From the Molecular Cell Biology Laboratory, Graduate
School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki,
Aoba-ku, Sendai 980-8578, Japan, § AGENE Research
Institute, 200 Kajiwara, Kamakura, Kanagawa 247-0063, Japan, and the
¶ Center for Basic Research, Kitasato Institute, Shirokane 5-9-1, Minato-ku, Tokyo 108-8642, Japan
Received for publication, January 23, 2001, and in revised form, March 28, 2001
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ABSTRACT |
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Werner's syndrome (WS) is a rare autosomal
recessive disorder characterized by premature aging. The gene
responsible for WS encodes a protein homologous to Escherichia
coli RecQ. Here we describe a novel Werner
helicase interacting protein
(WHIP), which interacts with the N-terminal portion of Werner protein
(WRN), containing the exonuclease domain. WHIP, which shows homology to
replication factor C family proteins, is conserved from E. coli to human. Ectopically expressed WHIP and WRN co-localized in
granular structures in the nucleus. The functional relationship between
WHIP and WRN was indicated by genetic analysis of yeast cells.
Disruptants of the SGS1 gene of Saccharomyces
cerevisiae, which is the WRN homologue in yeast, show
an accelerated aging phenotype and high sensitivity to methyl
methanesulfonate as compared with wild-type cells. Disruption of the
yeast WHIP (yWHIP) gene in wild-type cells and
sgs1 disruptants resulted in slightly accelerated aging and
enhancement of the premature aging phenotype of sgs1 disruptants, respectively. In contrast, disruption of the
yWHIP gene partially alleviated the sensitivity to methyl
methanesulfonate of sgs1 disruptants.
Werner's syndrome (WS)1
is a rare autosomal recessive disorder characterized by premature aging
and an early onset of age-related diseases including arteriosclerosis,
malignant neoplasms, melituria, and cataract (1). Somatic cells derived
from WS patients show chromosome instability, a shorter life span in
in vitro culture, and accelerated telomere shortening (2,
3). WS cells have subtle defects in DNA replication, resulting in a
reduced frequency of firing of replication origins (4). In addition, a
large number of reports have shown that many cellular events including DNA repair, transcription, and apoptosis are affected in WS cells (5-7). The gene responsible for WS encodes a protein (WRN) that is a
member of the RecQ family of DNA helicases (8). Most of the WS
mutations that have been identified are nonsense or frameshift mutations, resulting in the truncation of WRN (9, 10). The clinical
features and cellular phenotypes of most WS patients seem to be due to
an absolute lack of WRN in the nucleus because the nuclear localization
signal of WRN resides in its C-terminal end (11).
The RecQ family includes Escherichia coli RecQ, S. cerevisiae Sgs1, Shizosaccharomyces pombe Rqh1, and
five human RecQ helicases, namely DNA helicase Q1/RecQL (RecQL1), WRN,
Bloom's syndrome gene product (BLM), Rhusmund-Thomson's
syndrome gene product (RecQL4), and RecQL5 (12-19).
Rhusmund-Thomson's syndrome also shows some features of the premature
aging phenotype, and Bloom's syndrome is characterized by a
predisposition to various malignant neoplasms. In S. cerevisiae, mutations in the SGS1 gene caused premature aging and hyper-recombination phenotypes (20, 21). The sgs1 mutants showed higher sensitivity to MMS and hydroxyurea (22-25, 37).
Thus, sgs1 mutants exhibit some of the phenotypes of WS.
WRN has been shown to have DNA helicase and exonuclease activity
(26-29). Recent studies (30-32) have revealed that WRN
interacts with replication protein A, PCNA, DNA topoisomerase I, and
DNA polymerase Two-hybrid Assay--
The yeast strains and plasmids for
two-hybrid screening were described previously (36).
Cloning of Mouse and Human WHIP cDNA--
We cloned partial
mWHIP cDNA lacking the 5' region by two-hybrid
screening. To obtain the 5' region of mWHIP, we performed nested PCR using a Cap-site cDNA library of mouse testis
(Nippon Gene) as a template, appropriate primers, and the Advantage
GC2-PCR kit (CLONTECH). Based on the 5' sequence of
mWHIP obtained by nested PCR, full-length mWHIP
cDNA was amplified by a reverse transcriptase-mediated polymerase
chain reaction (RT-PCR) using the Advantage GC2-PCR kit and appropriate
primers containing BglII and BamHI sites on total
RNA from testes of C57BL/6 mice and cloned into the pGEM-T-Easy
vector (Promega). hWHIP cDNA was cloned by RT-PCR using
primers synthesized based on the hWHIP sequence as revealed
by Expressed Sequence Tag and the Cap-site hunting method.
Construction of Plasmids--
The bait plasmid pGBT9-mWRN,
plasmids containing variously truncated mouse WRN cDNA,
pGBT9-mouse BLM, pGBT9-mouse RECQL1 Northern Blot Analysis--
The expression of hWHIP
and WRN mRNA was studied using multiple tissue Northern
blots (CLONTECH). The filters were hybridized with
[ Expression of WRN and WHIP--
Human 293 EBNA cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum. Cells were grown to 70% confluence in 10-cm dishes,
transfected with plasmid DNA using LipofectAMINE (Life Technologies,
Inc.), and incubated for 48 h.
Immunoprecipitation and Western Blot Analysis--
Transfected
and nontransfected 293 EBNA cells were used to detect the interaction
of exogenous and endogenous proteins, respectively. The cells were
washed once with phosphate-buffered saline, lysed with 0.5% Triton
buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1 mM dithiothreitol) containing
Complete protease inhibitor mixture (Roche Molecular Biochemicals), and
left standing for 20 min on ice. The cell lysates were
centrifuged, and the supernatants were incubated with protein
A-Sepharose CL-4B (Amersham Pharmacia Biotech) for 1 h at 4 °C
and centrifuged. The resultant supernatants were incubated for 1.5 h at 4 °C with anti-FLAG M2 antibody (Sigma)-coated protein
A-Sepharose and WHIP antiserum-coated protein A-Sepharose to
immunoprecipitate exogenous and endogenous proteins, respectively. The
protein-bound beads were precipitated by centrifugation, washed three
times with lysis buffer, and then suspended in SDS-sample buffer.
Samples were fractionated in 7% SDS-polyacrylamide gels. The gels were
transferred to polyvinylidene difluoride membranes (Millipore). For the
detection of interaction between exogenous proteins the membrane was
immunoblotted with anti-FLAG M2 (Sigma) and anti-HA (MBL Laboratories)
antibody, followed by the secondary antibody, horseradish
peroxidase-conjugated anti-mouse IgG (Dako). Bands were visualized
using ECL detection reagents (Amersham Pharmacia Biotech). Interaction
between exogenous proteins was detected by immunoblotting with a WHIP
antiserum and hWRN monoclonal antibody (Santa Cruz Biotechnology)
followed by secondary antibody, horseradish peroxidase-conjugated
anti-rabbit IgG (New England Biolabs), or anti-goat IgG (Dako).
In Vitro Binding Assay--
The MBP-mWHIP and MBP were expressed
in E. coli BL21(DE3) and purified using amylose resin (New
Englands Biolabs). mWRN was synthesized in the presence of
[35S]methionine using the TNT SP6-coupled reticulocyte
lysate system (Promega). The lysate containing
[35S]methionine-labeled mWRN was incubated with amylose
resin bound with MBP or MBP-mWHIP in binding buffer (40 mM
Tris-HCl, pH 7.6, 100 mM NaCl, 10% glycerol, 0.1% Triton
X-100, 0.1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol) at 4 °C for 2 h. The beads were
washed once with binding buffer and twice with binding buffer devoid of
glycerol and Triton X-100 and then were suspended in SDS-sample buffer.
Samples were fractionated in 7% SDS-polyacrylamide gel. The gel was
fixed, soaked with Amplify (Amersham Pharmacia Biotech), dried, and
subjected to autoradiography.
Generation of Anti-WHIP Sera--
Rabbit polyclonal antisera
against mouse WHIP was generated by immunizing rabbits with purified
MBP-mWHIP expressed in E. coli. The antisera were confirmed
to cross-react with human WHIP and to be able to immunoprecipitate both
mWHIP and hWHIP (data not shown).
Immunofluorescence--
The 293 EBNA cells were grown on
poly-L-lysine coated 8-chamber culture slides and
transfected with plasmid DNA by lipofection. Cells cultured for 24 h after transfection were rinsed three times with PBS, fixed with 4%
paraformaldehyde in PBS containing 2% sucrose for 10 min, and then
permeabilized with Triton buffer (20 mM HEPES, pH 7.4, 0.5% Triton X-100, 50 mM NaCl, 3 mM
MgCl2, 300 mM sucrose) for 5 min. After being
rinsed three times with PBS, the cells in each well were overlaid with
blocking buffer (0.1 mM citrate buffer, pH 6.0, skim milk,
0.05% NaN3) for 3 h at 37 °C, and the blocking
buffer was removed. The cells were incubated with anti-FLAG M2
monoclonal antibody (Eastman Kodak Co.) in PBS containing 1% bovine
serum albumin for 12 h at 4 °C. After three washes with PBS,
cells were treated for 3 h at 37 °C with Texas
red-conjugated goat anti-mouse IgG (Vector Laboratories, Inc.)
and washed five times with PBS. Samples were mounted in Permafluor
(Lipshaw-Immunon, Inc.) and analyzed with a Bio-Rad MRC-1024 confocal microscope.
Yeast Strains and Plasmids--
Yeast strains used in this study
were derived from MR966 (MATa ura3-52 leu2-3, 112 trp-289
his1-7). The yWHIP (YNL218w) deletion strain 966whip
(MATa ura3-52 leu2-3, 112 trp-289 his1-7
whip::KanMX4) and ywhip sgs1 strain 966ws
(MATa ura3-52 leu2-3, 112 trp-289 his1-7
whip::KanMX4 sgs1 Life Span Analysis--
The life span of yeast strains was
measured using a micromanipulator as described by Kennedy et
al. (38).
Analysis of MMS Sensitivity To gain an insight into the cellular processes in which WRN is
involved, we tried to identify proteins that interact with WRN by a
yeast two-hybrid screening using cDNA encoding the mouse WRN as
bait. We identified three proteins: a novel protein, which we
designated as WHIP, and Ubc9 and SUMO-1 (36). Recently, it has been
established that Ubc9 is the enzyme conjugating SUMO-1 to other
proteins (39). We have recently obtained results showing that mWRN is
covalently attached to SUMO-1 (36). The mWHIP consists of 660-amino
acid polypeptides and shows partial homology to replication factor C family proteins, which are required for loading PCNA to the
end of elongating DNA (40). A search of DNA data bases indicated that
WHIP is conserved from E. coli to human (Fig.
1A), and in the N-terminal
region (20-39 aa), eukaryotic WHIPs have a conserved zinc finger motif
(CX2CX11H3C),
which is homologous to that in the post-replicational repair protein,
RAD18 (Fig. 1B).
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ABSTRACT
INTRODUCTION
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, indicating the involvement of WRN in some aspects of DNA replication. WRN also interacts with the p53 and Ku 70/86 heterodimer, suggesting that WRN is involved in apoptosis and the
repair of DNA double strand breaks (7, 33-35). Despite these observations, it is not clear how the dysfunction of WRN is related to
the observed phenotypes of WS cells. To obtain further insight into the
process in which WRN is involved, we performed a two-hybrid screening
using mouse WRN (mWRN) as bait and identified three interacting
proteins: Ubc9, SUMO-1 (small ubiquitin-related
modifier-1), and a novel protein, WHIP (Werner
Helicase Interacting Protein), which is conserved from E. coli to human (36). Here we
report that mWRN physically interacts with mWHIP, and the yeast
homologue of WRN, Sgs1, genetically interacts with yWHIP.
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and pGBT9-mouse RECQL1
, and
mammalian expression plasmid pFLAG-mWRN were prepared as described
previously (36). For in vitro translation, full-length
WRN cDNA was amplified by PCR using appropriate primers containing a SalI site and subcloned into a
SalI-digested pSPUTK vector (Stratagene). The bacterial
expression vector encoding MBP-fused mWHIP (pMAL-mWHIP) and the
mammalian expression vectors encoding N-terminal HA-tagged mWHIP
(pHA-mWHIP) and N-terminal GFP-tagged mWHIP (pEGFP-mWHIP) were
constructed by inserting the mWHIP cDNA in-frame at the
BamHI site of pMAL-c2 (New Englands Biolabs), pHA
(pCMV5-HA), and pEGFP-C1 (CLONTECH), respectively.
-32P]dCTP-labeled hWHIP and WRN
cDNA fragments, respectively, at 42 °C overnight in a 5× SSPE
buffer containing 50% formamide, 2% SDS, 10× Denhardt's solution,
and 100 µg/ml depurinated salmon sperm DNA. The washing was done
under highly stringent conditions: three washes with 2× SSC, 0.1% SDS
at room temperature and one with 0.2× SSC, 0.1% SDS for 30 min at
65 °C. The filters were analyzed using a BAS 1500 system (Fuji Film).
::AUR1) were constructed
by PCR-based gene replacement using oligonucleotides designed to delete
the whole coding sequence. pFA6a-KanMX4, kindly provided by Dr. P. Philippsen, was used as a template to generate an intact
kanMX4 gene flanked by sequences homologous to
yWHIP. The PCR product was used to transform MR966, and the
sgs1 strain and transformants were selected in YPAD
medium containing 200 µg/ml Geneticin. The oligonucleotides
used were as follows: oligo 1, 5'-CATCGGTTGCTTTCCTTGTGAAACTGTACGCGTTGTCTTAAACCGTACGCTGCAGGTCGAC-3'; oligo 2, 5'-GAATTGTAGACGCGGCATTGAGAGAAGTATGGAAGACCCTTGACATCGATGAATTCGAGCTCG-3'. The sgs1 strain 966 sgs1
(MATa ura3-52
leu2-3, 112 trp-289 his1-7 sgs1
::AUR1) was created
by transforming pNS1-27 digested with KpnI and
SacI (37). Transformants were selected in YPAD medium containing 0.5 µg/ml Aureobasidin A. All of the mutants were checked by PCR.
-Stationary phase YPAD grown cells were
diluted with distilled water and spotted at 10-fold dilutions (105-102 cells) onto YPAD plates containing
MMS, incubated at 30 °C for 3 days, and photographed.
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ABSTRACT
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Fig. 1.
Conservation of WHIP from prokaryotes to
higher eukaryotes and expression of WHIP mRNA in
various human tissues. A, amino acid alignment of mouse
(M.m., AB056151), human (H.s., AB056152), fission
yeast (S.p., SPAC26H5.02c), budding yeast (S.c.,
YNL218w), and E. coli (E.c., BAA35624) WHIPs.
Alignment was performed using the CLUSTALW program. The identity
of amino acids is 94, 38, 32, and 35%, for human, fission yeast,
budding yeast, and E. coli, respectively, compared with
mouse WHIP. The zinc-finger motif, Walker A and B motifs, and Sensor 1 and 2 motifs are underlined. B, alignment of the
conserved zinc-finger motifs of hWHIP and hRAD18. C,
expression of WHIP and WRN mRNAs in
various human tissues. Multiple tissue Northern blots containing 2 µg
of poly(A)+ RNA/lane were hybridized with cDNA fragments
of human WHIP and WRN genes as described under
"Experimental Procedures." The arrows show the major
species of WHIP and WRN transcripts.
pbl, peripheral blood lymphocyte; kb, kilobase
pair(s).
Fig. 1C shows expression of WHIP and WRN mRNAs in various human tissues. Although WRN was transcribed in a relatively tissue-specific manner, WHIP was transcribed ubiquitously.
To confirm the binding of WHIP to WRN, FLAG-tagged mWRN (FLAG-mWRN) and
HA-tagged mWHIP (HA-mWHIP) were expressed in human 293 EBNA cells and
immunoprecipitated with an anti-FLAG antibody. The immunoprecipitants
were analyzed by Western blotting using the anti-FLAG antibody and an
anti-HA antibody, revealing co-precipitation of mWRN and mWHIP (Fig.
2A). In addition, the direct
association between mWRN and mWHIP was confirmed by a pull-down assay
for in vitro translated mWRN using MBP-mWHIP (Fig.
2B). To address the interaction between endogenous WRN and
WHIP in the cell, we generated anti-WHIP antisera and performed
immunoprecipitation using the antisera. As shown in Fig. 2C,
endogenous WRN was co-immunoprecipitated with WHIP. We next determined
the region of WRN where WHIP binds by using the two-hybrid system.
Deletion mutants of mWRN were transfected into yeast cells,
and -galactosidase activity was assayed. Positive results were
obtained with constructs encoding polypeptides containing the
N-terminal portion of mWRN (1-271 aa) including the exonuclease domain
(78-219 aa) but not with the construct encoding the polypeptide
corresponding to the region where Ubc9 binds (272-514 aa) (36) (Fig.
2D).
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In addition to WRN, there are four recQ
homologues in human cells; RECQL1, BLM,
RECQL4, and RECQL5. We have previously shown that
mUbc9 interacts with both mWRN and mBLM but not with mRECQL1 isozymes
(36). Thus, we examined whether mWHIP interacts with RecQ family
proteins other than WRN. As shown in Fig.
3A, mWHIP did not interact
with either isomer of mRECQL1 or mBLM. In this context, it is
interesting that exogenously expressed mWHIP co-localized with
exogenously expressed mWRN in granular structures in the nucleus (Fig.
3B).
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Numerous studies have shown that many cellular events including
telomere maintenance and DNA replication, DNA repair, transcription, and apoptosis are affected in WS cells. Recently, the
focus-forming activity 1, which was found as a factor for recruiting
RPA to the pre-replicative foci in a cell-free system, was identified as the Xenopus leavis homologue for WRN (41). In addition,
it has been reported that WRN interacts with PCNA, DNA topoisomerase I,
and DNA polymerase , suggesting that WRN plays some role in DNA
replication (30-32). Thus, it is quite interesting that WHIP has
motifs similar to those in replication factor C. To examine the
functional relationship between WHIP and WRN, we took advantage of
yeast genetics, because WHIP is conserved from yeast to human.
In budding yeast, a sole recQ homologue was identified as SGS1 (13). An original mutant allele of SGS1 was identified as a suppressor of the slow growth phenotype of top3 mutants. Deletion mutants of the SGS1 gene showed pleiotropic phenotypes including premature aging of mother cells, poor sporulation, a reduction in the fidelity of chromosome segregation during mitosis and meiosis, and a mitotic hyper-recombination phenotype (13, 20-22, 37, 42, 43). In addition, the sgs1 mutants were shown to be hypersensitive to hydroxyurea and MMS (22-25, 37). Cells derived from WS patients show chromosome instability and have a shorter life span in vitro in culture (2). In Bloom's syndrome cells, the interchanges between homologous chromosomes are increased, and an abnormally large number of sister chromatid exchanges are present (44). Because similar phenotypes were observed between sgs1 disruptants and WS and Bloom's syndrome cells, sgs1 disruptants seem to be a good model for both WS and Bloom's syndrome cells.
The sgs1 disruptants showed an accelerated aging phenotype
(Fig. 4) and high sensitivity to MMS
(Fig. 5) as described previously (20,
37). In contrast, ywhip disruptants showed a slightly accelerated aging phenotype and no apparent sensitivity to MMS. We
constructed whip/sgs1 double gene disruptants. The
ywhip/sgs1 showed a slow growth phenotype (Fig. 5). Deletion
of the yWHIP gene intensified the accelerated aging
phenotype of the sgs1 disruptants (Fig. 4). In contrast,
disruption of the yWHIP gene partially alleviated the MMS
sensitivity of sgs1 disruptants (Fig. 5). Although these
results suggest that under normal growth conditions, yWHIP functions in
a pathway not involving Sgs1, whereas under DNA damage-induced conditions, it acts upstream of Sgs1 in a pathway involving Sgs1, these
issues must be clarified in a future study. In this context, the levels
of WRN mRNA and WHIP mRNA in various
human tissues were not necessarily correlated (Fig. 1B).
Recently, we and others (45, 46) found that Sgs1 plays dual functions,
that is, to suppress recombination under normal growth conditions and
to induce recombination under DNA damage-induced conditions. Thus, it
seems likely that yWHIP functions as a modulator for Sgs1 when DNA is damaged. The yWHIP gene was identified independently as
encoding a protein possessing Walker A and B motifs, which are
substantially homologous to those of the E. coli RuvB
protein. The protein encoded by this gene, MGS1
(yWHIP), was shown to possess DNA-dependent ATPase and single-stranded DNA annealing activity (47). Thus, Sgs1 and
yWHIP catalyze opposite reactions, the unwinding of double-stranded DNA
and annealing of single-stranded DNA, respectively. This fact may help
to explain the alleviation of the MMS sensitivity of sgs1
disruptants by disruption of the yWHIP gene.
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In conclusion, the physical interaction in mammalian cells and the
genetic interaction in budding yeast between WHIP and WRN (Sgs1)
indicate a functional link between WHIP and WRN that might be
conserved from yeast to human.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research and for scientific research on priority areas from the Ministry of Education, Science, Sports and Culture of Japan, health sciences research grants from the Ministry of Health and Welfare of Japan, and a grant from the Mitsubishi Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB056151 and AB056152 for mouse and human WHIP, respectively.
To whom correspondence should be addressed. Tel.:
81-22-217-6874; Fax: 81-22-217-6873; E-mail:
enomoto@mail.pharm.tohoku.ac.jp.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.C100035200
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ABBREVIATIONS |
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The abbreviations used are: WS, Werner's syndrome; Sgs1, slow growth suppressor 1; MMS, methyl methanesulfonate; PCNA, proliferating cell nuclear antigen; WHIP, Werner helicase-interacting protein; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-mediated PCR; MBP, maltose-binding protein; HA, hemagglutinin; GFP, green fluorescent protein; m, mouse; h, human; y, yeast; PBS, calcium- and magnesium-free phosphate-buffered saline; aa, amino acid; YPAD, yeast extract-peptone-adenine-dextrose..
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