,
protein to DNA interaction is mediated by the ‘‘DNA
recognition helix’’ of the helix-turn-helix, which makes
direct contact with the major groove of DNA as shown in the
DNA–protein co-crystal structures of a variety of proteins
involved in DNA metabolism (Clark et al., 1993; Fogh et al.,
1994; Gajiwala et al., 2000). Moreover, using similar
approaches in addition to solution nuclear magnetic
J.W. Lee et al./Mechanisms of Ageing and Development 126 (2005) 79–8683
resonance (solutionNMR),thewHTHmotifhasbeen shown
to mediate protein–protein interactions (Littlefield and
Nelson, 1999; Mer et al., 2000). Therefore, the presence
of the wHTH motif in the catalytic core of E. coli RecQ and
the possibility that the wHTH motif may also exist in the
Also, the binding activities of WRN RQC to various DNA
substrates, such as fork, holiday junction, bubble, and G4
tetraplex, support the existence of wHTH in the RQC
region for WRN–DNA interactions (Opresko et al., 2003).
Thus, it will be of interest to investigate the structure of
WRN and its RQC region through an alternative approach to
X-ray crystallography, such as solution NMR, which may
provide insights to understanding the details of how
interactions between WRN and numerous proteins occur
in addition to understanding WRN–DNA interactions (Mer
et al., 2000). Furthermore, understanding the mechanism of
function of the RQC region will greatly advance our
understanding of WRN functions in its prevention of aging.
It will also provide insightful clues about the function of
other family members of the RecQ family of helicases,
including human Rothmund-Thompson and Bloom syn-
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