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/primase
* Laboratory of Molecular Pathology, Division of Oral Pathology, and
Laboratory of Oral and Maxillofacial Surgery, Harvard University, School of Dental Medicine, Boston, Massachusetts 02115, USA; and
Laboratory of Immunobiology, DBS, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA
2Correspondence: Harvard University, School of Dental Medicine, 188 Longwood Ave., Boston, MA 02115, USA. E-mail: David_Wong{at}hms.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/primase (pol-:primase) in vitro
and in cells. The pol-:primase binding domain in
p12DOC-1 is mapped to the amino-terminal six amino acid
(MSYKPN). The biological effect of p12DOC-1 on
pol-:primase was examined using in vitro DNA
replication assays. Using the SV40 DNA replication assay,
p12DOC-1 suppresses DNA replication, leveling at ~50%.
Similar results were obtained using the M13 single-stranded DNA
synthesis assay. Analysis of the DNA replication products revealed that
p12DOC-1 affects the initiation step, not the elongation
phase. The p12DOC-1 suppression of DNA replication is
likely to be mediated either by a direct inhibitory effect on
pol-:primase or by its effect on cyclin-dependent kinase 2
(CDK2), a recently identified p12DOC-1-associated
protein known to stimulate DNA replication by phosphorylating
pol-:primase. p12DOC-1 suppresses
CDK2-mediated phosphorylation of pol-:primase. These data support
a role of p12DOC-1 as a regulator of DNA
replication by direct inhibition of pol-:primase or by negatively
regulating the CDK2-mediated phosphorylation of
pol-:primase.—Matsuo, K., Shintani, S., Tsuji, T., Nagata, E.,
Lerman, M., McBride, J., Nakahara, Y., Ohyama, H., Todd, R., Wong,
D. T. W. p12DOC-1, a growth suppressor,
associates with DNA polymerase /primase.
Key Words: pol-:primase complex • cell cycle regulator • DNA replication
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. It is a highly conserved cellular gene. We (1
, 2)
and others (3
, 4)
have cloned
p12DOC-1 cDNA from human, mouse, and hamster.
The full-length human and mouse p12DOC-1 cDNAs
are 1.6 kb and 1.2 kb, respectively. Human
p12DOC-1 has one additional codon at residue 19,
which encodes for an alanine, and differs from the mouse and hamster
p12DOC-1 at only two other amino acid
residues (Ala
Thr at residue 8 and GlySer at residue 100). The
identity of human and rodent p12DOC-1
polypeptides is 97% whereas the mouse and hamster
p12DOC-1 protein sequences are identical.
Human p12DOC-1 is a 115 amino acid peptide with a
molecular mass of 12.4 kDa (pI of 9.62). Transfection of
p12DOC-1 into malignant oral keratinocytes led to
the reversion of transformation phenotypes (anchorage independence,
doubling time, and morphology). The genetic sequence of
p12DOC-1 matched to a murine tumor necrosis
factor (TNF-) early-response murine transcript, TU-166,
inducible by TNF- (3)
, suggesting
p12DOC-1 is a downstream event in the TNF-
signaling pathway.
This paper presents data that provide insights into the
mechanisms whereby p12DOC-1 exerts its growth
suppressor function. We have previously shown that
p12DOC-1 associates with a phosphorylated 180 kDa
cellular protein (2)
. This
p12DOC-1-associated protein was determined to be
the large catalytic subunit, p180, of pol-:primase. Using a newly
raised polyclonal antibody raised against the entire pol-:primase
complex (p180, p70, p55, and p49), p12DOC-1 was
shown to associate with all four subunits of pol-:primase, in
vitro as well as under cellular conditions. The binding of
pol-:primase was shown to be mediated by the amino-terminal six
amino acids of p12DOC-1. The effect of
p12DOC-1 on pol-:primase was examined using
the SV40 and M13 single-stranded DNA replication assays. Input
p12DOC-1 protein significantly inhibits DNA
replication. p12DOC-1 affected primarily the
initiation step of DNA replication, not elongation. CDK2 is a recently
identified p12DOC-1-associated protein (S.
Shintani et al., unpublished results). CDK2 is known to phosphorylate
pol-:primase, stimulating DNA replication. In vitro
kinase experiments revealed that p12DOC-1
suppresses the CDK2-mediated phosphorylation of pol-:primase.
These results jointly suggest that p12DOC-1
is a negative regulator of DNA replication either by direct inhibition
of pol-:primase or by suppressing the CDK2-mediated phosphorylation
of pol-:primase.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of GST-p12DOC-1 fusion protein
The human p12DOC-1 cDNA was cloned into
the glutathione S-transferase (GST) fusion protein vector pGEX-4T-1
(Pharmacia Biotech, Piscataway, N.J.) at the SalI (5') and
NotI (3') sites containing the entire open reading frame to
be expressed in-frame with the GST protein
(GST-p12DOC-1).
GST-p12DOC-1 fusion protein was produced by
growing pGST-p12DOC-1 transformed DH5-
cells to OD600 between 1.0 and 2.0, and induced with 0.1 mM
isopropyl-ß-thiogalactopyranoside (IPTG; Stratagene, La Jolla,
Calif.). GST-p12DOC-1 fusion proteins were
purified with use of glutathione Sepharose 4B beads (Pharmacia
Biotech). The production of the GST-p12DOC-1
fusion protein was confirmed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot with p12DOC-1/Ab3, an anti-human
p12DOC-1 rabbit polyclonal antibody. For use in
experiments, the GST-p12DOC-1 fusion protein
bound to glutathione Sepharose 4B beads was either eluted with the
elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0) or
cleaved by thrombin (Sigma Chemicals, St. Louis, Mo.) to separate from
GST moiety. GST protein (28 kDa) produced by the parental pGEX-4T-1
vector was also purified in the same fashion and used as controls.
GST-p12DOC-1 and cellular lysate binding assay
Total cell lysate from 293 cells was prepared using a mild lysis
buffer (1% Nonidet P-40, 150 mM NaCl, 10 mM sodium phosphate [pH
7.4], 2 mM EDTA, 50 mM sodium fluoride, 200 µM sodium orthovanadate,
2 µg/ml aprotinin, and 100 µg/ml phenylmethylsulfonyl fluoride)
containing 1 µM of clast-lactacystin ß-lactone (Calbiochem, San
Diego, Calif.). Five hundred micrograms of total cell lysate was
diluted 1:1 in EL buffer (250 mM NaCl, 50 mM HEPES pH 7.0, 0.1%
Nonidet P-40, 200 µM sodium orthovanadate, 2 µg/ml aprotinin, 100
µg/ml phenylmethylsulfonyl fluoride, and 1 µM clast-lactacystin
ß-lactone) and mixed with purified GST-p12DOC-1
fusion protein bound to glutathione Sepharose 4B beads. GST alone was
used as negative control. After incubation on ice for 2 h, the
protein complexes were centrifuged at 500 g for 5 min at
4°C. The pellet was resuspended with cold EL buffer. This washing
procedure was repeated four times. The resulting complexes were
subjected to kinase assay, SDS-PAGE, and/or immunoblot analysis.
Preparation and purification of pol-:primase
The recombinant baculoviruses expressing pol-:primase
subunits were gifts from Teresa Wang (Stanford University) (p180) and
Ellen Fanning (Vanderbilt University) (p68, p55, and p49). High-Five
insect cells (Invitrogen, Carlsbad, Calif.) in suspension were
coinfected with recombinant baculoviruses expressing each of the four
subunits of human pol-:primase at a multiplicity of infection of 10.
Pol-:primase was purified by immunoaffinity column chromatography
with the monoclonal antibody SJK-237–37 (Teresa Wang, Stanford
University) specific for the p180 subunit (5)
.
Immunoprecipitations and immunoblottings
Immunoprecipitations and Western blottings were performed
using standard protocol (6)
. For
p12DOC-1 immunoprecipitation, the
p12DOC-1/Ab3 rabbit polyclonal antibody
was used. This rabbit polyclonal antibody was generated using a
GST-p12DOC-1 fusion protein. For Western blotting
to detect the p180/pol-:primase, a chicken IgY polyclonal antibody
(ID) directed against the p180 subunit of human pol-:primase was
used (gift from T. Wang). To detect all four subunits of
pol-:primase, the newly raised pol-:primase/Ab3 polyclonal
antibody was used. For signal detection, the Amersham ECL System was
used in conjunction with BioMax MR film.
p12DOC-1 deletion mutants
A series of p12DOC-1 carboxyl-terminal
deletion mutants were created by mutagenizing specific amino acids to a
stop codon at the following positions: 6, 16, 36, 56, 76, 96. The
Stratagene ‘QuickChange Site-Directed Mutagenesis Kit’ was used
(Stratagene). Each mutation was accomplished by two synthetic
oligonucleotide primers containing the desired mutation.
The p12DOC-1 amino-terminal deletion mutant [p12DOC-1 (1–6)] was created by inserting EcoRI sites from the amino-terminal between amino acids 5 and 6, again using the Stratagene ‘QuickChange Site-Directed Mutagenesis Kit’. The unique EcoRI site in the multiple cloning site of pGEX4–1 vector was used with newly created EcoRI site to remove the first six amino acids, thus creating the p12DOC-1 (1–6) mutant.
SV-40 in vitro DNA replication assay
DNA replication assay was performed according to published
procedures (7
8
9)
. pSV010 was used as the plasmid template
containing an SV40 origin of DNA replication. S100 lysate from human
293 cells and SV40 T antigen from baculovirus-expressing T antigen were
prepared as described by Dutta and Winchester (9)
.
Titration curves were done to optimize the amounts of S100 lysate and T
antigen for incorporation of 32P-dCTP: for a 25
µl reaction, 8 µl and 0.5 µg of S100 and T antigen, respectively.
GST-p12DOC-1 and GST control proteins were
produced as described and subjected to thrombin digestion. After
removal of the parental GST domain conjugated with the Sepharose 4B
beads by centrifugation, the thrombin-cleaved
p12DOC-1 and GST control protein (28 kDa) were
visualized by Coomassie blue-stained SDS-PAGE and the amount was
quantified. Twenty to 100 ng of GST-p12DOC-1 or
GST proteins resuspended in distilled water was added. Reaction
mixtures without S100 extract or T antigen were also included as
negative controls. Specific activity of
[-32P]dCTP in reaction (cpm/pmol) was
calculated by the following formula: cpm/pmol = cpm of 1.3 µl
master mix/125. The pmol of [-32P]dCTP
incorporated in each reaction was calculated as follows: pmol =
(cpm of DE 81 paper x 10)/specific activity. Each sample was
assayed in triplicate; the results were analyzed by Student’s
t test.
M13 ssDNA replication assay
DNA synthesis on single-stranded M13 DNA (M13mp18 ssDNA)
templates was carried out for 60 min at 37°C in the presence of 30
µM of M13mp18 ssDNA, 20 mM Tris acetate, pH 7.3, 5 mM magnesium
acetate, 20 mM potassium acetate, 1 mM dithiothreitol, 0.1 mg/ml of
BSA, 1 mM ATP, 0.1 mM each of CTP, GTP, UTP, dATP, dGTP, TTP, and
[-32P] dCTP (100 cpm/pmol). The polymerized
products were analyzed by 1.8% alkaline agarose-gel electrophoresis
for 16 h at 60V (10)
. In parallel, total
incorporation of [-32P] dCTP was determined
by scintillation counting after removal of unincorporated
32P-dCTP by G50 Sepharose columns.
In vitro phosphorylation assay
For kinase assay, cell lysates were prepared from 293
cells transfected for 48 h with FLAG or
FLAG-p12DOC-1. Immunoprecipitations were
performed with antibodies specific to CDK2 (M2; Santa Cruz
Biotechnology, Santa Cruz, Calif.), cyclin A (BF683; Santa Cruz
Biotechnology), cyclin E (M20; Santa Cruz Biotechnology), and protein
A/G agarose beads. The immune complexes were washed four times with
kinase buffer (50 mM Tris, pH 7.4, 0.1 mM EDTA, 1 mM DTT) and
resuspended in a final volume of 10 µl of kinase buffer. The kinase
reactions included 500 ng Hi-Five cell-expressed pol-:primase, 5
µM ATP, 10 mM Mg2+, and 10 µCi of
(
-32P) ATP and were incubated for 15 min at
37°C. At the end of the reaction, 10 µl of 2x SDS sample buffer
was added and proteins were loaded on 10% SDS-polyacrylamide gels. The
gels were stained with Coomassie blue prior to autoradiography.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/primase:primase (2)
. Using ID, a human
pol-:primase-specific chicken polyclonal antibody that detects the
p180 subunit of pol-:primase (11)
, and a newly
generated polyclonal antibody (pol-:primase/Ab3) raised against all
four subunits of human pol-:primase,
GST-p12DOC-1 was found to associate with
pol-:primase (Fig. 1A, B
, lanes 2). GST
alone did not complex with pol-:primase (Fig. 1A, B
, lane 1).
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p12DOC-1 interacts with pol-:primase in cells
To ascertain that p12DOC-1 binds to human
pol-:primase under cellular conditions, sf9 insect cells were
coinfected with recombinant baculovirus containing
p12DOC-1 and each of the four subunits of
pol-:primase. Insect cells are necessary to be host for these
experiments because they will permit optimal protein aggregation and
folding of each of the four subunits of pol-:primase. Dornreiter et
al. (12)
used this system to demonstrate the in
vivo interaction of SV40 large T antigen with pol-:primase.
Coprecipitation experiments were performed to demonstrate association
of p12DOC-1 with pol-:primase in cells (Fig. 1C
). Cells coinfected with p12DOC-1
and four subunits of pol-:primase produced all four subunits of
pol-:primase (Fig. 1C
, lane 3). Immunoprecipitation with
the anti-human p12DOC-1 polyclonal antibody
(p12DOC-1/Ab3) coprecipitated all four subunits
of pol-:primase (Fig. 1C
, lane 2), whereas the normal
rabbit serum (NRS) control did not (Fig. 1C
, lane 1). These
experiments demonstrated that p12DOC-1 interacts
with pol-:primase under both in vitro and cellular
conditions.
p12DOC-1 binds to pol-:primase via the
amino-terminal six amino acids
A panel of carboxyl-terminal deletion mutants was created by
site-specific mutagenesis of the parental
p12DOC-1 protein. The truncated proteins deleted
20, 40, 60, 80, and 100 amino acid residues, respectively, from the
carboxyl-terminal. The resultant p12DOC-1 mutants
were 95, 75, 55, 35, 15 amino acids in length. In
vitro binding experiments were performed using each of the five
truncated p12DOC-1 proteins with 100 ng of input
pol-:primase. The resultant bound complexes were analyzed by
SDS-PAGE followed by immunoblotting for pol-:primase using the
pol-:primase/Ab3 polyclonal antibody. Pol-:primase bound to all
of the truncated p12DOC-1 peptides (Fig. 2
, lanes 3–7). These results
indicate that the pol-:primase binding domain resides in the
amino-terminal fragment (20 amino acids) of
p12DOC-1. To further map the pol-:primase
binding domain, two amino-terminal mutants were created,
p12DOC-1 (1–6) and
p12DOC-1 (7–115), consisting only of the first
six amino acids or deleting the first six amino acids, respectively.
The first six amino acids were targeted because the first five amino
acids between p12DOC-1 and a closely related
protein DOC-1R are identical (13)
. Using these two
NH2-terminal mutants in pol-:primase binding
experiments revealed that the p12DOC-1 (7–115)
mutant failed to bind pol-:primase (Fig. 2
, lane 10), whereas the
p12DOC-1 (1–6) mutant retained binding to
pol-:primase (Fig. 2
, lane 9), suggesting the first six amino acid
(MSYKPN) is sufficient for pol-:primase association. GST alone did
not bind to pol-:primase (Fig. 2
, lane 2).
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p12DOC-1 inhibits DNA replication in cell-free DNA
replication assays
Pol-:primase is the principal polymerase in eukaryotic DNA
replication. Its function is to incorporate dNTPs into leading and
lagging strands of the replicative fork. We proceeded to examine
whether the association of p12DOC-1 with
pol-:primase in the DNA replicative enzyme complex affects DNA
replication. Two methods were used to examine the effect of
p12DOC-1 on DNA replication. Using a cell-free
SV40 DNA replication system (7)
, addition of purified
p12DOC-1 caused a dose-dependent suppression of
DNA replication (Fig. 3A
). The maximum level of
suppression was ~49%. The effect is pol-:primase specific. Use of
aphidicolin (30 µg/ml) or the omission of pol-:primase completely
abolished DNA replication (data not shown). In addition, the SV40
replication products were digested with restriction endonucleases to
distinguish replication products from repair products (data not shown).
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To examine whether p12DOC-1 affects DNA
replication at the initiation or elongation step, a M13mp18
single-stranded DNA replication assay was used (14)
.
Similar to the results obtained with the SV40 DNA replication assay,
increasing input p12DOC-1 suppressed DNA
replication in a dose-dependent manner (Fig. 3B
). The
maximum level of suppression was ~47%. Newly synthesized
32P-labeled DNA replication products were
analyzed by alkaline agarose-gel electrophoresis (Fig. 3C
).
Comparison of the DNA polymerization products in the presence of
increasing input p12DOC-1 protein showed that
whereas the size distribution was similar, with a range of distribution
of ~100 to 2500 nucleotides (Fig. 3C
, compare lane 4 with
lanes 5–7), the intensity of the DNA replication products is reduced
(Fig. 3C
, lanes 5–7). These results were confirmed by using
poly(dT) as templates in order to eliminate possible snap back
structure from nicked M13 circles (data not shown). Jointly, these
results suggest that the p12DOC-1-mediated
suppression of DNA replication affects primarily the initiation step,
not the elongation process.
p12DOC-1 suppresses CDK2-mediated phosphorylation of
pol-:primase
Phosphorylation of pol-:primase is known to stimulate DNA
replication (15
, 16)
. CDK2, complexed with cyclins E and
A, is the principal kinase known to phosphorylate the p180 and p70
subunits of pol-:primase (17)
. We have recently shown
that CDK2 is a p12DOC-1-associated protein that
suppresses CDK2-associated pRB and histone H1 kinase activities
(S. Shintani et al., unpublished results). These results prompted
us to examine whether p12DOC-1 can similarly
suppress CDK2-mediated phosphorylation of pol-:primase, which may be
partly responsible for the observed
p12DOC-1-mediated suppression of DNA replication.
Cellular CDK2 kinase activity at any moment inside a cell is largely a
reflection of intracellular levels of activated cyclin A-associated
CDK2 (S phase) and cyclin E-associated CDK2 (G1/S). We evaluated the
effect of ectopic p12DOC-1 expression on cellular
CDK2 kinase activity by transfecting p12DOC-1
into 293 cells, followed by immunoprecipitation of total CDK2, cyclin
A-associated kinases (CDK2 and CDC2), and cyclin E-associated CDK2. The
immunoprecipitated complexes were used to phosphorylate pol-:primase
(Fig. 4
). Figure 4
shows that
ectopic expression of p12DOC-1 reduces
phosphorylation of the p180 subunit of pol-:primase by CDK2
~threefold (lane 2). A similar reduction was observed for cyclin
A-associated kinases (~fourfold, lane 4) and cyclin E-associated CDK2
(~fourfold, lane 6). Cyclin E-associated CDK2 also phosphorylates the
p70 subunit (Fig. 4
, lane 5). Ectopic expression of
p12DOC-1 abolished the cyclin E/CDK2-mediated
phosphorylation of p70. The inhibitory effect of
p12DOC-1 for CDK2-associated pol-:primase
kinase activities is specific. In a similar experiment
immunoprecipitating CDK4 and CDK6, ectopic expression of
p12DOC-1 did not alter the phosphorylation
pattern of pol-:primase (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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:primase. p12DOC-1 complexes with
pol-:primase in vitro and under physiological conditions
in cells. We have mapped the p12DOC-1’s
pol-:primase binding domain to a region at the six amino-terminal
amino acids. p12DOC-1 suppresses DNA replication
in the SV40-based and M13mp18 ssDNA assays. The mechanism is likely to
be through a direct suppression effect on pol-:primase and/or by the
p12DOC-1-mediated suppression of pol-:primase
phosphorylation by CDK2. These data demonstrate a novel association of
p12DOC-1 with pol-:primase and provide a
potential mechanism whereby a growth suppressor can regulate the
activity of the key DNA replication enzyme.
Pol-:primase, the principal enzyme for DNA replication, is composed
of a 180 kDa catalytic subunit (p180), a 70 kDa subunit, and two
polypeptides of 49 and 55 kDa associated with DNA primase activity
(18)
. Although our data show that
p12DOC-1 associates with the entire
pol-:primase complex, it is likely that the actual association is
through one of the four subunits. Note that the same six amino acids
responsible for pol-:primase binding to
p12DOC-1 are conserved in a
p12DOC-1-related protein, DOC-1R
(13)
, suggesting the DOC-1R may similarly associate with
pol-:primase.
Pol-:primase gene expression is induced during activation of
quiescent cells to proliferate, elevated in tumor cells and
down-regulated in terminated differentiated cells (19)
. In
actively proliferating cells, pol-:primase is expressed
constitutively at both the transcriptional and translational levels
throughout the cell cycle (20)
. Phosphorylation has been
shown to play a role in the regulation of pol-:primase. Wong et al.
(21)
have shown that both catalytic subunits of human
pol-:primase (p180 and p70) are phosphorylated at serine and
threonine residues. Donaldson and Gerner (16)
demonstrated
that pol-:primase is phosphorylated in culture cells by a serine
kinase, and suggest that phosphorylation/dephosphorylation reactions
modulate the activity of the enzyme. Cripps-Wolfman et al.
(15)
showed that there is a correlation of pol-:primase
phosphorylation and activity in human diploid fibroblasts in
replicative DNA synthesis. These data also suggest that fluctuations in
pol-:primase activity are not due to transcriptional or
translational modifications but to posttranslational phosphorylation,
stimulating its catalytic activity.
Data from these laboratories provide insight into our current findings.
Our data showed that p12DOC-1 suppresses the
CDK2-mediated phosphorylation of the p180 and p70 subunits of
pol-:primase. In particular, ectopic expression abolished the cyclin
E/CDK2-mediated phosphorylation of p70 subunit. Although the role of
p70 in DNA replication is not yet defined, it is likely that the
p12DOC-1-mediated suppression of CDK2
phosphorylation of pol-:primase is in part responsible for the
observed suppression of DNA replication. We propose that
p12DOC-1’s association with pol-:primase
suppresses its activity whereas p12DOC-1’s
association with CDK2 suppresses the CDK2-mediated phosphorylation of
pol-:primase. Consistent with this model is that ectopic expression
of p12DOC-1 is associated with significant
reduction of [3H]-thymidine uptake by 239 cells
(data not shown).
We have not yet been able to obtain evidence of endogenous interaction
of p12DOC-1 and DNA polymerase-/primase. A
reason for this is that most of the cell models we have examined do not
express an appreciable amount of p12DOC-1.
Efforts are currently under way to identify cellular models that
express sufficient levels of p12DOC-1 so as to
permit the function role of this growth suppressor to be examined under
endogenous physiological conditions. These cellular models can also
allow us to further examine the detailed role of
p12DOC-1 in DNA replication. Our data indicate
that the maximal suppression of DNA replication by
p12DOC-1 is at ~50%. It is unclear why there
is no further suppression beyond 50%. In view of our recent finding
that p12DOC-1 associates with CDK2 and can
negatively regulate the CDK2-mediated phosphorylation of p180 and p70
(Fig. 4)
, perhaps we can speculate that whereas ectopic expression of
p12DOC-1 completely shut off the CDK2-mediate
phosphorylation of p70, the phosphorylation of the p180 subunit is only
partially inhibited and thus may allow partial initiation of the DNA
replication process.
Pol-:primase is the only eukaryotic enzyme that can initiate
DNA replication de novo. While
p21WAF1/CIP1/CAP20 has been shown to associate
with proliferating cell nuclear antigen and inhibits DNA replication
(22)
, our data demonstrate a proximal control mechanism
whereby the first step of DNA replication, initiation, can be regulated
on interaction with p12DOC-1. We propose that
p12DOC-1 is a regulator of DNA replication,
negatively regulating the activity of pol-:primase.
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ACKNOWLEDGMENTS |
|---|
:primase (ID) and the baculovirus
containing the p180 cDNA. We thank Dr. Ellen Fanning (Vanderbilt
University) for providing the baculovirus containing the p68, p55, and
p49 subunits to human pol-:primase. We also thank Dr. Heinz-Peter
Nasheuer for providing us with the CL-22 and Pol SIGMA
anti-pol-:primase antibodies (Inst. F. Molekulare Biotechnologie
E.V, Jena Germany). In addition, we are grateful to Dr. Yi-Ling Lin her
input into this project. We are grateful for the conceptual and
technical assistance from the members’ head and neck/oral cancer
research group at Harvard Medical School. This include Drs. Karl
Münger and Philip Hinds (Harvard Medical School), Dr. James G.
Rheinwald (Brigham & Women’s Hospital), Dr. Anil Rustgi (Massachusetts
General Hospital), Drs. Marshall Posner and Arthur Pardee (Dana Farber
Cancer Institute), and Dr. Ralph Kent (Forsyth Institute). |
FOOTNOTES |
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Received for publication August 31, 1999.
Revision received December 21, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 R. Todd, P.W. Hinds, K. Munger, A.K. Rustgi, O.G. Opitz, Y. Suliman, and D.T. Wong CELL CYCLE DYSREGULATION IN ORAL CANCER Critical Reviews in Oral Biology & Medicine, January 1, 2002; 13(1): 51 - 61. [Abstract] [Full Text] [PDF]
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S. Shintani, M. Mihara, N. Terakado, Y. Nakahara, T. Matsumura, Y. Kohno, H. Ohyama, J. McBride, R. Kent, R. Todd, et al. Reduction of p12DOC-1 Expression Is a Negative Prognostic Indicator in Patients with Surgically Resected Oral Squamous Cell Carcinoma Clin. Cancer Res., September 1, 2001; 7(9): 2776 - 2782. [Abstract] [Full Text] [PDF]
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M. T. Hartsough, S. E. Clare, M. Mair, A. G. Elkahloun, D. Sgroi, C. K. Osborne, G. Clark, and P. S. Steeg Elevation of Breast Carcinoma Nm23-H1 Metastasis Suppressor Gene Expression and Reduced Motility by DNA Methylation Inhibition Cancer Res., March 1, 2001; 61(5): 2320 - 2327. [Abstract] [Full Text]
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)
or an equivalent amount of thrombin-digested GST protein (pale circles)
was used as an input protein. DNA replication is measured as pmol of
32P-dCMP incorporated into a 25 µl reaction in 1 h.
Triplicates were determined at each data point. B) M13
ssDNA replication assays. p12DOC-1 significantly suppresses
DNA replication at all concentrations tested (P<0.05).
C) Analysis of DNA replication products by alkaline
agarose-gel electrophoresis. PC: positive control (lane 1). Lanes 2 and
3 are positive controls in the presence of the pol-