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Mol Cell Biol, June 1998, p. 3552-3562, Vol. 18, No. 6
The Institute of Physical and Chemical
Research,
Received 23 June 1997/Returned for modification 15 December
1997/Accepted 2 March 1998
DNA polymerase Chromosomal DNA replication in
eukaryotes is a highly regulated process that requires a large number
of replication factors, including distinct three types of DNA
polymerases, Mouse DNA polymerase In Saccharomyces cerevisiae, p68 is encoded by a single and
essential gene called POL12 (12, 32). Recently,
it was reported that p68 specifically plays an essential role at the
initial stage of DNA synthesis, before the hydroxyurea-sensitive step,
and that the subunit is phosphorylated and dephosphorylated in a cell
cycle-dependent manner (11, 12). Furthermore, formation of
the p180-p68 subcomplex appears to be a prerequisite for p68
phosphorylation (10). However, the physiological role of p68
phosphorylation has been refractory to investigation.
To explore the structure-function relationship of DNA polymerase
In this report, we describe a novel function for p68, identified by
using a cDNA expression system. Coexpression of p68 with p180 markedly
increased the protein level of p180, and as a result, exogenously
expressed DNA polymerase activity increased considerably. In addition,
coexpression of p68 with p180 markedly altered the subcellular
distributions of these subunits; coexpressed p68 and p180 were
exclusively colocalized in the nucleus, whereas p68 or p180 expressed
alone was localized in the cytoplasm. Using several mutants containing
deletion or substitution constructs, we found that mutual interaction
is essential for the p180-p68 heterodimer to translocate into the
nucleus. These results indicate that p68 plays a crucial and dual role
in the function of p180 by allowing both its protein synthesis and
translocation into the nucleus.
Materials.
All restriction enzymes and Klenow fragment were
purchased from Takara (Ohtsu, Japan); phenylmethylsulfonyl fluoride
(PMSF) was from Sigma; Pfu DNA polymerase was from
Stratagene; horseradish peroxidase-conjugated goat anti-rabbit and
anti-mouse immunoglobulin G (IgG) antibodies were from MBL (Nagoya,
Japan); fluorescein isothiocyanate (FITC)- or Texas red-conjugated goat
anti-rabbit or anti-mouse IgG antibodies were from Vector Inc.; fetal
bovine serum was from Nipro (Osaka, Japan); calf serum was from
HyClone. The expression vector pcDEB Construction of expression vector.
cDNAs for the four
subunits of the mouse DNA polymerase
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Second-Largest Subunit of the Mouse DNA
Polymerase
-Primase Complex Facilitates Both Production and Nuclear
Translocation of the Catalytic Subunit of DNA Polymerase
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ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-primase is a replication enzyme necessary for
DNA replication in all eukaryotes examined so far. Mouse DNA polymerase
is made up of four subunits, the largest of which is the catalytic
subunit with a molecular mass of 180 kDa (p180). This subunit exists as
a tight complex with the second-largest subunit (p68), whose
physiological role has remained unclear up until now. We set out to
characterize these subunits individually or in combination by using a
cDNA expression system in cultured mammalian cells. Coexpression of p68
markedly increased the protein level of p180, with the result that
ectopically generated DNA polymerase activity was dramatically
increased. Immunofluorescence analysis showed that while either singly
expressed p180 or p68 was localized in the cytoplasm, cotransfection of
both subunits resulted in colocalization in the nucleus. We identified
a putative nuclear localization signal for p180 (residues 1419 to 1437)
and found that interaction with p68 is essential for p180 to
translocate into the nucleus. These results indicate that association
of p180 with p68 is important for both protein synthesis of p180 and
translocation into the nucleus, implying that p68 plays a pivotal role
in the newly synthesized DNA polymerase
complex.
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INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
,
, and
. Studies on the in vitro replication of
simian virus 40 (SV40) DNA have made possible the functional
identification of several DNA replication factors, including DNA
polymerases (4, 17, 35). To date, DNA polymerase
-primase
has been considered to provide RNA-DNA primers for the initiation of
leading-strand synthesis and Okazaki fragment synthesis on the lagging
strand during SV40 DNA replication (36, 43, 44).
-primase complex was isolated from FM3A cells
as a protein complex consisting of four subunits. The apparent
molecular masses of these subunits based on their migration on sodium
dodecyl sulfate (SDS) polyacrylamide gels are 180, 68, 54, and 46 kDa
(39, 40). The two smaller subunits, p46 and p54, can be
dissociated from the other subunits by treatment with 50% ethylene
glycol (38) and possess DNA primase activity, as demonstrated by the synthesis of unit-length oligoribonucleotides (7, 27). The largest subunit, p180, is the catalytic subunit of DNA polymerase and exhibits intrinsic DNA polymerase activity, as
revealed in the baculovirus expression system (6). When the
human p180 subunit is expressed alone in insect cells, it displays
similarities to the holoenzyme such as an identical
Km for the primer-template and deoxynucleoside
triphosphate and similarities with respect to sensitivity to
inhibitors, thermostability, DNA synthetic processivity, and fidelity
(6). In contrast, the function of the second-largest
subunit, p68, which is tightly bound to p180, remains unclear. To date,
no enzymatic activity has been found for p68, and biochemical
approaches have failed to dissociate p68 in a native form from p180 in
mammalian cells. However, in all eukaryotes analyzed so far, the DNA
polymerase
-primase complex consists of four subunits, including
p68, and these subunits display significant homology in organisms
ranging from yeasts to humans (5, 8, 23). Therefore, p68 has
been considered to have a regulatory function conserved throughout evolution.
-primase, we previously constructed a cDNA overexpression system in
cultured mammalian cell lines and analyzed a temperature-sensitive mutant which has a defective DNA polymerase
-primase complex (19). Using this system, we identified the nuclear
localization signal (NLS) of DNA primase in the amino terminus of p54
and a piggyback binding transport mechanism of DNA primase
(24). The finding that DNA primase possesses an independent
NLS within its own sequence and that it can translocate into the
nucleus in the absence of DNA polymerase
prompted us to investigate
the subcellular distribution of the other subunits of DNA polymerase
, p180 and p68.
![]()
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
was a gift from Y. Nakabeppu
(25). DNA polymerase
-specific hybridoma SJK132-20 was
purchased from the American Type Culture Collection. Unless otherwise
noted, all other chemicals and reagents were obtained from Wako
Chemicals (Osaka, Japan).
-primase complex were
introduced into pcDEB
, which contains the SR
promoter
(41), to generate plasmids pSR
46, pSR
54, pSR
68, and
pSR
180 as described previously (19, 24).
1-97
(5'-ATTGTTTCTAGAATGGAGCTAATTGAA-3'), p68
1-157
(5'-CCGAGTTCTAGAATGTCCCAGAAATAC-3'), andp68
1-208
(5'-CTGGCAGCTATCTAGAGATGTTTCAGCA-3'). PCR-amplified products
were digested with XbaI and StuI and were used to
replace the original fragment in the p68 sequence.
557-600, a
stop codon and restriction enzyme sites were introduced by PCR using
the amino-terminal primer 5'-CAGGTGGTCTAGATGCAGTTC-3' and
carboxy-terminal primer 5'-CATGATATCCTATTCCCGAAGG-3'. The PCR products were then digested with XbaI and
EcoRV and subcloned into
XbaI-EcoRV-digested pSR
68.
1-191, the
initiation methionine and restriction enzyme sites were introduced by
PCR with primers 5'-CAGTAGATCTCATTGAGCTCGGTATCAATACA-3' and
5'-TCCAAGCTCCTCGAGGAACT-3'. The PCR product was digested
with XhoI and BglII and subcloned into
XhoI-BglII-digested pSR
180. Then the
SacI/SacI fragment of the subcloned plasmid was
removed to adjust the initiation methionine.
Deletion mutants p180
1417-1465 and p180
1442-1465 were constructed
by PCR with a common 5' primer (5'-AAGCCGGGACACCATTG-3') and
different 3' primers: p180
1417-1465
(5'-CTTCTTCAATAGATCTTACTCATGTTCAGT-3') and p180
1442-1465
(5'-CAGGACAAGATCTACTCTGCTATGTT-3'). PCR-amplified fragments
were digested with BglII and BamHI and were used
to replace the original fragment in pSR
180.
Mutants with substitutions were constructed by PCR using the overlap
extension technique (18). The following primer sets were
used to introduce mutations: p180KK(45,46)QQ,
5'-GGAAGGATCCAGAACAATCTGCGATTG-3' and
5'-CTTTGGTAAAATACTGAA-3'; p180KK(1421,1422)QQ,
5'-TATGAGCCACAGCAACAGGACCACATC-3' and
5'-AGACTTTCCTTCCTCTGA-3'; and p180RKVK(1434-1437)NNVN,
5'-TATGAGCCACAGCAACAGGACCACATC-3' and 5'-AGACTTTCCTTCCTCTGA-3'.
The double mutant p180KK(1421,1422)QQ RKVK(1434-1437)NNVN was
constructed by PCR using p180RKVK(1434-1437)NNVN as a template DNA
and primers for p180KK(1421,1422)QQ.
The p180-green fluorescent protein (GFP) fusion constructs were
produced by introducing the cDNA for GFP into the carboxyl terminus of
pSR
180. The 1.0-kb NheI-MluI fragment of
pEGFP-N1 (Clontech) was blunted by Klenow enzyme and subcloned into
MluI site of pSR
180, which was generated by replacement
of a 1.6-kb BamHI fragment of pSR
180 with PCR-amplified
products, using a 5' primer (5'-AAGCCGGGACACCATTG-3') and a
3' primer
(5'-GCAGATCTACGCGTAGTCTGGTACGTCGTACGGGTAGGACTTCCCAGCGT-3').
The identity of each construct was confirmed by double-strand DNA
sequencing using an ALF DNA sequencer (Pharmacia).
Cell culture and transfection. COS-1 and COS-7 cells, which were derived from the African green monkey kidney cell line CV-1 by transformation with an origin-defective SV40 isolate, were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a 5% CO2 incubator. Transfection was performed by electroporation as described previously (33). The level of protein expression was analyzed 48 h after transfection unless otherwise indicated.
To synchronize G2/M phase, cells at 4 h after transfection with p180-GFP and pSR
68 were incubated with 10 mM
thymidine for 22 h and then released with culture medium for
6 h. Cells enriched in S phase were then incubated in the presence
of nocodazole (45 ng/ml) for 8 h and allowed to traverse the cell
cycle.
Antibodies.
The anti-p68 and anti-p180 polyclonal antibodies
were generated in rabbits against glutathione S-transferase
(GST) fusion proteins expressed in Escherichia coli. The
2.2-kb SmaI fragment of p68 cDNA or the 1.5-kb
HindIII fragment of p180 was inserted into the
expression vector pGEX2T (Pharmacia). The GST-p180 fusion construct was
further excised and inserted into the vector pET22b (Novagen). These
expression vectors encoding the fusion proteins were introduced into
E. coli BL21 cells, and after induction by 0.5 mM
isopropyl-1-thio-
-D-galactopyranoside for 4 h, GST
fusion proteins were precipitated as inclusion bodies, purified, and used as antigens.
80°C. The
anti-p180 antiserum was directly used for Western blot analysis,
immunofluorescence analysis, and immunoprecipitation studies. The
anti-p54 and anti-p46 polyclonal antibodies were as described
previously (24).
Indirect immunofluorescence staining. Cells were grown in chamber slides (Nunc) coated with poly-L-lysine, washed with PBS, and fixed with 3.7% formaldehyde in PBS for 10 min on ice. Cells were then washed with PBS and permeabilized sequentially with 50, 75, and 95% ethanol on ice for 5 min each. The slides were then blocked with PBS containing 5% normal goat serum (blocking buffer) for 30 min at room temperature, incubated with anti-p68 (1.3 µg/ml in blocking buffer) or anti-p180 (diluted 1:3,000 in blocking buffer) antibody for 1 h at room temperature, and washed three times with PBS for 5 min each time. Cells were then incubated with FITC-conjugated secondary antibody for 1 h at room temperature, washed three times with PBS, and preserved in Vectorshield (Vector Inc.). DNA staining was performed by adding 1 µg of bisbenzimide (Hoechst 33258) per ml into the final PBS wash. The samples were examined under an Olympus PROVIS AX70 fluorescence microscope. For double-staining studies, monoclonal antibody SJK132-20 (ascitic fluid), antihemagglutinin (anti-HA) antibody, and Texas red-conjugated secondary antibody were used at a 1:400 dilution, 5 µg/ml, and 1.5 µg/ml, respectively.
For visualization of cells transfected with a GFP fusion construct, cells were fixed, permeabilized, and simultaneously stained with antibodies and Hoechst 33258 as described above.Preparation of cell extracts and Western blot analysis. After transfection, COS-1 cells were washed with PBS, scraped from plates in PBS, centrifuged for 5 min, and resuspended in either Laemmli sample buffer (21), for whole-cell extracts, or extraction buffer as described previously (40). The samples were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred electrophoretically onto 0.45-µm-pore-size polyvinylidene difluoride membranes (Millipore). After incubation of the membranes with either anti-p46 (0.13 µg/ml), anti-p54 (0.3 µg/ml), anti-p68 (0.3 µg/ml), or anti-p180 (1:3,000) antibodies in TBS (Tris-buffered saline; 50 mM Tris-HCl [pH 7.5], 150 mM NaCl) containing 5% (wt/vol) dried milk for 1 h at room temperature, the membranes were washed three times with TBS containing 0.05% Tween 20. The membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG in TBS containing 5% dried milk and washed again. Detection of the protein bands was performed with an enhanced chemiluminescence reagent (SuperSignal; Pierce) as instructed by the manufacturer. Kaleidoscope prestained standards (Bio-Rad) were used as molecular weight standards.
Northern blot analysis.
Total RNA was isolated by using
Isogen (Nippongene) from COS-1 cells which were transfected with
expression plasmids. Ten micrograms of total RNA per lane was subjected
to gel electrophoresis using a 1% agarose-6% formaldehyde gel,
stained with SYBR green II (Molecular Probes), and transferred to a
nylon membrane as described previously (23). The probe was
generated by random priming with a 1.5-kb HindIII
fragment of pSR
180.
Pulse-chase experiment. Transfected COS-1 cells were divided into two sets; half of the cells were used for Northern blot analysis, and the other half were used for pulse-chase experiments. Cells at 24 h after transfection were incubated for 1 h in methionine-cysteine-deficient Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% dialyzed calf serum and then labeled with 200 µCi of Tran35S-label (ICN Pharmaceuticals, Inc.) per ml for either 10 or 30 min. The cells were subsequently washed in complete medium, chased with complete medium for indicated times, and lysed at 4°C in buffer A (50 mM Tris-HCl [pH 8.0]; 300 mM KCl; 10% glycerol; 1% Nonidet P-40; 1 mM PMSF; 0.2 µg of aprotinin, 0.2 µg of leupeptin, 0.1 µg of antipain, and 0.1 µg of pepstatin A per ml; 1 mM EDTA). In the case of pulse-labeling for 10 min, a chased medium contained a large excess of unlabeled methionine (300 µg/ml). After centrifugation, the protein concentrations of the supernatants were determined by the Bradford assay (Bio-Rad). Thirty micrograms of the supernatant was immunoprecipitated with 1 µl of anti-p180 polyclonal antiserum which had been preadsorbed to protein A-Sepharose CL-4B (Pharmacia) at 4°C for 6 h. After being washed with buffer A, precipitates were dissolved with 2× Laemmli sample buffer and then subjected to SDS-PAGE and fluorography using En3Hance (Dupont NEN).
Coimmunoprecipitation analysis. Fifty-microgram aliquots of COS-1 cell extracts were immunoprecipitated with 1 µl of anti-p180 monoclonal antibody SJK132-20 (ascitic fluid) which had been preadsorbed to protein G-Sepharose (Pharmacia) for 4 h at 4°C in 200 µl of NET buffer (50 mM Tris-HCl [pH 7.5]; 150 mM NaCl; 0.1% Nonidet P-40; 1 mM EDTA; 0.25% gelatin; 1 mM PMSF; 0.2 µg of aprotinin, 0.2 µg of leupeptin, 0.1 µg of antipain, and 0.1 µg of pepstatin A per ml). After being washed with NET buffer, precipitates were dissolved with 30 µl of 2× Laemmli sample buffer and then subjected to SDS-PAGE and Western blot analysis using anti-p180 and anti-p68 polyclonal antibodies.
DNA polymerase assay. The DNA polymerase assay was carried out as described previously (38). Briefly, 5-µg aliquots of proteins were incubated with DNase I-activated calf thymus DNA (0.5 mg/ml) in a buffer containing 20 mM Tris-HCl (pH 8.0), 10% glycerol, 60 mM KCl, 5 mM MgCl2, 3.3 mM 2-mercaptoethanol, 0.2 mg of bovine serum albumin per ml, 100 µM each dATP, dCTP, and dGTP, and 50 µM [3H]dTTP (0.1 Ci/mmol). Incorporated [3H]dTMP was measured by using a Whatman DE81 paper disc as described previously (37).
Glycerol density gradient sedimentation.
COS-7 cells were
transfected with either pcDEB
(16 µg), pSR
68 (16 µg),
pSR
180 (16 µg), or pSR
180 (10 µg) and pSR
68 (6 µg),
incubated for 72 h, and then harvested as described above. Extracts containing 250 µg of protein in 100 µl were layered onto a
2-ml linear 15 to 35% glycerol gradient in a buffer containing 25 mM
potassium phosphate (pH 7.5), 300 mM KCl, 1 mM MgCl2, and 0.5% Triton X-100. Centrifugation was for 16 h at 55,000 rpm at 4°C (Beckman TLS-55), and the gradient was collected from the top and
divided into 29 fractions. DNA polymerase activity was assayed, and
Western blot analysis of each fraction was performed.
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RESULTS |
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Expression of mouse p68 and p180 subunits in COS-1 cells.
To
explore the functions of the four subunits of the DNA polymerase
-primase complex, we developed a cDNA expression system using
cultured mammalian cells as hosts. Various combinations of cDNAs for
the four subunits of the mouse complex were transiently transfected
into monkey COS-1 cells, and the cells were cultured for 48 h.
Proteins were extracted from the transfected cells, and DNA polymerase
activity in the whole-cell extract was determined by using activated
calf thymus DNA as a template. When pSR
180 alone was transfected,
DNA polymerase activity increased moderately compared with the control
extract in which only vector plasmid had been transfected (Fig.
1A). When pSR
180 was cotransfected with pSR
46 or pSR
54, a similar moderate increase in DNA
polymerase activity was observed. However, cotransfection of pSR
180
with pSR
68 resulted in a marked increase in DNA polymerase activity. The effect of pSR
68 on the increase of DNA polymerase activity was
dose dependent, as shown in Fig. 1A. Since there have been no reports
about any contribution of p68 to DNA polymerase activity, we decided to
further investigate this novel effect of p68. To determine the protein
levels of exogenously expressed subunits, we prepared polyclonal
antibodies against mouse p180 and p68 expressed in bacteria. The
specificity of each of these antibodies was demonstrated by Western
blot analysis of purified DNA polymerase subunits from mouse FM3A cells
(data not shown) and with exogenously overexpressed mouse DNA
polymerase subunits (Fig. 1B). The antiserum against mouse p180
scarcely cross-reacted with monkey p180. This was also the case for the
antibody against p68, which cross-reacted even more weakly with the
monkey subunit, as shown by Western blot analysis and
immunofluorescence analysis (described below). The anti-p180
antibody reacted with a 180-kDa protein, corresponding to
full-length p180, as well as with a 160-kDa protein which corresponds to the truncated p180 molecule. This truncated molecule was
characterized previously (16, 40). Therefore, these
antibodies were considered to be suitable for the detection of
ectopically expressed mouse DNA polymerase
-primase subunits in
monkey COS-1 cells. The anti-p54 and anti-p46 antibodies have been
characterized previously (24). These antibodies enabled us
to estimate the protein levels of DNA polymerase
-primase subunits
in transfected COS-1 cells. When p180 was cotransfected with p68, an
intense signal for p180 was detected (Fig. 1B). The intensity of the
p180 signal correlated with that of p68, showing that the effect of p68
was again dose dependent. Truncated 160-kDa protein was generated in
proportion to the level of full-length p180. These results suggested
that coexpression of p68 with p180 increased the protein level of p180, resulting in a marked increase in exogenous DNA polymerase activity. These findings were reproducible in other cell lines, including COS-7
and CV-1 cells (data not shown).
|
-primase tetrameric complex in COS-7 cells was shown in
the gradient containing the control extract in which only vector
plasmid had been transfected. Heterotetrameric DNA polymerase
sedimented at fractions 23 to 25, slightly faster than the heterodimer.
The sedimentation coefficient of coexpressed p180 and p68 was 7.4S,
which is close to the value of 7.1S observed for heterodimeric DNA
polymerase purified from the tetrameric complex of FM3A cells
(38). These results showed that coexpressed p180 and p68
were assembled into a heterodimeric complex, while singly transfected
p180 existed as a monomer. An additional signal for p68 in the presence
of p180 was found around fractions 13 to 17. Singly expressed p68
sedimented at a similar position, indicating that coexpressed p68
occurred in two forms, one as a heterodimeric complex with p180 and the
other as free p68 whose sedimentation coefficient was 4.4S.
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Posttranscriptional control of the p180 protein level by coexpression of p68. To determine whether the accumulation of p180 caused by cotransfection of p68 occurs at the transcriptional level, we analyzed the mRNA level of the p180 gene in transfected cells. Transiently transfected COS-1 cells were divided into two parts; one half was harvested for the isolation of total RNA, and the other half was used for Western blot analysis and the pulse-chase experiment. Northern blot analysis revealed that the levels of p180 mRNA transcribed in p180-transfected cells were equivalent in the presence or absence of p68 coexpression (Fig. 3B). When the levels were normalized by comparison with 28S rRNA stained with SYBR green II, the mRNA levels of p180 were found to be 1 and 0.8 in the presence and absence of p68, respectively (Fig. 3A and B). In sharp contrast, the level of p180 protein as measured by Western blot analysis was 10 times higher in cells coexpressing both p180 and p68 than in cells expressing p180 alone (Fig. 3C). Since the marked increase of p180 levels could not be explained by the slight difference in the mRNA level, we conclude that the effect of p68 on the p180 protein level does not occur at the transcriptional level but involves some later step of either cotranslational or posttranslational control.
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Subcellular distribution of p180 and p68 in transiently transfected COS-1 cells. The finding that coexpression of p68 and p180 markedly increased the protein level of p180 prompted us to examine the subcellular distribution of transiently transfected p68 and p180 by indirect immunofluorescence analysis. The affinity-purified anti-p68 antibody reacted with endogenous p68 in mouse NIH 3T3 cells and showed bright nuclear staining (data not shown). However, monkey p68 in COS-1 cells could be scarcely detected and was observed only after longer exposures (not shown). The anti-p180 monoclonal antibody SJK132-20 (1) reacted with monkey p180 as well as with mouse p180. The transiently transfected COS-1 cells were fixed 48 h after transfection, permeabilized, and incubated with anti-p68 polyclonal antibody or SJK132-20. In our transfection experiments, ectopic proteins were overexpressed in 10 to 20% of the cells. Unexpectedly, we found that p68 and p180 showed diffused cytoplasmic staining when each subunit was transfected individually (Fig. 5A, a and c). We scored 408 cells which displayed overproduction of ectopic p180 proteins. Among these cells, 403 cells showed cytoplasmic localization. In the case of p68, 329 of 441 transfected cells showed cytoplasmic localization. In sharp contrast, coexpression of both subunits markedly changed their subcellular distributions. As shown in Fig. 5A, e and f, coexpressed p68 and p180 were localized predominantly in the nucleus: 389 of 408 cotransfected cells showed nuclear localization. These findings were reproduced with HA-tagged p180 and anti-HA monoclonal antibody used in a double-staining experiment and in other cell lines, including COS-7, CV-1, and NIH 3T3 (data not shown). These results indicate that nuclear translocation of p180 and p68 depends on their mutual interaction.
|
. Thus, transiently overexpressed
subunits were translocated into the nucleus in the same manner as the
endogenous polymerase
.
Identification of the domains of p68 implicated in the increase of
p180 and nuclear translocation of the p180-p68 heterodimer.
To
date, the amino acid sequence of the second-largest subunit of DNA
polymerase
-primase complex has been identified in S. cerevisiae, Drosophila, mouse, and human cells.
Alignment of the p68 sequences from these species reveals several
features of the structure of p68. First, whereas the amino-terminal
region is diverse and exhibits little homology, conserved amino acid sequences are distributed from the center to the carboxy-terminal region. Second, clusters of -Ser/Thr-Pro- motifs, which are considered to be putative phosphorylation sites for the cdc2 kinase family, are
located in the amino-terminal region, as shown in Fig.
6A. However, the precise roles of these
regions remain unclear. To confirm the role of p68 in regulating p180
protein levels and the nuclear translocation of the heterodimer, in
addition to elucidating the structure-function relationship of p68, we
constructed several deletion mutants of p68 (Fig. 6A). These mutant
constructs were cotransfected into COS-1 cells with pSR
180, and the
resultant mutant proteins were characterized by immunofluorescence
analysis (Fig. 6B), DNA polymerase activity (Fig. 6C), and Western blot analysis (Fig. 6D). The amino-terminal deletion mutant p68
1-97 was
almost identical to the wild type in terms of the increase in p180
protein level, DNA polymerase activity, and translocation into the
nucleus. In contrast, the amino-terminal deletion mutant p68
1-208
and carboxy-terminal deletion mutant p68
557-600 lost the ability to
increase the protein level of p180 and were localized in the cytoplasm.
The amino-terminal deletion mutant p68
1-157, which lacks the
clusters of -Ser/Thr-Pro- motifs, was found to be expressed in the
cytoplasm even though it increased the p180 protein level more than the
mutant p68
1-208. Taken together, these results indicate that
p68-promoted increases in the p180 protein level and translocation of
the heterodimer into the nucleus are supported by a broad region of
p68, stretching from the center to the carboxyl terminus, which is
highly conserved among eukaryotes.
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Identification of the domain of p180 essential for nuclear
translocation by using deletion and point mutants.
Our finding
that p68-promoted overproduction of p180 led to colocalization of both
subunits in the nucleus prompted us to identify the domain of p180
essential for entry into the nucleus. Several motifs comprised of
clusters of basic residues for the targeting of proteins to the
nucleus, such as -KKKRK- for SV40 large T antigen (20, 22)
and KR---------KKKK for nucleoplasmin (30), have been
identified. A search for sequences in p180 suggested that an authentic
NLS might be located at residues 31 to 46 (23). To determine
whether this putative NLS really functions as the NLS, we constructed a
set of deletion mutants (Fig.
7A).
Expression of each mutant in COS-1 cells was detected by Western blot
analysis as shown in Fig. 7B. We found that all mutants of p180 singly transfected were localized in the cytoplasm in the absence of p68 (data
not shown). However, the cells expressing p180
1-191 or
p180
1442-1465, in which the amino terminus or carboxyl terminus, respectively, was truncated, showed intense nuclear localization similar to that seen with the wild-type p180 in the presence of coexpressed p68 (Fig. 7D, h and q). Substitution mutant
p180KK(45,46)QQ, which lacked the basic residues of the putative NLS,
was also exclusively localized in the nucleus in the presence of p68
(Fig. 7D, e). In contrast, cells expressing p180
1417-1465 exhibited diffuse cytoplasmic staining for both p68 and p180 (Fig. 7D, s and t).
These findings suggest that (i) nuclear translocation of p180 is
absolutely dependent on an interaction with coexpressed p68 and (ii)
the amino-terminal region and a putative NLS found previously are not
necessary for translocation of p180 into the nucleus.
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DISCUSSION |
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Since baculovirus-derived recombinant p180 protein has been shown
to possess intrinsic DNA polymerase activity not different from that of
the four-subunit holoenzyme (6), the role of p68 has been an
enigma. We used a cDNA expression system in cultured mammalian cells to
study the influence of the noncatalytic subunit p68 on the subunit
assembly and activity of DNA polymerase
. Our finding that p68 plays
a role in the maintenance of the p180 concentration instead of in
enzymatic regulation provides insight not only into the function of the
p68 subunit but also into the mechanism of biosynthesis of this
multisubunit complex.
The mechanism through which p68 increases the protein level of p180 is
still speculative. We first found that coexpression of p180 with p68
markedly increased DNA polymerase activity in COS-1 cells. However,
using Northern blot analyses and pulse-labeling experiments with
Western blot analyses, we observed that the mRNA level and the turnover
rate of recombinant p180 were not significantly affected by the
presence of p68. In contrast, newly synthesized p180 was rapidly
assembled into a heterodimeric complex with p68, and this resulted in
the generation of much more p180 during the pulse-labeling period.
Therefore, the p68-promoted increase in the p180 protein level probably
occurs not at the transcriptional or posttranslational level but at the
cotranslational level. In eukaryotic cells, although initiation control
of transcription is the major step in the regulation of most genes, a
variety of controls can intervene at the posttranscriptional level.
Among these controls, translational control enables a cell to adjust the concentration of a protein rapidly and reversibly without rapid
turnover of its mRNA. Such a mechanism has been reported for
proto-oncogenes (28), ferritin (15), and many
proteins during fertilization (46). In addition, pausing of
translation elongation has been reported recently for eukaryotic cells.
It was shown by van Wijk and Eichacker that translation elongation of
chlorophyll-binding photosystem II center protein D1 was controlled strongly by light, and pausing resulted in the accumulation of translational intermediates in the dark (42). Young and
Andrews reported that the
subunit of the signal recognition
particle receptor has a strong pause site on its mRNA and that
translation pausing facilitates the cotranslational membrane binding of
this subunit (47). These are novel cases of cotranslational
pausing, but such a regulatory mechanism may occur widely in eukaryotic cells (47). In the light of these data, it is tempting to
speculate that p180 mRNA possesses a putative pausing site and that
interaction with p68 facilitates progression of the translational
apparatus through the pause site, resulting in the accumulation of a
mature heterodimer complex containing p180 and p68.
The characterization of mammalian p68 was previously reported by
Collins et al., who used bacterially expressed human p68, which was
shown to serve as a molecular tether between DNA polymerase
and
SV40 large T antigen in the SV40 replication system (5). On
the other hand, mouse p68 expressed in the baculovirus system did not
show any significant effect on the polyomavirus replication system
(3). In both studies, the effect of p68 on production of
p180 was not considered. In addition, the subcellular distribution of
p68 has never been studied in mammalian cells. Collins et al. (5) demonstrated that the domain of p68 for interaction with the SV40 large T antigen was located in the amino-terminal region from
amino acids 1 to 240. From these findings taken together with our
results, the following domain structure of p68 emerges: the
amino-terminal region of p68 (amino acids 1 to 240) serves as a binding
site for other replicative proteins, whereas the center and the
carboxy-terminal regions (157 to 600) are essential for interaction
with p180. Interestingly, putative phosphorylation sites for cdc2
kinase are located in the amino-terminal region (115 to 157), implying
that phosphorylation(s) of p68 may affect its interaction with other
proteins, rather than being involved in intrinsic DNA polymerase
activity or the biosynthetic pathway.
Our results strongly suggest that translocation of p68 and p180 into the nucleus is dependent on their mutual interaction. We tried to identify the essential domain required for nuclear translocation by deletion and substitution mutants and found that basic residues along with zinc finger motifs of p180 were critical for nuclear translocation. Although these basic charged residues were predicted by Dingwall and Laskey (9) to form a typical NLS, the function of the NLS is strictly dependent on interaction with p68. In general, an NLS is considered to consist of comparatively short sequences rich in basic amino acid residues, and such short sequences have been shown to be necessary and sufficient for translocation of proteins into the nucleus (9, 13, 14, 31). However, our result showing that the NLS of p180 is dependent on interaction with p68 is an exception to this rule. Since none of the constructs of p180 were able to enter the nucleus in the absence of p68, and all of the mutants of p68 were retained in the cytoplasm without p180 (data not shown), the NLS of p180 may not be able by itself to present the requisite molecular surface for nuclear entry. For this to occur, binding to p68, and resultant conformational changes in the carboxyl terminus of p180, may be necessary to allow the cryptic NLS to appear on the molecular surface and bind to the NLS receptors. However, these results do not eliminate the possibility that p68 has a cryptic NLS of its own and that a conformational change in p68 on interaction with p180 activates this cryptic NLS. There is another possibility: that the p180-p68 heterodimer exhibits a new class of NLS which may be different from the authentic NLS composed of basic charged residues, such as the M9 domain of hnRNPA1 (14). To confirm that the NLS of p180 is localized in the carboxy-terminal domain and dependent on an interaction with p68 for activation, it will be necessary to examine the molecular conformation of the carboxyl terminus of p180 in the presence of p68 and binding of p180 to an NLS receptor such as mPendulin, mSRPI, or Rch1 (14, 29) in the presence of p68.
It was reported that in S. cerevisiae, a mutant strain
overexpressing p68 contained this subunit in the nucleus
(12). In addition, the NLS in p180 of
Schizosaccharomyces pombe has recently been shown to be
located in the amino-terminal region (2). However, compared
with human and mouse p180, the corresponding region of the putative NLS
of S. pombe p180 displays few conserved amino acid residues.
Therefore, the nuclear translocation process of DNA polymerase
may
be different between mammals and other eukaryotes, including S. cerevisiae, S. pombe, and Drosophila melanogaster.
In conclusion, we have demonstrated that the noncatalytic subunit p68
plays a crucial role in the biosynthesis of catalytic subunit p180.
Since characterization of the in vivo function of p68 was hampered by
the limited amount of DNA polymerase
in cells, the overexpression
system provided a useful tool to understand the in vivo function of the
four subunits of DNA polymerase
-primase. Continuation of this
approach should allow a detailed analysis of the roles of the different
subunits of DNA polymerase
in DNA replication in vivo and the
nature of the protein-protein interactions between the subunits or with
other replicative factors involved in the control of cell
proliferation.
| |
ACKNOWLEDGMENTS |
|---|
We thank Yusaku Nakabeppu for providing the pcDEB
expression
vector and Masako Izumi, Chiaki Maruyama, Aiji Sakamoto, and Kaoru
Sugasawa for helpful discussions.
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan and a special grant for promotion of research from the Institute of Physical and Chemical Research (RIKEN) and the Biodesign Research Program of RIKEN. T.M. is a special postdoctoral researcher of RIKEN.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565, Japan. Phone: 81-6-879-7975. Fax: 81-6-877-9382. E-mail: fhanaoka{at}imcb.osaka-u.ac.jp.
| |
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