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INTRODUCTION |
Polyomavirus DNA replication has been studied extensively because
of the ease with which viruses of this family, which include SV40 and
mouse polyoma virus (PyV),1
can be grown in cell culture and moreover because of the availability of an in vitro replication system, which allows detailed
investigation of the individual factors that play a role in the
replication process (1, 2). Polyomavirus DNA replication has been found to be largely dependent upon factors involved in replication of the
host DNA but does contribute one essential trans-acting
factor known as large T antigen (TAg) to the replication complex. TAg is responsible for recognition of the viral origin of replication, at
which it forms a double hexamer (3). In the presence of the
single-stranded DNA-binding protein, RPA (replication protein A), and
topoisomerase I, TAg proceeds to unwind the origin DNA and recruits the
DNA polymerase 
-primase heterotetramer, which then initiates
bidirectional DNA synthesis (4-11). DNA polymerase -primase
initiates DNA replication through the action of its smallest subunit,
p48, which synthesizes RNA primers that are subsequently elongated by
the large subunit, p180 (12-15). The bulk of DNA synthesis is,
however, carried out by a more processive enzyme complex consisting of
DNA polymerase 
and its processivity factor, PCNA (16-18).
The transition between DNA synthesis by DNA polymerases and is
mediated by the PCNA loading factor replication factor C (16, 19).
Throughout the viral replication cycle, TAg double hexamers are thought
to act as the replicative helicase (20). For a more extensive account
of the DNA replication process and its participating factors see Refs.
1, 2, and 21-23.
SV40 and PyV are very similar with regard to DNA replication; their
respective TAgs are 36% identical, have largely the same biochemical
activities, and recognize very similar pentanucleotide motifs at their
replication origins, which also exhibit great similarity (21).
Nevertheless, both viruses show clear differences with respect to the
host cells in which DNA replication can be initiated successfully.
Indeed, this appears to be the basis for the strict species specificity
observed with SV40 and PyV. The former lytically infects only primate
cells, and the latter infects only murine cells (24). Whether this is a
common factor in determination of species specificity of polyomaviruses
in general remains to be seen, but the similarity in replication
mechanisms throughout this virus family would make it a likely
possibility. Species specificity of replication can be reproduced in
cell-free systems, and this led to the demonstration that DNA
polymerase -primase is the host factor that determines the species
specificity of viral initiation of DNA replication of both SV40 and PyV
(25-27). However, studies with hybrid DNA polymerase -primase
complexes showed that the subunit requirement differs between these two viruses, PyV specifically needing p48 to be of murine origin and SV40
requiring a human p180 protein (28, 29). In the case of PyV, further
resolution was achieved with studies of DNA polymerase -primase
complexes containing chimeric p48 subunits consisting of various
combinations of human and murine sequences. It was concluded that a
lesser conserved stretch of amino acids (residues 257-288) from the
murine subunit was necessary for successful PyV DNA replication, and it
is likely that further study of the function of this region will shed
light on the mechanism involved in this phenomenon (30, 31). In this
study we applied a similar approach in an attempt to determine which
domains of human p180 are essential for initiation of SV40 DNA replication.
Initiation of DNA replication requires the interaction between various
proteins at the origin of replication. Interactions between TAg and
both RPA and DNA polymerase -primase have been shown to be
important, and SV40 TAg is known to bind the p180, p68, p58, and p48
subunits of DNA polymerase -primase independently (4, 32-34). For
human (H) p180, the site of interaction was mapped to an N-terminal
stretch spanning residues 195-313, and it was shown that this
interaction is required for the establishment of a functional
initiation complex (35). This led us to investigate whether this
interaction plays a part in the species specificity of SV40 DNA
replication. To this end we constructed various chimeric p180 subunits
that consist of regions derived from both the mouse and the human
proteins. Using the baculovirus system these mutants were expressed in
complex with the other human DNA polymerase -primase subunits, and
their activities were investigated in both a cell-free replication
assay and in an initiation assay consisting of purified proteins only.
In addition, we tested our mutant complexes for their ability to be
stimulated by SV40 TAg in a system that assays coupled primer and DNA
synthesis on single-stranded DNA (ssDNA) in the presence of RPA (33,
34, 52). We show that the N-terminal 488 amino acids of human p180,
comprising the TAg-binding domain, are not responsible for the species
specificity of DNA replication nor for efficient stimulation of DNA
polymerase activity by TAg on ssDNA. These results are supported by
data obtained with surface plasmon resonance (BIAcore), which shows that SV40 TAg shows little difference in affinity for DNA polymerase -primase from either human or murine origin.
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MATERIALS AND METHODS |
Construction of Baculoviruses Expressing Chimeric p180
Proteins--
For the construction of recombinant baculoviruses, we
used the Bac-to-Bac® system (Invitrogen) according to the
manufacturer's protocol. Briefly, p180 wild type and mutants were
cloned into the plasmid pFastBac1®, and this was introduced into the
Escherichia coli strain DH10Bac®. This strain harbors
baculovirus DNA as a single copy F episome (Bacmid) as well as a
plasmid encoding transposase. The insert in pFastBac1® is located on a
Tn7 transposable element that can recombine in vivo with an
acceptor site on the Bacmid, resulting in recombinant baculovirus DNA
for which there is a selection procedure. This DNA is recovered from
the E. coli strain and transfected into Sf9 insect
cells, where it gives rise to recombinant baculoviruses expressing the
desired protein.
pFB/Hp180 (36) was constructed by cloning an
EcoRI-XbaI Hp180 cDNA fragment from
pUC19/Hp180 into pFastBac1® digested with EcoRI-XbaI at the polylinker. pFB/Mp180 was
constructed by cloning an Mp180 cDNA
EcoRI-PstI fragment from pVL1393/Mp180 (37) into pFastBac1® digested with EcoRI-PstI. pFB/M257H
and pFB/H257M were made by digesting pFB/Hp180 and pFB/Mp180 at the
NcoI sites, located at the ATG start codon and internally,
followed by reciprocal exchange and ligation of the resulting
fragments. The orientation of the NcoI fragments in the
resulting clones was checked by restriction analysis. pFB/H488M was
created by digesting pFB/Hp180 and pFB/Mp180 with both EcoRI
and PpuMI and ligating the 1513-bp fragment containing the
5'-end of the Hp180 gene onto the 3'-end of Mp180 in pFastBac. The
reciprocal exchange resulting in pFB/M488H was made by first digesting
pFB/Mp180 with EcoRI, followed by partial digestion with
PpuMI and exchanging the resulting 1513-bp fragment with that of pFB/Hp180 fully digested with EcoRI and
PpuMI.
To create pFB/H671M, the PflMI site at position 2011 in the
Hp180 open reading frame (CCAAAGCTTGG) was mutated by site-directed mutagenesis to render it compatible with the corresponding site in
Mp180 (CCAAAACTTGG). The overlap extension PCR method (38) was used to
introduce the mutation, which is silent at the protein level. Modified
pFB/Hp180 and pFB/Mp180 were then both fully digested with
PflMI (both the Hp180 and Mp180 open reading frames have additional PflMI sites at positions 809 and 3688, respectively, which are mutually incompatible and incompatible with the
mutated site). pFB/H671M was produced by ligating the four resulting
fragments. pFB/M1141H and pFB/H1141M were constructed by exchange of
BamHI fragments from pFB/Hp180 and pFB/Mp180 using the
internal site and the one located in the polylinker beyond the stop
codon. pFB/M1395H and pFB/H1395M were produced by digesting pFB/Hp180
and pFB/Mp180 with EcoRI and AgeI, located before
the start codon and internally at position 4196/4184, respectively, and
exchanging the resulting fragments.
All of the constructs were transferred to baculovirus using the
Bac-to-Bac® (Invitrogen) system, amplified in Sf9 insect cells, and then used to infect High Five insect cells (ITC Biotechnology GmbH,
Heidelberg) to test for expression of the desired proteins by Western blotting.
Proteins--
SV40 TAg and the DNA polymerase -primase
complex (p180-p68-p58-p48) were purified from baculovirus-infected
insect cells as described (28, 37, 39, 40). The complexes containing hybrid p180 subunits were purified using monoclonal antibody SJK237-71 if they contained human sequence between amino acids 257 and 488, and
otherwise monoclonal antibody SJK287-38 was used. During purification of hybrid complexes containing murine p68, the wash with 150 mM KCl was omitted.
RPA was bacterially expressed and purified as outlined before (41, 42).
Human topoisomerase I expressed in insect cells and purified as
described by Søe et al. (43) was a generous gift of K. Søe
(Institut für Molekulare Biotechnologie, Jena, Germany). The
monoclonal antibodies SJK237-71 and SJK287-38 (44), specific for DNA
polymerase -primase, were purified by affinity chromatography
(45).
Protein Manipulations--
Protein concentration was determined
according to Bradford (46) using a commercial reagent with bovine serum
albumin as a standard. SDS gel electrophoresis was carried out as
described (47) with 10-kDa ladders (Invitrogen) as molecular mass markers.
Preparation of S100 Extracts and Replication of SV40 in
Vitro--
S100 extracts were prepared from logarithmically growing
FM3A cells as described previously (29, 37). The cells were harvested by centrifugation and then washed twice with phosphate-buffered saline
and once with hypotonic buffer. The cells were resuspended in hypotonic
buffer, incubated for 10 min. on ice, and broken by 12 strokes in a
Dounce homogenizer. The extracts were centrifuged at 4 °C and
11,000 × g. The supernatant was then adjusted to 100 mM NaCl and clarified by a second centrifugation at
100,000 × g (S100 extract).
The replication of SV40 DNA in vitro was performed as
described previously (28, 37, 39). Briefly, the assay contained 0.6 µg of SV40 TAg, 250 ng of pUC-HS DNA (SV40 origin DNA) (27), and 200 µg S100 in 30 mM HEPES/NaOH, pH 7.8, 1 mM
dithiothreitol, 7 mM magnesium acetate, 1 mM
EGTA, pH 7.8, 4 mM ATP, 0.3 mM CTP, GTP, and
UTP, 0.1 mM dATP and dGTP, 0.05 mM dCTP and
dTTP, 40 mM creatine phosphate, 80 µg/ml creatine kinase,
and 5 µCi each of [-32P]dCTP and
[-32P]dTTP (3000 Ci/mmol; Amersham Biosciences). DNA
polymerase -primase was added as indicated. The incorporation of
radioactive dNMP was measured by acid precipitation of DNA and
scintillation counting. The total radioactivity was measured after
spotting 5 µl of a 200-fold dilution of the replication assay onto
GF52 filters (Schleicher & Schüll).
Initiation of Replication on SV40 DNA--
Initiation reactions
were performed essentially as described previously (12, 28, 39).
Briefly, the SV40 initiation assay (40 µl) was assembled on ice and
contained 0.25 µg of pUC-HS DNA (SV40 origin DNA), 0.6 µg of SV40 T
antigen, and 0.5 µg of RPA in 30 mM HEPES-KOH, pH 7.8, 7 mM magnesium acetate, 1 mM EGTA, 1 mM dithiothreitol, 0.2 mM UTP, 0.2 mM GTP, 0.01 mM CTP, 4 mM ATP, 40 mM creatine phosphate, 1 µg of creatine kinase, 0.3 µg of topoisomerase I, 0.25 mg/ml heat-treated bovine serum albumin, and
20 µCi of [-32P]CTP (3000 Ci/mmol; PerkinElmer Life
Sciences). Recombinant DNA polymerase -primase was added as
indicated in the figure legends. After incubation for 2 h at
37 °C, 
th of the reaction mixture was used to estimate the
amount of incorporated nucleotides by spotting it onto DE81 paper (48).
The reaction products were precipitated with 0.8 M LiCl, 10 µg of sonicated salmon sperm DNA (Sigma), 10 mM
MgCl2, and 120 µl of ethanol for 1 h on dry ice,
washed twice with 75% ethanol with water, dried, redissolved in 45%
formamide, 5 mM EDTA, 0.05% xylene cyanol FF, 0.05%
bromphenol blue at 65 °C for 30 min, heated for 3 min at 95 °C,
and electrophoresed in denaturing 20% polyacrylamide gels for 3-4 h
at 600 V as described (27, 28). The reaction products were visualized
by autoradiography.
DNA Synthesis on 
X174 ssDNA with RPA and TAg--
DNA
synthesis was carried out according to Kautz et al. (30).
Briefly, in an assay mixture (40 µl) containing 30 mM
HEPES-KOH, pH 7.8, 7 mM MgAc, 0.1 mM EGTA, 1 mM dithiothreitol, 0.25 mg/ml bovine serum albumin, 80 µg/ml creatine kinase, 40 mM creatine phosphate, 4 mM ATP, 0.2 mM each CTP, GTP and UTP, 0.1 mM each dATP, dGTP, and dTTP, 0.002 mM dCTP,
0.5 µl of [-32P]dCTP (specific activity, 3000 Ci/mmol; 10 µCi/µl; Amersham Biosciences), and 250 ng (0.76 nmol of
nucleotides) of X174 ssDNA. Where appropriate this mixture was
preincubated with 1 µg of SV40 TAg for 2 min on ice and then with
0.3-1.2 µg RPA for a further 5 min on ice. The reaction was started
by the addition of 100-400 ng (depending on specific activity) DNA
polymerase -primase. After incubation for 90 min at 37 °C, 10 µl of the reaction mixture was spotted onto GF-52 filters and
precipitated in 10% trichloroacetic acid. The amount of DNA synthesis
was measured by liquid scintillation counting.
Biomolecular Interaction Analysis--
Interaction analysis was
performed using the BIAcore 2000 apparatus from BIAcore AB (Freiburg,
Germany) as described previously (34, 49). Sensor chips CM5, surfactant
P20, and the amine coupling kit were purchased from BIAcore AB. The
antibodies were immobilized by amine coupling according to the
supplier's protocol. For a final ligand immobilization yield of 1,000 relative resonance units, about 1800 resonance units of the antibody
was initially attached to the flow cell surface. The
anti-DNA-polymerase monoclonal antibody 2CT25 (50 µg/ml) was
loaded at a flow rate of 5 µl/min in 0.03 M sodium
acetate buffer, pH 5.0. The ligands, four subunit DNA polymerase
-primases, and the analyte, SV40 TAg, were microdialyzed against the
binding buffer 20 mM HEPES-KOH, pH 7.5, containing 100 mM NaCl, and 0.005% P20 before use. The ligands were
loaded to a 8-100 µg/ml concentration and cross-linked to the
antibody by using the amino coupling kit. For the studies between 1,500 and 10,000 resonance units of the ligands were immobilized. The binding
studies were usually performed with 5-70 µg/ml of SV40 TAg as an
analyte at 25 °C and a flow rate of 40 µl/min. After recording of
the association and dissociation phases, the remaining non-cross-linked
protein-protein contacts were dissociated by regenerating the flow
cells with 0.1 M K3PO4, pH 12, for
30 s. A control cell contained antibody without loaded ligand to
correct for nonspecific binding. The data were collected at 1 Hz and
analyzed using the BIAevaluation program 3.0.
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RESULTS |
Construction of Hybrid p180 Polypeptides--
Human and murine
p180 show 88% identity at the amino acid level, and the corresponding
similarity at the DNA level allowed us to construct exchange mutants
between the two genes using conserved restriction sites (see
"Materials and Methods" and Fig. 1).
Initially four mutants were constructed in which either part of or the
entire TAg-binding site was exchanged. The mutants were named
HnM or MnH, where the first and last letters
denote the origin (human or murine) of the N and C termini,
respectively, and the number (n) denotes the amino acid
residue at the transition between the human and murine sequences. These
mutants were able to form complexes with the three smaller human DNA
polymerase -primase subunits (p48, p58, and p68), and furthermore,
these complexes possessed both specific DNA polymerase and primase
activities in the range of those of the wild type, indicating that the
exchanges had not disrupted the basic enzymatic functions of the
various DNA polymerase -primase complexes (Table
I and Fig.
2). Indeed, all of the complexes could
efficiently carry out coupled primer synthesis and elongation on ssDNA
(data not shown).
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Fig. 1.
Wild-type and mutant DNA polymerase
polypeptides used in this study.
Shaded and unshaded regions are of human and
murine origin, respectively. For details see "Materials and
Methods." At the top the sites of reported interaction of
Hp180 with SV40 TAg and with p68 are indicated (35, 51). Roman
numerals I-VI indicate regions of conservation among DNA
polymerases of the B family (59). The TAg-binding site is indicated in
each construct as a box. Conserved restriction sites used
for construction of the hybrids are shown at the corresponding fusion
points in the protein.
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Fig. 2.
SDS-polyacrylamide gel electrophoresis of
indicated purified DNA polymerase -primase
complexes consisting of human or hybrid p180 subunits and human p68,
p58, and p48. Approximately 4 µg of each protein complex was
loaded, and the gel was stained with Coomassie Brilliant Blue. The
additional protein band of ~55 kDa in lane 5 is probably
the large chain of murine IgG eluting from the antibody resin.
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The N-terminal 488 Amino Acids of Hp180 Do Not Determine the
Species Specificity of Initiation of SV40 DNA Replication--
The
mutant protein complexes were compared with the wild type both in the
cell-free DNA replication assay and in the initiation assay composed of
purified proteins. Because the various purified complexes show some
variation in their specific enzymatic activities, equal levels of DNA
polymerase units of each complex were compared in DNA replication
assays. As shown in Fig. 3A,
both (M257H)H3 and (M488H)H3 are active, albeit
less so than H4, in the replication of DNA containing an
SV40 replication origin to form a hemimethylated (DpnI-resistant) product. In contrast, (H257M)H3
and (H488M)H3 are almost inactive. Therefore, the ability
to replicate SV40 DNA appears to be not absolutely dependent upon the
presence of an SV40 TAg-binding site of human origin. This result was
reproducible, although absolute incorporation values varied between
experiments because of the use of different batches of cell extracts
and purified enzymes (TAg and RPA).
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Fig. 3.
A, in vitro SV40 DNA
replication assay with 0.25, 0.5, and 1.0 DNA polymerase units of the
indicated DNA polymerase -primase complexes. Enzyme activities were
determined beforehand with a DNA polymerase assay on activated calf
thymus DNA. Pairs of reciprocal exchange mutants are separated by
vertical dashed lines. The standard deviations are indicated
as error bars. B, in vitro SV40 DNA
replication assay with 0.5 DNA polymerase units of
(M257H)H3 in the absence (column 2) and in the
presence of increasing amounts of MH3 (0.25, 0.5, and 1.0 DNA polymerase units).
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Although the specific DNA polymerase and primase activities of the
various purified mutant and wild-type complexes are in the same range,
they do differ to some extent (Table I). To exclude the possibility
that inactive protein present in purifications with low specific
enzymatic activity is somehow acting as a (competitive) inhibitor in
the replication assay, we performed an experiment where replication
inactive MH3 was added to active (M257H)H3. This did not result in any inhibition of SV40 replication activity (Fig. 3B).
Previous work demonstrated that the species-specific function of human
p180 was executed at the initiation stage of DNA replication (27, 29).
To verify that this was again the case in these experiments, the mutant
complexes were tested in an initiation assay where the ability to form
RNA primers at the replication origin is tested ("Materials and
Methods"). In this case, equal levels of primase units were compared.
Fig. 4 shows that the activity of the
complexes in the DNA replication assay is reflected in the initiation
assay. In conclusion, the region(s) of Hp180 that determine SV40
species specificity must be C-terminal of the TAg-binding site.
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Fig. 4.
Autoradiogram of an in vitro
SV40 DNA replication initiation assay with 0.2 primase units of
various DNA polymerase -primase
complexes. Specific primase activities were determined beforehand
with a primase assay on poly(dT). Lanes 1 and 2,
control reaction with DNA polymerase -primase (H4)
lacking TAg and with TAg but lacking DNA polymerase -primase,
respectively; lanes 3 and 4, human
(H4) and murine DNA polymerase -primase
(M4), respectively; lanes 5-9, the hybrid
complexes MH3 (murine p180) in complex with three small
human subunits), (H257M)H3, (M257H)H3,
(H488M)H3, and (M488H)H3 (chimerical
human-murine p180, for explanation see Fig. 1, together with three
small human subunits), respectively. The approximate sizes of the
reaction products are indicated on the right in nucleotides
(nt).
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Quantitation of the Interactions between SV40 TAg and Human DNA
Polymerase -Primase--
We used surface plasmon resonance to
obtain quantitative data for the strength of interaction between SV40
TAg and human DNA polymerase -primase, the p180-p68 subcomplex, and
p180 alone. The various proteins were immobilized with a monoclonal
antibody, 2CT25, directed against p180 (50). SV40 TAg binds human p180 with an association constant (Ka) = 
109 M
1 (Table
II). Binding could only be detected if
TAg was used as the analyte and was dependent upon the presence of
Mg2+. The hetero-oligomeric complexes p180-p68 and
p180-p68-p58-p48 did not display significantly greater binding
constants than did p180 alone.
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Table II
Interactions between SV40 TAg and human DNA polymerase -primase
Standard deviations were calculated from multiple experiments.
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Interactions of SV40 TAg with Both Human and Murine DNA Polymerase
-Primase Are of the Same Order of Magnitude--
Human DNA
polymerase -primase (H4), murine DNA polymerase
-primase (M4), and a hybrid enzyme complex
(H2M2) consisting of the human two large
subunits combined with the murine primase subunits were immobilized on
a BIAcore chip with the antibody 2CT25, and purified SV40 TAg was used
as the analyte. Table III shows that the
Ka values for H4 are
approximately two to three times as great as those for M4.
Variations in the experimental conditions such as changing the
temperature over the range of 25-37 °C, the addition of 1 mM ATP, or changes in the buffer system (HEPES or Tris
acetate) did not alter the relative binding of human and murine DNA
polymerase to SV40 TAg nor did the use of a different monoclonal
antibody for immobilization (data not shown).
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Table III
Species-specific interactions between SV40 TAg and human or murine
DNA polymerase -primase
Standard deviations were calculated from multiple experiments.
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Construction of Further p180 Exchange Mutants--
So far we had
mapped the region of human p180 necessary for SV40 DNA replication as
being C-terminal of amino acid residue 488. As an attempt to define
this region more precisely, we created further reciprocal exchange
mutants using conserved BamHI (amino acid 1141) and
AgeI (amino acid 1395) sites and a partially conserved PflMI (amino acid 671) site. The exchange mutants formed
complexes with the human p68, p58, and p48 subunits, which were active
both in DNA polymerase assays on activated DNA and in primase assays (Fig. 2 and Table I). One mutant, M671H, proved to be unstable for
unknown reasons and could not be purified. Fig.
5 shows the results of in
vitro replication assays using DNA polymerase -primase complexes containing mutant subunits created from pairs of reciprocal exchanges. All complexes are partially active, but the presence of
murine sequences within the C-terminal 974 amino acids of p180 is
deleterious to SV40 replication ((H1141M)H3,
(H1395M)H3, (M1395H)H3, and
(H671M)H3; see Fig. 5 and
Fig. 6A, columns 11 and 12).
Furthermore, the human C-terminal 321 amino acids contribute
significantly toward SV40 replication activity but are insufficient for
full activity ((M1141H)H3). The exchanges made do not allow
for the clear definition of a single domain within p180 responsible for the species specificity of SV40 DNA replication; rather, various sequences C-terminal of amino acid 488 contribute to activity either
severally or in combination.
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Fig. 5.
In vitro SV40 DNA replication
assay with 0.5, 1.0, and 2.0 DNA polymerase units of the indicated DNA
polymerase -primase complexes. The
activities shown are the averages from three experiments performed with
identical batches of cell extracts and RPA. Specific DNA polymerase
activities were determined beforehand with a DNA polymerase assay on
activated calf thymus DNA.
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Fig. 6.
In vitro SV40 DNA replication
assay with DNA polymerase -primase
complexes. In the following experiments we compare the hybrid
enzyme complexes containing all small subunits p68, p58, and p48 from
human origin (indicated by H3) or containing the
human primase subunits p58 as well as p48 but murine p68 (indicated by
MH2). Specific DNA polymerase activities were
determined beforehand with a DNA polymerase assay on activated calf
thymus DNA. A, 0.5 and 1.0 DNA polymerase units of the
indicated DNA polymerase -primase complexes were added to murine
cell extracts. Pairs of complexes differing in the nature of p68 are
separated by vertical dashed lines. The standard deviations
are indicated as error bars. B, in
vitro SV40 DNA replication assay with 0.5 and 1.0 DNA polymerase
units of human (H4) and two independent purifications of
hybrid DNA polymerase -primase (HMH2) consisting
of murine p68, human p180, p58, and p48. The means of the incorporation
values obtained with these purifications are shown, and the standard
deviations are indicated as error bars.
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Influence of the Murine p68 Subunit on the SV40 Replication
Activity of Hybrid Complexes--
The region of p180, which is
necessary for SV40 replication, encompasses the p68-binding site, which
has been shown to be located between residues 1275 and 1462 (51).2 Furthermore, binding
of p68 to p180 induces a conformational change in the latter
polypeptide (51). This prompted us to investigate whether the lack of
activity of complexes containing a murine p180 C terminus stems from a
suboptimal interaction of the mutant p180 subunit with human p68.
Therefore, the complexes were purified containing murine p68, and we
investigated whether its presence could suppress the low replication
activity of some of the mutant complexes. Fig. 6A shows that
this is, to some extent, the case. The activities of the
(H/Mp180)MH2 complexes are reproducibly higher than those
of the corresponding (H/Mp180)H3 complexes. H671M
complexes (lanes 11-14) show little effect of the p68
substitution; nevertheless this minor effect was reproducible with
several independently purified batches of these complexes. Substitution
of Mp68 for Hp68 in the human polymerase -primase complex containing
wild-type Hp180 did not enhance activity (Fig. 6B and Ref.
29), indicating that a murine p68-binding site on p180 is required for
the stimulatory effect of murine p68.
Interaction of SV40 TAg, RPA, and DNA Polymerase -Primase on
ssDNA--
So far we have demonstrated species specificity of SV40
replication only on double-stranded DNA containing an SV40 origin of
replication. A functional interaction between SV40 TAg, RPA, and DNA
polymerase -primase has also been shown to occur on natural ssDNA.
Low concentrations of RPA inhibit the activity of DNA polymerase -primase on M13 ssDNA, and this inhibition can be relieved by the
addition of SV40 TAg (27, 33, 34, 52, 53). We investigated how our
hybrid DNA polymerase -primase complexes would behave in this system
to draw conclusions concerning the requirements for a functional
interaction between p180 and SV40 TAg.
Fig. 7A shows the effect of
RPA and SV40 TAg on the activity of human DNA polymerase -primase in
a coupled priming and DNA synthesis assay on unprimed single-stranded
X174 DNA. Increasing amounts of RPA reduced DNA synthesis by DNA
polymerase -primase to about 22% (Fig. 7A, columns
2 and 5). The addition of 1 µg of TAg completely
reversed this inhibition (Fig. 7A, columns 6 and
9). We performed this assay with our hybrid DNA polymerase -primase complexes and found that all of the complexes efficiently synthesized DNA in the absence of RPA (see also Table I) and that all
were inhibited by the addition of RPA. However, the ability of TAg to
reverse this inhibition varied significantly between the complexes.
Fig. 7B shows the maximal stimulation achieved with each
pair of reciprocal exchange mutants. Firstly, we observed that a
complex containing a murine p180 subunit, MH3, fails almost entirely to be stimulated by SV40 TAg. Secondly, we observed that there
exists among all complexes a qualitative correlation between those that
show a strong TAg-mediated stimulation in this assay and those that
function well in the SV40 DNA replication assay on double-stranded DNA
carrying an SV40 origin of replication (Figs. 3 and 5). However, a
difference between the two assays is seen in the effect of murine p68.
The complex (H1395M)MH2, which shows much greater activity
than (H1395M)H3 in the SV40 DNA replication assay (Fig.
6A, columns 15-18), is not subject to greater
stimulation by SV40 TAg on ssDNA (Fig. 7B). This probably reflects differences in the interactions between DNA polymerase -primase and SV40 TAg in the initiation complex and the simpler complex on ssDNA studied here.
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Fig. 7.
A, stimulation of human DNA polymerase
-primase (H4) by TAg on X174 ssDNA in the presence of
RPA. 200 ng of human DNA polymerase -primase H4 was
added to the reaction mixture in the presence or absence of 1 µg SV40
TAg and increasing amounts of RPA (0, 0.6, 0.9, and 1.2 µg) where
indicated. B, a summary of the results of experiments as in
A performed with the human DNA polymerase -primase
H4 and the chimerical enzyme complexes MH3
(with murine p180 and the three small human subunits),
(M257H)H3, (H257M)H3, (M488H)H3,
(H488M)H3, (M1141H)H3, (H1141M)H3,
(M1395H)H3, (H1395M)H3, and
(H1395M)MH2 (with chimerical human-murine p180 (for
explanation see Fig. 1) and three small human subunits indicated as
H3 or murine p68 together with human p58 as well as p48
indicated as MH2). The activity of each complex in the
presence of 1.2 µg of RPA was set at an arbitrary value of 100%
(horizontal dotted line). The degree of stimulation of DNA
synthesis in the presence of 1.2 µg of RPA and 1 µg of SV40 TAg
(this was the maximal stimulation obtained in each case) is shown for
each complex as the mean value from three or more assays. The standard
deviations are indicated as error bars. Pairs of reciprocal
exchange mutants are separated by vertical dashed
lines.
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DISCUSSION |
Initiation of SV40 replication requires the formation of an active
quaternary complex between DNA polymerase -primase, TAg, RPA, and
the viral origin of replication. DNA polymerase -primase interacts
directly with each of the three other components of the complex, and
any of these interactions could, in principle, form the basis of the
observed species specificity of DNA replication (4, 5, 34). However,
the functional cooperation of RPA and DNA polymerase -primase from
mammalian origin has been shown not to be species-specific (27, 29,
54). Therefore, we concentrated on the functional interactions between
SV40 TAg and DNA polymerase -primase and, more specifically, on the
p180 subunit known to control SV40 species specificity (29).
Because residues 295-313 of human p180 are involved in binding the
viral TAg and because the N terminus of the protein exhibits slightly
reduced conservation between the human and murine p180 subunits (35,
37), we constructed mutants in which parts of the p180 N termini were
reciprocally exchanged between the human and murine counterparts.
Although all mutants were capable of forming enzymatically active DNA
polymerase -primase (Fig. 2 and Table I), only those enzymes
carrying sequences, which lie C-terminal of human amino acid residue
488, were capable of initiating DNA replication at an SV40 origin of
replication (Figs. 3 and 4). This was a surprising result because it
indicates that the interaction of p180 with SV40 TAg is not responsible
for species specificity. However, this agrees with quantitative surface
plasmon resonance data, which show that SV40 TAg binds human DNA
polymerase -primase only 2-3-fold more strongly than the
replication-incompetent murine analogue (Table III). Because a 10-fold
increase in the concentration of murine enzyme complex does not allow
murine cell extracts to replicate SV40 DNA (data not shown), the
measured difference in binding constants for the interaction of SV40
TAg with human versus murine proteins is insufficient to
explain species specificity. Therefore, the mapped N-terminal
TAg-binding site of p180 (35) may be the primary site of interaction
between the proteins; but formation of an active initiation complex may require additional interactions involving other regions of the DNA
polymerase -primase. This notion has some experimental support because an N-terminal fragment of p180 can competitively inhibit formation of the initiation complex and thus DNA replication only during the first 15 min of the reaction (35). This indicates that the
establishment of an active initiation complex may be a dynamic process
where interactions of the p180 N terminus may play an important role
only during the initial recruitment of DNA polymerase -primase to
the complex. The existence of an SV40 TAg-binding site at the N
terminus of the murine protein has not been excluded by this study.
This putative murine binding site may compensate for the absence of the
human counterpart in our hybrid p180. Possibly secondary interaction
sites, which only occur in a quaternary initiation complex and which
are consequently not detected by binary interaction studies, may
contribute to the initiation reaction.
Further exchange mutants indicate that the substitution of C-terminal
p180 sequences with those of murine origin, especially beyond amino
acid 1141, has an inhibitory effect on SV40 replication. This part of
the polypeptide contains the DNA-binding domain necessary for
interaction with the replication template but is also involved in
interactions with the p68 subunit, which has no known catalytic activity but induces a conformational change into p180 (1, 2, 51, 55).
We investigated whether the inhibitory effect of the murine p180 C
terminus on SV40 replication could be (in part) the consequence of a
suboptimal interaction between murine p180 and human p68. Substitution
of murine p68 for its human counterpart shows that the interactions
between p180 and p68 appear to contribute to species specificity to
some extent, perhaps by changing the conformation of p180.
Alternatively, because p68 itself binds SV40 TAg (32, 34, 56), active
complex formation may require a particular coordination between the
interactions of p180 and p68 with TAg, which might be disturbed when
the two subunits are derived from different species. Abundant evidence
exists that p68 acts as a regulator of DNA polymerase -primase (36,
55-58), but the suppression of Mp180 C terminus-induced inhibition of SV40 DNA replication by Mp68 is only partial and is not evident in
complexes such as (H671M)MH2 and
M2H2 (Fig. 6A and Ref. 29). It
appears therefore that multiple functions contribute to the species-specific requirement for human p180 and that the sequences involved are spread over a large part of the p180 protein. This situation differs from that found in polyomavirus DNA replication, where a short defined stretch of Mp48 controls the species specificity, and biochemical data in conjunction with protein structure predictions gave some insight into possible functions for this region (30). With
p180 the situation is more complicated, and therefore detailed conclusions concerning the mechanism underlying species specificity will very likely require resolution of the three-dimensional structure of the initiation complex.
In addition to a species-specific effect of p180 on the initiation of
replication at the double-stranded SV40 origin, we describe a similar
effect on TAg-mediated stimulation of DNA polymerase -primase on
RPA-coated ssDNA. Although this system is not strictly species-specific, and the mechanism behind stimulation of primer synthesis by TAg is not entirely understood, it requires specific interactions of TAg with multiple sites on several or all RPA and DNA
polymerase -primase subunits (27, 30, 53). Substitution of Mp180 for
Hp180 prevents stimulation by SV40 TAg. The regions of Mp180 that
inhibit this stimulation appear largely to coincide with those that
prevent initiation of SV40 DNA replication (Figs. 3-5 and
7B). Because all tested DNA polymerase -primase complexes are inhibited to comparable degrees by RPA, it appears that differences in the interactions of the DNA polymerase -primase hybrids with TAg
affect the levels of stimulation. Notwithstanding the fact that the
protein-DNA complex under study here differs fundamentally from the
initiation complex, we note the importance of the presence of human
C-terminal regions for TAg-mediated stimulation and, moreover, the lack
of a simple correlation between effective stimulation and the presence
of the mapped N-terminal TAg-binding site (35). This reinforces our
conclusion that this region is not sufficient for the functional
interactions of DNA polymerase -primase with TAg and for this reason
probably is not the determinant of SV40 species specificity.
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ABSTRACT
INTRODUCTION
Present address: PerkinElmer Life Sciences, Imperiastraat 8, BE-1930 Zaventem, Belgium.
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