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J. Biol. Chem., Vol. 277, Issue 21, 18291-18302, May 24, 2002
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From the
Received for publication, December 3, 2001, and in revised form, March 7, 2002
Human Werner Syndrome is
characterized by early onset of aging, elevated chromosomal
instability, and a high incidence of cancer. Werner protein (WRN) is a
member of the recQ gene family, but unlike other
members of the recQ family, it contains a unique 3' Werner syndrome (WS)1 is
a human autosomal recessive disorder characterized by early onset
of premature aging characteristics including graying and loss of hair,
wrinkling and ulceration of skin, atherosclerosis, osteoporosis, and
cataracts. In addition, WS patients exhibit an increased incidence of
diabetes mellitus type 2, hypertension, and malignancies (1). The gene
(WRN), defects in which are responsible for WS, encodes a
1,432-amino acid protein (WRN) (2) that has both 3' Cells from patients with WS show premature replicative senesence
compared with cells derived from normal individuals (8). The WS
cellular phenotype suggests correlations among faulty DNA metabolism,
genomic instability, and senescence. WS cells show hypersensitivity to
selected DNA-damaging agents including 4-nitroquinoline-1-oxide (4NQO)
(9), topoisomerase inhibitors (10), and certain DNA cross-linking
agents (11). Compared with normal cells, WS cells also exhibit
increased genomic instability including higher levels of DNA deletions,
translocations, and chromosomal breaks (12, 13). These studies suggest
that WRN plays an important role in DNA metabolism possibly by
participating in DNA repair, replication, and/or recombination pathways.
We have previously identified a physical and functional interaction
between WRN and the Ku heterodimer (14). This finding has been
confirmed recently by an independent laboratory (15). Together with the
catalytic subunit of the DNA-dependent protein kinase
(DNA-PKcs), Ku is required for repair of DNA double strand breaks (dsb)
generated during recombination and by reactive oxygen species resulting
from endogenous metabolism or treatment with ionizing radiation and
certain mutagens (16). We have previously shown that Ku significantly
stimulates the 3' DNA dsb can be created by ionizing irradiation or during V(D)J
recombination, a process that generates immunological diversity. One of
the major pathways for the repair of dsb involves an end-rejoining reaction that requires DNA-PK. DNA-PK is composed of a 465-kDa catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer (20). The
precise role of DNA-PK in DNA dsb repair in mammalian cells is not
clear, but both Ku and DNA-PKcs are essential for the processing of DNA
ends. Current models predict that Ku binds to ends of double-stranded DNA and then recruits DNA-PKcs to form the active protein kinase complex. DNA-PK is essential for efficient DNA dsb repair because cells
lacking either DNA-PKcs or Ku are sensitive to ionizing radiation and
defective in V(D)J recombination (20). DNA-PK has been shown to
phosphorylate a variety of proteins in vitro, including SP1,
SV40 T antigen, p53, and replication protein A (20, 21).
Although the kinase activity of DNA-PK is required in vivo
(22), its physiological substrates are not known.
Because WRN has been shown to interact with Ku, we have explored its
role in the DNA-PK damage response pathway. In this study we have
examined functional interactions among WRN, Ku, and DNA-PKcs. We find
that DNA-PKcs, Ku, and WRN interact on a DNA substrate and that DNA-PK
down-regulates the exonuclease activity of WRN. Correspondingly,
dephosphorylation of WRN increases its helicase and exonuclease
activities. We also show that WRN is phosphorylated in vitro
by DNA-PK and that DNA damage-induced phosphorylation of WRN is absent
from a human cell that lacks DNA-PKcs. Our results would suggest that
WRN may play a role in DNA-PK-mediated end rejoining.
Proteins
Amplified baculovirus was used to infect Sf9 insect cells
for overexpression of WRN protein as described before (7). The protein
was purified by DEAE-Sepharose (Amersham Biosciences), Q-Sepharose
(Amersham Biosciences), and nickel-nitrilotriacetic acid (Invitrogen)
chromatography as described previously (7). Human Ku heterodimer (23)
and DNA-PKcs (24) were purified as reported. The purity of these
proteins was checked routinely, and they specifically did not contain
any ATM protein (data not shown). Baculovirus constructs for
hexahistidine-tagged full-length WRN protein were kindly provided by
Dr. Mathew Gray (University of Washington, Seattle).
Materials
Wortmannin and LY294002 were from Alexis Biochemicals. 4NQO was
from Janssen Chemical. Bleomycin sulfate was from Sigma. T4 polynucleotide kinase was from New England Biolabs. Protein phosphatase 1 (PP1) was from Roche Molecular Biochemicals.
[ DNA Substrates
Exonuclease Substrates--
Single-stranded DNA oligomers
(72-mer and 53-mer) were obtained from Invitrogen. 7 pmol of 53-mer was
5'-labeled with 60 µCi of [ Helicase Substrates--
PAGE-purified 44-mer and 19-mer
oligonucleotides were purchased from the Midland Certified Reagent
Company. The 19-bp duplex substrate with a 25-nucleotide 3'-tail
(3'-overhang DNA substrate) was constructed by labeling 10 pmol
of 19-mer oligonucleotide (5'-GTAAAACGACGGCCAGTGC-3') at its 5'-end
using T4 polynucleotide kinase and [ Exonuclease Assay
The assay to measure the 3' Band Shift Assay
For DNA binding activity the solution conditions were the same
as those used in the exonuclease assay. Approximately 3 fmol of
32P-labeled exonuclease substrate was incubated on ice for
10 min with the indicated amount of Ku, DNA-PKcs, and WRN in buffer
containing 40 mM Tris (pH 7.8), 5 mM
MgCl2, 1 mM dithiothreitol, 0.1 mg/ml BSA, and
10 mM ATP. 0.1% glutaraldehyde was added to the reaction, and incubation continued for 5 min more. 10 µl of DNA protein mixture
was analyzed by 4% nondenaturing gel electrophoresis at 4 °C at 250 V with 1× Tris acetate buffer for 1.5 h. DNA was visualized using
a PhosphorImager and analyzed using ImageQuant software.
In Vitro Phosphorylation
In vitro phosphorylation reactions were carried out
either with the same exonuclease buffer and substrate or with kinase
buffer (50 mM KCl, 1 mM dithiothreitol, 25 mM Hepes-KOH (pH 7.6), 5 mM MgCl2,
50 µM ATP) with 100 ng of sonicated calf thymus DNA and 10 µCi of [ Phosphatase Reaction
2 nM WRN (final concentration) was incubated with
the indicated amounts of PP1 in a 15-µl reaction (40 mM
Hepes (pH 7.5), 7% glycerol, 50 mM KCl, 130 ng/µl BSA,
11 mM MgCl2, and 3 mM ATP). The
reaction was incubated for 10 min at 24 °C.
DNA Helicase Assay
Immediately after the phosphatase reaction, 10 fmol
of the DNA helicase substrate (5 µl) was added to the reaction for a
final volume of 20 µl (30 mM Hepes (pH 7.5), 5%
glycerol, 40 mM KCl, 100 ng/µl BSA, 8 mM
MgCl2, and 2 mM ATP). The mixture was then incubated for 15 min at 37 °C. The reaction was terminated by the
addition of 10 µl of 50 mM EDTA, 40% glycerol, 0.9%
SDS, 0.1% bromphenol blue, and 0.1% xylene cyanol. The products of
the helicase reaction were resolved by electrophoresis on 12% 1× TBE
nondenaturing polyacrylamide gels. The radiolabeled DNA products were
visualized using a PhosphorImager as described above. To determine the
percentage of helicase substrate unwound, the following formula was
used: % displacement = 100 × P/(S + P), where P is the product volume, and
S is the substrate volume. The values for P and
S were corrected by subtracting background values in the
no-enzyme and heat-denatured controls, respectively.
ATPase Assay
123 fmol of WRN was incubated with 1 microunit of PP1 in a
phosphatase reaction buffer (40 mM Hepes (pH 7.5), 7%
glycerol, 50 mM KCl, 130 ng/µl BSA, and 11 mM
MgCl2). The reaction was incubated for 10 min at 24 °C.
In control reactions, phosphatase was omitted. Immediately after the
phosphatase reaction, 100 ng of M13mp18 single-stranded DNA and 16 nmol
of [3H]ATP were added to the reaction for a final volume
of 20 µl (30 mM Hepes (pH 7.5), 5% glycerol, 40 mM KCl, 100 ng/µl BSA, 8 mM MgCl2). The reaction was then incubated for 15 min at
37 °C and subsequently quenched with the addition of 10 µl of stop
solution (100 mM EDTA, 20 mM ATP, 20 mM ADP). Products of the ATPase reaction were resolved by
thin layer chromatography and quantitated by scintillation counting.
Immunoprecipitation
For the in vitro reaction, purified WRN, Ku, and
DNA-PKcs were incubated with cold exonuclease substrate and buffer at
37 °C for 15 min. The reaction mixture was then incubated with
either goat polyclonal anti-WRN (Santa Cruz Biotechnology) or
polyclonal anti DNA-PKcs (Santa Cruz Biotechnology) for 1 h at
room temperature followed by a 1-h incubation with G protein. After
washing the complex with 50 mM Hepes (pH 7.4) and 0.5%
Triton X-100, the immunoprecipitated complex was separated in a 4-12%
acrylamide Tris-glycine gel and transferred to a polyvinylidene
difluoride membrane. The membrane was then incubated with mouse
monoclonal anti-Ku70/Ku80 (Santa Cruz Biotechnology, 1:500), mouse
monoclonal anti-WRN (PharMingen, 1:250), and mouse monoclonal
anti-DNA-PKcs (Oncogene, 1:1000) antibody overnight at 4 °C followed
by a 1-h incubation with horseradish peroxidase-conjugated goat
anti-mouse IgG (1:10,000). The resulting signal was visualized with ECL
plus a Western blotting detection system (Amersham Biosciences).
Immunoprecipitation with total cell lysate was essentially the same as
above; however, instead of purified proteins, cell lysate was used.
Cell lysate was precleared with G protein-agarose prior to immunoprecipitation.
In Vivo Phosphorylation
HeLa cells were grown for 24 h in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum. The human
glioblastoma cell lines M059K (containing normal levels DNA-PKcs) and
M059J (lacking expression of DNA-PKcs) were grown in Dulbecco's
modified Eagle's medium and F-12 medium (Invitrogen) with 15% fetal
bovine serum. After 24 h, ~2-5 × 106 cells
were incubated with phosphate-free Dulbecco's modified Eagle's medium
(Invitrogen) supplemented with 5% dialyzed fetal bovine serum for
1 h to exhaust the pool of endogenous phosphate. The cells were
then freshly supplemented with phosphate-free medium containing 100 µCi of 32Pi (DuPont) for 5 h. In some
experiments, DNA-damaging agents bleomycin (1 or 5 µg/ml) or 4NQO
(0.1 or 0.5 µg/ml) were added during the incubation period. In some
experiments, cells were incubated with 25 µM wortmannin.
The radiolabeling was terminated by washing the cells with ice-cold
phosphate-buffered saline. The cells were then lysed with RIPA buffer
(150 mM NaCl, 1% Triton, 0.1% SDS, 10 mM Tris
(pH 8), 0.5% sodium deoxycholate, 0.2 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 10 µg/ml
leupeptine, 1 mM sodium orthovanadate, 10 units/ml DNase)
supplemented with phosphatase inhibitors (1:1,000, Sigma). After
centrifugation at 12,000 rpm for 10 min at 4 °C, the supernatant was
precleared with G protein-agarose beads (Calbiochem) and incubated with
4 µg of polyclonal goat anti-WRN (from Santa Cruz Biotechnology) for
4 h. The immune complexes were collected by adding G
protein-agarose beads and washed three times with RIPA buffer.
Immunoprecipitated proteins were analyzed on 8-16% acrylamide
Tris-glycine gels. The gels were washed and visualized by autoradiography.
DNA-PKcs Alone Does Not Affect WRN Exonuclease
We have shown previously that Ku can interact physically with WRN
and functionally stimulate WRN 3' Inhibition of WRN Exonuclease Activity by DNA-PKcs in the Presence
of Ku
Because Ku functionally modulates the activities of both WRN and
DNA-PKcs, we assayed for the modulation of WRN exonuclease activity by
DNA-PK. In Fig.
1A, 15 nM Ku and 15 nM WRN were incubated in the
presence of different amounts of DNA-PKcs (2.5, 5, 10, and 15 nM) and exonuclease substrate in kinase buffer. 15 nM Ku alone or 15 nM DNA-PKcs alone has no
effect on the substrate (lanes 2 and 3,
respectively). WRN exonuclease activity was not altered by equimolar
amounts of DNA-PKcs (lane 5). In lane 6, it was
observed that Ku stimulated WRN exonuclease activity, as the
degradation products were smaller in molecular size. We have reported
this stimulation previously (14). To address the possibility that Ku
heterodimer stimulates WRN exonuclease activity by enhancing the
processivity of WRN exonuclease, we conducted unlabeled DNA competitor
experiments. Unlabeled DNA substrate added in increasing amounts to
ongoing WRN exonuclease reactions effectively quenched the WRN
exonuclease reactions in the absence of Ku; however, the presence of Ku
rendered the WRN exonuclease reaction resistant to quenching by a
10-fold molar excess of unlabeled DNA substrate (data not shown). These
results suggest that the effect of Ku on WRN exonuclease is to render
the enzyme more processive.
When DNA-PKcs was added to the reactions containing WRN and Ku, WRN
exonuclease activity was inhibited markedly (Fig. 1A, lanes 7-10). At the highest concentration of DNA-PKcs used
(corresponding to equimolar amounts of DNA-PKcs, Ku, and WRN) the WRN
exonuclease activity was inhibited strongly (Fig. 1, lane
10). Similar results were also observed when kinase buffer was
replaced by exonuclease buffer (data not shown). In Fig. 1B,
we have scanned the lanes from the gel in Fig. 1A. It is
evident from the patterns of digestion products shown by the scan
analysis that DNA-PK inhibited WRN exonuclease activity. We also
calculated the exonuclease activity based on the amount of undigested
(full-length) substrate. It was 0.184 ± 0.12% fmol in the
presence of 15 nM WRN. When 15 nM Ku was
present this became 0.1 ± 0.5%, and in the additional presence of 5, 10, and 15 nM DNA-PKcs, it became 0.55 ± 0.14, 0.8 ± 0.15, and 1.38 ± 0.28%, respectively. The fraction
of digested product ( Incubation with Kinase Inhibitors Can Restore WRN Exonuclease
Activity
DNA-bound Ku is required for maximal stimulation of the protein
kinase activity of DNA-PKcs (26), and the protein kinase activity of
DNA-PK is inhibited by 50-100 nM wortmannin or 50-100 µM LY294002 (27). The presence of wortmannin or LY294002
in the Ku/DNA-PKcs/WRN incubation reaction prevented the
DNA-PK-dependent inhibition of WRN exonuclease activity
(Fig. 2A, lanes 6 and 7). DNA-PKcs was preincubated with 100 nM
wortmannin and 100 µM LY294002 to inhibit the DNA-PKcs
kinase activity completely. In lane 3, it is shown again
that DNA-PKcs itself did not affect the WRN exonuclease activity,
whereas the inhibition of the exonuclease in the presence of Ku and
DNA-PKcs was observed (lane 5). Neither wortmannin nor
LY294002 had any effect on WRN exonuclease activity or the Ku
stimulation of the WRN exonuclease activity (data not shown). Thus, it
appears that the protein kinase activity of DNA-PK is essential for the
inactivation of WRN exonuclease activity.
Werner Protein Is a Target of DNA-dependent Protein
Kinase in Vivo and in Vitro, and Its
Catalytic Activities Are Regulated by Phosphorylation*
,,,,,
,, and**
Laboratory of Molecular Gerontology, NIA,
National Institutes of Health, Baltimore, Maryland 21224, the
§ Department of Biochemistry and Molecular Biology,
University of Calgary, Calgary, Alberta T2NIN4, Canada, and the
Lineberger Comprehensive Cancer Center, the Department
Biochemistry and Biophysics, and the Curriculum in Genetics and
Molecular Biology, University of North Carolina,
Chapel Hill, North Carolina 27599-7295
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5'
exonuclease activity. We have reported previously that human Ku
heterodimer interacts physically with WRN and functionally stimulates
WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic
subunit of DNA-dependent protein kinase (DNA-PK), form a
complex at DNA ends, we have now explored the possibility of functional
modulation of WRN exonuclease activity by DNA-PK. We find that although
DNA-PKcs alone does not affect the WRN exonuclease activity, the
additional presence of Ku mediates a marked inhibition of it. The
inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity
of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this
phosphorylation requires the presence of Ku. We also find that
treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN
exonuclease and helicase activities and that WRN catalytic activity can
be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level
of phosphorylation of WRN appears to regulate its catalytic activities.
WRN forms a complex, both in vitro and in vivo,
with DNA-PKC. WRN is phosphorylated in vivo after treatment
of cells with DNA-damaging agents in a pathway that requires DNA-PKcs.
Thus, WRN protein is a target for DNA-PK phosphorylation in
vitro and in vivo, and this phosphorylation may be a
way of regulating its different catalytic activities, possibly in the
repair of DNA dsb.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' helicase and
3'5' exonuclease activities (3-7). Although WRN appears to play an important role in DNA metabolism, the precise cellular roles of both
the helicase and exonuclease activities of WRN remain to be determined.
5' exonuclease activity of WRN (14), suggesting
that WRN and Ku could act in a common pathway of DNA metabolism
involving the exonuclease function of WRN. This hypothesis is supported
by the observation that mice lacking the Ku80 subunit show a premature
aging phenotype similar to that of WS patients (17). Furthermore, cells
deficient in WRN, Ku70, or Ku80 all show genomic instability and
undergo premature replicative senescence (18, 19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was from PerkinElmer Life Sciences. ATP was
purchased from Amersham Biosciences.
-32P]ATP (3,000 Ci/mmol)
and 10 units of polynucleotide kinase using standard conditions. To
construct a double-stranded DNA substrate with one blunt end and one
3'-recessed (5'-overhang) end, labeled oligomer was mixed with a 2-fold
excess of unlabeled 72-mer, heated together at 90 °C for 5 min, then
cooled slowly to 25 °C. Annealed double-stranded substrates were
then separated from unannealed and excess single-stranded oligomers by
nondenaturing 12% PAGE. Intact double-stranded DNA substrates were
recovered using a Qiaex II gel extraction kit (Qiagen) and stored at
4 °C.
-32P]ATP. The
labeled 19-mer was purified from unincorporated nucleotide by using a
Microspin G-25 column from Amersham Biosciences. The 19-mer was mixed
with 25 pmol of 44-mer oligonucleotide
(5'-GCACTGGCCGTCGTTTTACGGTCGTGACTGGGAAAACCCTGGCG-3') in 50 mM NaCl, 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, heated at 100 °C for 5 min, then placed at
70 °C and allowed to cool slowly (~2 h) to 24 °C. The 19-bp
duplex substrate with a 26-nucleotide 5'-fork and 25-nucleotide 3'-tail
(forked DNA substrate) was prepared using the same protocol except 10 pmol of 45-mer oligonucleotide (5'-TTTTTTTTTTTTTTTTTTTTTTCCAAGTAAAACGACGGCCAGTGC-3') was
annealed to 25 pmol of the 44-mer oligonucleotide described above.
5' exonuclease activity of WRN was
carried out in 10 µl at 37 °C with 40 mM Tris (pH
8.0), 5 mM MgCl2, 1 mM
dithiothreitol, 0.1 mg/ml BSA, 10 mM ATP (25). 3 fmol of
exonuclease substrate was incubated with the indicated amount of Ku,
DNA-PKcs, and WRN. The reactions were initiated by the addition of WRN
and incubated at 37 °C for 60 min. Reactions were quenched by the
addition of an equal volume of formamide loading buffer (80%
formamide, 0.5× TBE, 0.1% bromphenol blue). The digestion products of
these reactions were separated on denaturing 14% polyacrylamide gels
and visualized using a PhosphorImager and quantitated using ImageQuant
software (Molecular Dynamics).
-32P]ATP. The reactions (10 µl) were
initiated by the addition of DNA-PKcs and carried out at room
temperature for 10 min. The reactions were stopped with the addition of
1 µl of 0.25 M EDTA. The resulting products were analyzed
by electrophoresis on 4-12% acrylamide Tris-glycine gel (Invitrogen).
The gel was washed and visualized as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' exonuclease activity (14).
Because Ku and DNA-PKcs are subunits of DNA-PK, which is involved in
the repair of DNA dsb, we explored the possibility that WRN exonuclease
activity is modulated by DNA-PKcs. The exonuclease activity of WRN was
assayed using a duplex DNA substrate containing a 5'-labeled, recessed
3'-end and one blunt end. DNA-PKcs alone has no effect on WRN
exonuclease activity even at higher molar ratios (3.5:1) of DNA-PKcs to
WRN (data not shown).
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Fig. 1.
WRN exonuclease activity is inhibited by
DNA-PKcs only in the presence of Ku. A, 15 nM Werner protein was incubated with 3 fmol of exonuclease
substrate in kinase buffer with 15 nM Ku and 0, 2.5, 5, 10, or 15 nM DNA-PKcs. The products were separated by 14%
polyacrylamide gels under denaturing conditions and visualized by
phosphorimaging. B, quantitative scan of the
lanes in A using ImageQuant software (Molecular
Dynamics). H.I., heat-inactivated DNA-PKcs.
20-mer) for the reaction containing WRN alone or
WRN + DNA-PKcs was 42 ± 4 and 44 ± 6%, respectively. In
the presence of Ku and WRN this fraction became 64 ± 6%. The
addition of 5, 10, and 15 nM DNA-PKcs to the exonuclease
reactions containing WRN and Ku yielded values of 57 ± 7, 48 ± 5, and 36 ± 6%, respectively, for products 20-mer. These
data, representing an average of three experiments, clearly demonstrate
that DNA-PKcs and Ku together effectively inhibit the WRN exonuclease
reaction. The addition of DNA-PKcs inhibits the ability of Ku alone to
stimulate WRN exonuclease activity, instead reducing exonuclease
activity to levels less than observed with WRN alone.
View larger version (78K):
[in a new window]
Fig. 2.
A, the phosphatidylinositol 3-kinase
inhibitors wortmannin and LY294002 restore WRN exonuclease
activity. Where indicated, 15 nM DNA-PKcs was preincubated
for 10 min at room temperature with either 100 nM
wortmannin or 100 µM LY294002 before addition to the
reaction mixtures. Reaction mixtures contained 15 nM WRN
protein, 15 nM Ku, 3 fmol of exonuclease substrate, and
exonuclease buffer. The reactions were incubated for 1 h at
37 °C. The products were separated by 14% denaturing PAGE and
visualized by phosphorimaging. B, Ku interaction with
DNA-PKcs is necessary for inhibition of WRN exonuclease activity. 15 nM recombinant Ku70/Ku80 
C in which the C-terminal 162 amino acids of Ku80 were deleted was incubated with 15 nM
WRN and 5, 10, or 15 nM DNA-PKcs with exonuclease substrate
(lanes 2-4, respectively). The products were
separated on 14% polyacrylamide gels under denaturing electrophoresis,
and the gels were visualized by phosphorimaging
C-terminal Ku80 Is Required for DNA-PK Inhibition of WRN Exonuclease Activity
To confirm that the protein kinase activity of DNA-PKcs was
essential for inhibition of WRN exonuclease activity, we tested a form
of DNA-PK which is defective in protein kinase activity. The C-terminal
12 amino acids of Ku80 have been shown to be essential for the
interaction of DNA-PKcs with Ku (28). A mutant form of the Ku
heterodimer with full-length Ku70 but missing the C-terminal 162 amino
acids of Ku80 (Ku70/80C) is fully able to bind DNA but does not
support DNA-PK protein kinase
activity.2 When this mutant
form of Ku was incubated with WRN and DNA-PKcs, there was no inhibition
of WRN exonuclease activity (Fig. 2B). These data support
the hypothesis that the active DNA-PK (DNA-PKcs + Ku) is required for
abrogation of the exonuclease activity of WRN.
WRN Is Phosphorylated by DNA-PKcs Only in the Presence of Ku
We next tested whether WRN is a substrate of DNA-PK. Different
combinations of WRN, Ku, and DNA-PKcs were incubated with sonicated calf thymus DNA as an effector and with [-32P]ATP (Fig
3). Fig. 3 shows that WRN is
phosphorylated only in the presence of both Ku and DNA-PKcs but not by
either Ku or DNA-PKcs alone (lanes 1 and 2). The
presence of wortmannin inhibited the phosphorylation of the four
proteins (lane 5). DNA-PK phosphorylated Ku and
autophosphorylated itself only when Ku was present (lane 3).
Thus, we show that WRN can be phosphorylated by DNA-PK in vitro and that Ku is required for this phosphorylation.
Phosphorylation of DNA-PKcs, Ku70, and Ku80 (Fig. 3, lane
3) represents autophosphorylation of DNA-PK (29). The WRN
phosphorylation by DNA-PK was also confirmed under the reaction
conditions for the exonuclease activity assay (data not shown),
providing further evidence for the correlation between WRN
phosphorylation and inhibition of its exonuclease activity.
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Ku Mediates the Interaction between WRN and DNA-PKcs
To explore further the potential interactions among WRN, Ku, and
DNA-PKcs, we conducted electrophoresis mobility shift assays, using the
same buffers and substrate conditions as in the WRN exonuclease assay
(Fig. 4). 5 nM Ku and 5 nM WRN were incubated with 1, 2.5, and 5 nM
DNA-PKcs in the presence of exonuclease DNA substrate on ice for 10 min, and the resulting products were separated on 4% native
polyacrylamide gels. Many studies have shown that Ku interacts with DNA
substrates to form protein·DNA complexes in electrophoresis mobility
shift assays. However, DNA-PKcs does not interact with DNA in this
assay, and the interaction among DNA-PKcs, Ku, and DNA is weak in the
absence of cross-linkers (30). Here, we used 0.1% glutaraldehyde to
stabilize the complex. As expected, WRN or DNA-PKcs, separately or
together, did not alter the mobility of the DNA probe (Fig. 4,
lanes 2, 3, and 5, respectively),
whereas the Ku·DNA substrate complex displayed a gel mobility shift
(lane 4). When Ku was present with either DNA-PKcs or WRN, a
supershift was observed (lanes 6 and 7,
respectively). When all three proteins were present, a further mobility
shift was observed. With increasing amounts of DNA-PKcs, the intensity of the supershifted bands was increased (lanes 8,
9, and 10). Interaction of the 156-kDa Ku
heterodimer, 465-kDa DNA-PKcs, plus 167-kDa WRN formed a large
protein·DNA complex that migrated a short distance into the gel. When
treated with 1% SDS, the proteins dissociated, and the substrate was
released (lane 11). The results suggest mutual bindings
among WRN, Ku, and DNA-PKcs, and the possible interaction between
DNA-PKcs and WRN would be mediated through Ku.
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Physical Association between WRN and DNA-PKcs in Vivo and in Vitro
In vitro association of WRN with DNA-PK was
investigated by mixing 5 nM purified WRN, Ku, and DNA-PKcs
(final concentration of each) in vitro in the presence of
exonuclease buffer and substrate, followed by immunoprecipitation with
an antibody against WRN. The presence of WRN, Ku, and DNA-PKcs in the
immunoprecipitated complex was determined by conventional Western
blotting using the respective antibodies. As shown in Fig.
5A, WRN was physically associated with Ku (lane 1). Under our reaction conditions
DNA-PKcs was not physically associated with WRN (lane 2).
However, WRN was physically associated with DNA-PKcs in the presence of
Ku (lane 4). Moreover, in the presence of DNase,
the physical association between WRN and DNA-PK was still present and
unchanged (lane 5). Also, goat IgG did not immunoprecipitate
any proteins from the purified mixture (lane 8). This
experiment suggests that the interaction between WRN and DNA-PKcs is
mediated by Ku.
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To address whether this in vitro physical association also occurs in vivo, we immunoprecipitated this complex from a HeLa cell lysate. As seen in Fig. 5B, any of the three proteins can be immunoprecipitated by any of the three antibodies against WRN, Ku, or DNA-PKcs, indicating that they form a complex in vivo. As a control, we used WRN mutant skin fibroblasts cells, AG11395, where WRN antibody could not precipitate any protein (lane 7). However, in these mutant cells, Ku immunoprecipitated DNA-PKcs, and thus, as expected, the association between Ku and DNA-PKcs takes place in the absence of WRN (lane 8). Input was ~10% of the lysate used in each immunoprecipitation experiment. As a control we used goat IgG, which does not immunoprecipitate either of the proteins mentioned above from HeLa cell lysate (Fig. 5B, lane 2).
WRN Is Phosphorylated when Expressed in Insect Cells
Because DNA-PK phosphorylates substrates at Ser/Thr residues, we
first determined whether recombinant WRN has such phosphorylation sites. Purified WRN recombinant protein was treated with PP1, which
specifically removes phosphate from serine or threonine residues. As
seen in Fig. 6A, the bands
corresponding to WRN as detected by antibody to Ser/Thr phosphate
disappeared with increasing amounts of PP1. Thus, recombinant WRN
protein, when expressed in insect cells, contained phosphorylated
residues at Ser/Thr.
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Because it was possible that dephosphorylation of WRN could remove phosphate groups that were essential for recognition of WRN by DNA-PK, we first treated WRN (directly purified from insect cells) with PP1, then inhibited the protein phosphatase by adding sodium orthovanadate, and then determined whether DNA-PK could rephosphorylate the dephosphorylated WRN. As shown in Fig. 6B, pretreatment with PP1 (lane 2) did not affect the ability of DNA-PK to phosphorylate WRN at Ser/Thr residues (compare lanes 3 and 4). The antibody against Ser/Thr phosphate also detects bands corresponding to the phosphorylation of Ku and DNA-PKcs. The presence of an equal amount of WRN or Ku was verified (Fig. 6, C and D). We also show that PP1 can remove the phosphate, which is incorporated by DNA-PK by treating the WRN·DNA-PK complex with PP1 after the kinase reaction was stopped with wortmannin. As seen in Fig. 6E, the intensity of the incorporated radioactive phosphate decreased after the PP1 treatment. This experiment clearly demonstrated that PP1 specifically removes phosphate, which was incorporated by DNA-PK, from WRN.
Given that DNA-PK phosphorylates WRN protein, we were next interested in whether this phosphorylation might affect any of the WRN catalytic activities. WRN has three characterized catalytic activities: an exonuclease function, a helicase function, and a DNA-dependent ATPase activity.
Dephosphorylation Enhances WRN Exonuclease Activity
We have shown that WRN is phosphorylated by DNA-PK in vitro and that the exonuclease activity of WRN is inhibited under conditions in which DNA-PK is active. PP1 has been shown to remove serine and threonine phosphate incorporated into DNA-PKcs and Ku by autophosphorylation (31). Protein phosphatase regulates DNA-PK activity. PP1 was shown to remove endogenous phosphate from baculovirus-expressed WRN (Fig. 6A) and from WRN that had been phosphorylated by DNA-PK in vitro (Fig. 6E). These data therefore suggested that PP1 could be used as a tool to characterize the effect of phosphorylation on the activities of WRN.
We next examined whether PP1 was able to modulate the exonuclease
activity of WRN. As shown in Fig.
7A, it was observed that treatment of purified, recombinant WRN with increasing amounts of PP1
enhanced the WRN exonuclease activity significantly (lanes 3-6). In the presence of WRN the percent of digested product
20-mer was 33 ± 5. When WRN was preincubated with PP1 the
percent of digested product 20-mer increased with the amount of PP1
used (0.1 unit, 36 ± 7%; 0.25 unit, 39 ± 7%; 0.5 unit,
45 ± 4%; 1 unit, 56 ± 7%). Heat-inactivated PP1 did not
affect WRN exonuclease (lane 8). Thus, dephosphorylation of
baculovirus expressed WRN enhanced its exonuclease
activity.
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The stimulating effect of PP1 treatment on WRN exonuclease activity may be caused by the enhancement of enzyme processivity. To address this possibility, we tested the effect of adding unlabeled DNA substrate to an ongoing WRN exonuclease reaction. The exonuclease reaction was initiated by the addition of WRN that was either untreated or treated with 10 nM PP1 to 3 fmol of radiolabeled DNA substrate. After 2 min of incubation at 37 °C, specific amounts of unlabeled DNA substrate (3, 6, and 15 fmol) were added to the reaction, and the reactions were allowed to incubate for an additional 8 min. The products were resolved on denaturing polyacrylamide gels. As seen in Fig. 7B, the exonuclease activities of both WRN or PP1-treated WRN were inhibited by the presence of increasing amounts of unlabeled DNA substrate in a dose-dependent manner. Quantification of the scanned lanes yielded the percent of undigested substrate <43-mer for WRN with increasing amounts of unlabeled substrate DNA; at 0, 3, 6, and 15 fmol, the percents were 80 ± 3, 84 ± 1.8, 93 ± 2.6, and 95 ± 5, respectively, whereas the corresponding values for PP1-treated WRN were 53 ± 9, 63 + 2.4, 71 ± 8, and 86 ± 1.8%. These results suggest that the processivities of WRN and PP1-treated WRN are similar. A similar effect of unlabeled DNA competitor on WRN exonuclease activity was observed when a higher concentration of WRN (30 nM) was used (data not shown). Thus, PP1 treatment of WRN did not enhance its processivity. Within the range of WRN phosphorylation status that we have been studying (before and after PP1 treatment), we found no change in DNA binding as observed by band shift assay (data not shown).
We have shown that DNA-PK could rephosphorylate WRN that had been PP1-treated, and we then asked whether rephosphorylation of WRN would affect the exonuclease function. As seen in Fig. 7C the exonuclease activity of WRN was increased after dephosphorylation (compare lanes 2 and 3). After rephosphorylation with DNA-PK (lane 4) the WRN exonuclease activity was strongly inhibited. In lane 5, we show that the Ku heterodimer stimulated the exonuclease activity of the dephosphorylated WRN. Thus, the presence of DNA-PKcs in the WRN-Ku reaction was responsible for the inhibition of WRN exonuclease activity. This experiment along with the experiment shown in Fig. 6B demonstrate that phosphorylation by DNA-PKcs can regulate WRN exonuclease activity.
Dephosphorylaton of WRN Enhances Its Helicase Activity on Duplex DNA Substrates
3'-Tailed Substrate--
We next examined whether WRN Ser/Thr
phosphorylation status affected its helicase activity. 1.5 nM WRN (final concentration) was incubated with 1 microunit
of PP1 or PP1 storage buffer and analyzed for helicase activity. As
shown on the gel (Fig. 8A, lane 3) WRN that was not treated with PP1 unwound 35% of
the 3'-overhang helicase substrate. When WRN was pretreated with PP1
the unwinding increased to 58% (lane 4). This represents a
1.7-fold increase in WRN helicase activity on the 3'-overhang substrate
for phosphatase-treated WRN compared with the untreated WRN protein.
Importantly, PP1 did not destabilize the DNA substrate (Fig.
8A, lane 2), and heat-denatured PP1 was unable to
enhance WRN helicase activity (data not shown). Also, 1 microunit of
PP1 was unable to enhance the helicase activity of a WRN ATPase mutant
protein (WRN K577M) that lacks helicase activity (32 and data not
shown). Quantification of WRN helicase activity on the 3'-tailed duplex
as a function of PP1 treatment is shown in Fig. 8C.
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Forked Substrate-- WRN was preincubated with PP1 or PP1 storage buffer and analyzed for helicase activity on a forked duplex substrate. In the absence of PP1, WRN catalyzed 8 and 12% unwinding of the forked duplex substrate at WRN concentrations of 100 and 380 pmol/liter, respectively (Fig. 8B, lane 2 in each gel). 1 microunit of PP1 enhanced WRN helicase activity ~3.5-fold (28% substrate unwound) and 4.8-fold (57% substrate unwound) at WRN protein concentrations of 100 and 380 pmol/liter, respectively (Fig. 8B, lane 3 in each gel). A quantitative representation of the data is shown in Fig. 8D.
The DNA-dependent ATPase activity of WRN was also tested after PP1 treatment. Although the other catalytic activities of WRN are enhanced significantly after PP1 treatment, there is no marked change in its ATPase activity (data not shown). Thus, the enhanced helicase activity of dephosphorylated WRN is not the result of a major change in its ATP-hydrolyzing activity.
Presence of Kinase Inhibitor or PP1 Treatment Does Not Dissociate the WRN·Ku·DNA-PKcs Complex
We next determined whether the presence of kinase inhibitor or PP1
treatment could affect the complex formation among WRN, Ku, and
DNA-PKcs using electrophoresis mobility shift assays. The results are
shown in Fig. 9A. Lane
2 represents the WRN·Ku·DNA-PKcs complex alone. With
increasing concentrations of wortmannin (50, 100, 150 nM)
the band pattern did not change (lanes 3-5). Also, the
presence of 0.25, 0.5, and 1 unit of PP1 in the reaction mixture did
not change the complex formation (lanes 6-8). Treatment
with 1% SDS dissociates the complex from the substrate (lane
9). We also found that the WRN·Ku·DNA-PKcs complex formed
in vitro was unaffected by PP1 treatment (Fig.
9B). These results suggest that the physical association
among WRN, Ku, and DNA-PKcs does not change in the presence of kinase
inhibitor or after PP1 treatment.
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WRN Is Phosphorylated in Vivo after DNA Damage
DNA-PK is required for the repair of DNA dsb induced by ionizing radiation, bleomycin, or other agents (20). Because WS cells are hypersensitive to 4NQO (9), an agent that produces oxidative DNA damage by the formation of radicals, which can ultimately cause DNA dsb, it is likely that WRN participates in this DNA damage response pathway. Indeed, the presence of both helicase and exonuclease activities within the WRN protein makes it an ideal candidate for processing of damage-induced DNA ends. We therefore tested whether WRN is phosphorylated in vivo in response to induced DNA damage.
HeLa cells were treated with bleomycin or 4NQO. As shown in Fig.
10, treatment with bleomycin induced
phosphorylation of WRN in a dose-dependent manner (Fig.
10A). Treatment of the cells in culture with 4NQO also
induced WRN phosphorylation (Fig. 10B). The upper
panels in Fig. 10, A and B, show the silver
staining of the gel, demonstrating that equal amounts of proteins were loaded. When the HeLa cells were preincubated with 25 µM
wortmannin for 1 h before the addition of bleomycin, the amount of
incorporated phosphate decreased significantly, suggesting that the
activity of kinases belonging to the phosphatidylinositol 3-kinase
family were responsible for phosphorylation of WRN in vivo
(Fig. 10C). To demonstrate that DNA-PKcs is a major kinase
that phosphorylates WRN, we used human cell lines with (M059K) or
without (M059J) DNA-PKcs (33). M059J are highly radiosensitive and fail
to express DNA-PKcs because of a message instability (34). Bleomycin
treatment of the cells induced phosphorylation of WRN only in M059K
cells (Fig. 10D) and not in the DNA-PKcs-deficient cell
line, M059J. Silver staining of the protein showed similar loading in
the respective lanes. Thus, WRN phosphorylation occurs in
vivo after DNA damage and is deficient in a cell line with loss of
DNA-PKcs function.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We show that WRN is phosphorylated by DNA-PK in vitro and that DNA damage-induced phosphorylation of WRN is absent in a human cell line that lacks DNA-PKcs. We also show that the phosphorylation of WRN inhibits its exonuclease activity, whereas dephosphorylation of WRN enhances both its exonuclease and helicase activities. Thus, the Ser/Thr phosphorylation status of WRN plays a role in the regulation of its catalytic activities. Furthermore, a complex is formed between DNA-PK and WRN in vivo and in vitro, and this complex formation does not appear to be dependent upon phosphorylation status of these proteins. Thus, WRN protein is a target of DNA-PK phosphorylation, and the catalytic activities of WRN are regulated by phosphorylation. These observations suggest that WRN participates in a DNA-PK pathway of DNA metabolism. We find that the presence of kinase inhibitors or PP1 does not change the physical association between WRN and DNA-PK, and we conclude that physical association between DNA-PK and WRN is not critical for this inhibition of WRN exonuclease activity. Incubation with kinase inhibitors restores WRN exonuclease from DNA-PK-mediated inhibition.
DNA-PK is required for the repair of DNA dsb via the nonhomologous end
joining (NHEJ) pathway, and cells that lack DNA-PK are defective in DNA
dsb repair. Although the precise mechanism of NHEJ has not been
elucidated it is proposed that Ku binds to a DNA dsb and that DNA-PKcs
is subsequently recruited to form an active protein kinase complex.
Other proteins such as XRCC4 and DNA ligase IV are then recruited to
ligate the DNA ends together. Although the physiological substrates of
DNA-PK are not known, the protein kinase activity of human DNA-PKcs is
required in DNA dsb rejoining (22), and ATP is required for NHEJ
in vitro (35). Studies in cell-free extracts suggest that
proteins in addition to DNA-PKcs, Ku, XRCC4 and DNA ligase IV are
required for NHEJ. It is likely that such additional proteins may play
a role in processing DNA ends prior to ligation. Based on current
models this processing is thought to involve both helicase and 3'5' exonuclease activity. The Mre11 component of the Mre11·Rad50·Nbs1 complex has 3'5' exonuclease activity and may function in NHEJ. It
has been suggested that Ku might function as a helicase in NHEJ (26)
based on previous observations that Ku can have helicase activity (36).
However, in this laboratory, we have tested Ku activity on a variety of
helicase substrates, and we have not found unwinding ability of the
heterodimer on any
substrate.3 Because WRN has
both helicase and 3'5' exonuclease activity and interacts with Ku
and DNA-PK, WRN could potentially accomplish both of the putative
processing steps of NHEJ. If WRN is essential in NHEJ, WS(
/) cells
would be expected to be more sensitive than normal cells to treatment
with agents that introduce lesions that are removed via the NHEJ
pathway. Some studies have shown a hypersensitivity of WS cells to
-irradiation, but others have not (37). Recently, using a
fluorescence in situ hybridization technique, it was
demonstrated that x-rays could induce more DNA fragmentation in WS cells than in normal cells (38). WS cells are also
hypersensitive to DNA cross-linking agents which ultimately can give
rise to dsb (11). These studies suggest that WRN may have a role in
repair of DNA damage which is repaired by NHEJ.
Recently Yannone et al. (39) reported that DNA-PKcs inhibited WRN exonuclease and helicase activity, and the additional presence of Ku alleviated this inhibition. The apparent discrepancy of those data with ours may be caused by the DNA substrates or purity of the proteins used in the two studies. Our results would suggest that contamination of the WRN or DNA-PKcs protein preparation with Ku results in the inhibition of WRN exonuclease activity mediated by DNA-PK phosphorylation of WRN. The concentration of DNA-PKcs used in the study by Yannone et al. (39) was 5-10-fold greater than that of WRN, raising the possibility that the inhibitory effect was nonspecific. We have shown that Ku-stimulated WRN exonuclease activity is inhibited markedly by the presence of DNA-PKcs and that the mutant Ku heterodimer (C-terminal 162-amino acid deletion of Ku80 subunit with full-length Ku70) that does not activate DNA-PK is also unable to inhibit WRN exonuclease activity in presence of DNA-PKcs (Fig. 2B). This particular Ku mutant retains its ability to stimulate WRN exonuclease activity (Fig. 2). Moreover, addition of kinase inhibitor to the WRN/Ku/DNA-PKcs reaction restored normal WRN exonuclease activity to the DNA substrate in the presence of Ku. This result suggests that catalytically active DNA-PK is required for inhibition of WRN exonuclease activity.
We show that WRN is phosphorylated in vivo after treatment of cells in culture with DNA-damaging agents. We have used two DNA-damaging agents, 4NQO and bleomycin. 4NQO is a carcinogen that introduces adducts, which are removed primarily by nucleotide excision repair, and WS cells are hypersensitive to this agent (9). Bleomycin is an x-ray mimetic agent, and some sensitivity to it has been reported for WS cells (38). Bleomycin is known to cause various DNA modifications including DNA adducts, single strand breaks, and DNA dsb (40). We find that bleomycin induces more WRN phosphorylation in vivo than does 4NQO (Fig. 10). This supports our hypothesis that WRN plays a role in dsb repair. In vivo phosphorylation of WRN was ablated by the DNA-PK inhibitor wortmannin or in the cell line M059J, which lacks normal DNA-PKcs function. Because the M059J cells are also very low in ATM activity (41) we have used A-T cells (an ATM mutated cell line) to examine if ATM is responsible for the inhibition of WRN phosphorylation in the M059J cells. We find that the level of WRN phosphorylation appeared to be similar between the A-T cells and wild type fibroblast cells (data not shown). Yannone et al. (39) reported that WRN is phosphorylated by DNA-PK in undamaged Jurkat cells and human skin fibroblasts. As mentioned, we were not able to detect significant phosphorylation without exposing the cells to stress. This discrepancy may be caused by differences in cell type or procedure.
In addition to its role in recombination and DNA dsb repair, DNA-PK has also been implicated in several nuclear processes including transcription and DNA replication. Mutant Chinese hamster ovary cells lacking the Ku80 subunit or DNA-PKcs showed a reduced rate of transcription (42). Interaction of Ku with proteins involved directly in transcription initiation has also been observed (43, 44). Recently, it has been shown that Gcn5, a putative transcriptional adapter in human and yeast, is phosphorylated both in vitro and in vivo by DNA-PK and that this phosphorylation down-regulates the histone acetyltransferase activity of Gcn5 (45). We have shown previously that human WRN is directly associated with RNA polymerase II-mediated transcription and that WS cells have reduced transcription rates (46). Modulation of WRN function by phosphorylation could be part of the transcription process.
DNA-PKcs phosphorylates various proteins in vitro. These include p53, the large subunit C-terminal domain of RNA polymerase II, and replication protein A (20, 47). Phosphorylation by DNA-PK of the above proteins has only been shown in vitro, and we now demonstrate that WRN is a target for this phosphorylation in vivo. Physical and functional interactions between WRN and replication protein A (32) and WRN and p53 (48) have been demonstrated. It is possible that phosphorylation by DNA-PK regulates WRN and proteins that are associated with it. Further studies are required to investigate this, especially under conditions when cells have been exposed to DNA damage.
It is not uncommon that protein catalytic activities are regulated by phosphorylation. For example, the replication initiation protein MCM is phosphorylated by cdk2/cyclinA, which in turns inactivates its helicase activity (49). The intrinsic helicase function of NS1, the major nonstructural protein of the parvovirus minute virus in mice, is dependent on its phosphorylation (50). Phosphorylation of SV40 T antigen promotes double hexamer formation, a process to unwind the SV40 origin of replication (51). It appears that there is no uniform mode of regulation by phosphorylation, and some protein functions may be stimulated, whereas others decrease with phosphorylation. This regulation may depend upon the specific phosphorylation sites that are involved locally in different protein interactions, and there are three phosphorylation sites in WRN. We found the greatest increase in WRN helicase activity by dephosphorylation on the forked duplex substrate, a preferred substrate for WRN (52). Our finding that the phosphorylation state of WRN protein affects its helicase activity on that substrate suggests that WRN engages this structure during specific events such as replication fork block. Fine tuned regulation of DNA unwinding at the fork by WRN phosphorylation is an attractive hypothesis, but further studies are required to address the functional importance of phosphorylation on WRN helicase activity in vivo.
Our finding that the level of phosphorylation of WRN greatly affects
its catalytic activities has important implications for studies using
this purified protein and perhaps for other helicase or exonuclease
studies. It is possible that differences in observations regarding
characteristics of the unwinding of different substrates by WRN could
be explained by differences in the level of phosphorylation of the
recombinant protein(s). This aspect should be considered in the
biochemical studies of WRN and related proteins.
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ACKNOWLEDGEMENTS |
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We thank Yaping Yu for purification of DNA-PK and Carl Anderson for comments.
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FOOTNOTES |
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* This work was supported in part by Public Health Service Grant CA84442-01 (to D. A. R. and C. M. S.).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.
¶ Supported by grants from the Alberta Heritage Foundation for Medical Research and the Canadian Institutes of Health Research.
** To whom correspondence should be addressed: Laboratory of Molecular Gerontology, Box 1, NIA, National Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8162; Fax: 410-558-8157; E-mail: vbohr@nih. gov.
Published, JBC Papers in Press, March 11, 2002, DOI 10.1074/jbc.M111523200
2 C. M. Snowden and D. A. Ramsden, unpublished observations.
3 P. Karmakar, J. Piotrowski, R. M. Brosh, Jr., J. A. Sommers, S. P. Lees Miller, W.-H. Cheng, C. M. Snowden, D. A. Ramsden, and V. A. Bohr, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: WS, Werner Syndrome; BSA, bovine serum albumin; DNA-PK, DNA-dependent protein kinase (Ku heterodimer and DNA-PKcs); DNA-PKcs, catalytic subunit of DNA-PK; dsb, double strand break(s); NHEJ, nonhomologous end rejoining; 4NQO, 4 nitroquinoline-1-oxide; PP1, protein phosphatase 1; WRN, Werner protein; ATM, ataxia telangiectasia-mutated protein.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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| 1. | Martin, G. M., Austad, S. N., and Johnson, T. E. (1996) Nat. |
