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J Virol, August 1998, p. 6893-6897, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Papillomavirus E1 Protein Forms a DNA-Dependent
Hexameric Complex with ATPase and DNA Helicase Activities
Juhan
Sedman and
Arne
Stenlund*
Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York 11724
Received 10 March 1998/Accepted 28 April 1998
 |
ABSTRACT |
The E1 protein from bovine papillomavirus has site-specific DNA
binding activity, DNA helicase activity, and DNA-dependent ATPase
activity consistent with the properties of an initiator protein. Here
we have identified and characterized a novel oligomeric form of E1 that
is associated with the ATPase and DNA helicase activities and whose
formation is strongly stimulated by single-stranded DNA. This
oligomeric form corresponds to a hexamer of E1.
| |
TEXT |
DNA helicases constitute an
interesting class of proteins with important functions in a large
number of biological processes. These proteins, by virtue of their
ability to separate the two complementary strands of the double helix,
are essential for all processes that require exposure of the base
sequence, including transcription, DNA replication, and recombination
(for reviews, see references 12 and
16). The initiator proteins of the small DNA tumor
viruses simian virus 40 (SV40) and polyomavirus form an interesting
subgroup of hexameric DNA helicases. These proteins, in addition to
helicase activity, also have other DNA replication-related activities,
such as sequence-specific DNA binding activity for ori
recognition (for reviews, see references 3 and
8). While most DNA helicases require a region of
single-stranded DNA for entry, these proteins can initiate unwinding
from completely double-stranded DNA, presumably by causing helix
melting and entry of the DNA helicase onto a single-stranded region. A
consequence of these three activities is the fact that the initiator
proteins have the unique capability of initiating unwinding on a
circular double-stranded template in a sequence-specific manner.
Bovine papillomavirus (BPV) has been studied intensively as a model
system for papillomavirus DNA replication (for a review, see reference
25). In vivo DNA replication studies have
demonstrated that two viral proteins are required for viral DNA
replication (27). These proteins, encoded from the E1 and E2
open reading frames, bind to the viral origin of replication, which
contains specific binding sites for both proteins (28, 31,
32). The BPV E1 protein belongs to the group of viral initiator
proteins whose best-studied member is SV40 large T antigen (1, 6, 14, 17, 22, 26, 33). Thus, BPV E1 has ori-specific
DNA-binding activity, DNA-dependent ATPase activity, and DNA helicase
activity (11, 13, 22, 26, 28, 31, 33).
Here, we describe the characterization of a novel oligomeric E1
complex. This complex forms spontaneously in the presence of
single-stranded DNA and appears to contain six molecules of E1 together
with one molecule of single-stranded DNA. The dependence on DNA for the
formation of this complex may reflect a dependence on DNA for the
assembly of the individual E1 subunits into a specific configuration
required for enzymatic activity.
DNA-dependent ATPase resides in an oligomeric form of E1.
A
hallmark of DNA helicases is the fact that the helicase activity is
invariably associated with DNA-dependent nucleoside triphosphatase
activity. Consistent with this finding, the BPV E1 protein has been
demonstrated to have DNA-dependent ATPase activity as well as DNA
helicase activity (22, 33). To determine in which form the
ATPase and helicase activities resided, we assembled reactions under
the conditions used for ATPase assays. Complexes were formed in 200 µl of buffer A (25 mM HEPES-Na [pH 7.6], 100 mM NaCl, 7 mM
MgCl2, 100 µM ATP, 1 mM dithiothreitol) containing 4 mM
ATP and 0.1 mg of bovine serum albumin (BSA)/ml, with 12 µg of E1
protein (21) and 2 µg of a single-stranded 28-mer
oligonucleotide. The mixture was incubated for 10 min at room
temperature, mixed with marker proteins, loaded onto a 15 to 30%
glycerol gradient, and centrifuged in an SW55 Ti rotor (Beckman
Instruments) at 50,000 rpm for 10 h at 4°C. Forty fractions were
collected and assayed for the presence of E1 by Western blotting with a
monoclonal antibody directed against E1 as well as for ATPase activity
by a standard ATPase assay as described previously (30). The
purified E1 protein in the absence of ATP, magnesium, and
oligonucleotide sedimented close to the BSA marker protein, indicating
that the protein was monomeric (data not shown). Strikingly, incubation
in the presence of the oligonucleotide and ATP resulted in a
quantitative conversion of E1 from the monomeric form to a much larger
form that sedimented as a sharp peak between the marker proteins
catalase (240 kDa) and thyroglobulin (650 kDa), as judged by Western
blotting (Fig. 1A). These same fractions
also contained all the ATPase activity that we could detect in the
gradient. Under these conditions, no E1 could be detected elsewhere in
the gradient.
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FIG. 1.
Incubation of E1 in the presence of single-stranded DNA
and ATP results in the quantitative conversion of E1 into an oligomeric
complex. (A) E1 was incubated under conditions used for DNA-dependent
ATPase assays and was subsequently loaded onto a 15 to 30% glycerol
gradient and sedimented as described in Materials and Methods. The
gradient was fractionated into 40 fractions, and every other fraction
was analyzed for ATPase activity. The glycerol gradient fractions were
analyzed for the presence of E1 by Western blotting with a monoclonal
antibody directed against E1. ALD, aldolase; CAT, catalase; THR,
thyroglobulin. (B) The ATPase activity of the oligomeric E1 complex is
not stimulated by DNA. Material from the oligomeric peak in the
glycerol gradient in panel A was analyzed for ATPase activity in the
absence or presence of poly(dT) over a 60-min time course. In parallel,
0.2 µg of purified, unfractionated E1 protein was analyzed under the
same conditions.
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|
ATPase activity associated with the oligomeric E1 complex is not
stimulated by DNA.
We were interested in whether the ATPase
activity recovered from the glycerol gradient had properties similar to
those of the unfractionated ATPase activity. Specifically, we wanted to determine if the ATPase activity of the oligomeric form of E1 was
stimulated by the addition of DNA, like that of the unfractionated, monomeric E1 protein. We therefore performed ATPase assays in the
absence and presence of added DNA with monomeric E1 as well as with the
oligomeric E1 from the peak fraction in the gradient, as shown in Fig.
1B. As expected, purified E1 showed a low level of ATPase activity that
was significantly stimulated by the addition of poly(dT). However, the
ATPase activity of the oligomeric E1 isolated from the gradient was not
stimulated upon addition of DNA. Several explanations for this
difference are possible. Sufficient quantities of oligonucleotide may
be present nonspecifically throughout the gradient to mask the DNA
dependence of the ATPase, or the DNA may be required for the formation
of the oligomeric complex and not directly for activity.
Single-stranded DNA is strongly stimulatory for E1 oligomerization
and is stably associated with the E1 oligomer.
To address these
questions, we monitored both E1 and the oligonucleotide in the
gradient. We assembled reaction mixtures as described above but
included small quantities of 32P-labeled oligonucleotide.
After sedimentation, as shown in Fig. 2A,
the 32P-oligonucleotide was present in two discrete peaks,
one at the top of the gradient corresponding to free oligonucleotide
and one peak that coincided with the oligomeric E1 peak that we had observed previously. Western analysis of the gradient fractions demonstrated that these fractions indeed corresponded to the oligomeric E1 peak (Fig. 2B). These results demonstrated that the oligonucleotide was specifically and stably associated with the E1 oligomer and not
distributed throughout the gradient. The association with oligonucleotide did not exhibit any obvious sequence specificity: several different oligonucleotides of similar sizes but with different sequences appeared to function equally well for oligomer formation, although a shorter, 17-mer oligonucleotide was less active for E1
oligomerization (data not shown).
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FIG. 2.
The oligonucleotide is stably associated with the
oligomeric E1 complex. (A) E1 was incubated in the presence of a 27-mer
oligonucleotide and sedimented on a gradient as described in the legend
to Fig. 1, except that a small fraction of 32P-labeled
27-mer oligonucleotide was added. The gradient fractions were analyzed
for the presence of 32P counts. ALD, aldolase; CAT,
catalase; THR, thyroglobulin. (B) Western blot analysis of the
oligomeric E1 peak. The E1 protein in the oligomer peak was quantitated
by comparison to a titration of known quantities (50, 25, 12, 8, 6, and
4 ng) of purified E1 protein. (C) E1 was incubated in the presence of
oligonucleotide as described in the legend to Fig. 1, except that
10-fold lower quantities of E1 protein (1.2 µg) and oligonucleotide
(0.2 µg) were used, followed by glycerol gradient sedimentation. The
fractions were analyzed by Western blotting.
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|
We used the results from this experiment to estimate the stoichiometry
of binding of E1 and oligonucleotide. We determined
the approximate
quantity of E1 in the oligomeric peak by comparing
the intensity of the
signal in the Western blot to those of standard
quantities of the
purified E1 that was loaded onto the gradient
(Fig.
2B). The
chemiluminescent signal was detected by exposure
to X-ray film, and
light exposures were used for quantitation
with a digital imaging
system. The oligomeric peak accounts for
at least 90% of the input E1
protein. We also determined the fraction
of input radioactive
oligonucleotide that was present in the oligomer
peak, from which we
could calculate the molar quantity of oligonucleotide
present in the
oligomer peak. Using these quantitations, we could
calculate a molar
ratio of E1 to oligonucleotide. In this experiment
this ratio was found
to be 5.4 mol of E1 per mol of oligonucleotide.
When identical reaction mixtures were assembled in the absence of
oligonucleotide, we failed to detect an oligomeric peak
of E1 by
Western analysis, demonstrating that at least under these
specific
conditions, the presence of oligonucleotide was required
for the
formation of the E1 oligomer (data not shown). Instead,
in the presence
of ATP and magnesium, but in the absence of oligonucleotide,
the
majority of the E1 protein could be recovered from the pellet,
consistent with the formation of very large E1 complexes (data
not
shown). At high E1 concentrations the oligonucleotide may
therefore
stimulate oligomer formation partly by preventing extensive
aggregation. At lower E1 concentrations, the oligonucleotide appears
to
stimulate oligomer formation by nucleation. At low E1 concentrations,
in the presence of limiting concentrations of oligonucleotide,
two E1
peaks corresponding to monomeric E1 and the oligomer can
be observed
simultaneously in the gradient (Fig.
2C). These were
the only
conditions under which we could observe both these forms
of E1
simultaneously.
The oligomeric E1 complex has DNA helicase activity.
To
determine if the oligomeric form of E1, isolated from glycerol
gradients, was active in a DNA helicase assay, we analyzed the material
from the gradient peak in an oligonucleotide displacement assay, as
described by Seo and Hurwitz (23). A 50-mer oligonucleotide with partial complementarity to the plasmid pET 11C was synthesized, generating a substrate with a 28-nucleotide-long double-stranded region
and a 22-nucleotide-long single-stranded 3' tail. The oligonucleotide was 5' labeled and annealed to the single-stranded plasmid at 55°C
for 30 min in a buffer containing 20 mM Tris-HCl (pH 7.5), 1 mM EDTA,
and 0.5 M NaCl. The substrate was separated from free oligonucleotide
by gel filtration on a Sepharose 6B CL column. Gradient fractions, or
purified E1, were incubated with substrate in a buffer containing 50 mM
Tris-HCl (pH 7.9), 3 mM MgCl2, 2 mM dithiothreitol, 1 mM
ATP, and 0.2 mg of BSA/ml at 37°C for 15 min. After the incubation,
sodium dodecyl sulfate was added to 0.1% and the sample was loaded
onto a 1.5% agarose gel in TAE buffer (0.04 M Tris-acetone, 1 mM
EDTA).
In the absence of added E1, only a very small fraction of free
oligonucleotide could be detected in the sample (Fig.
3A, lane
6). When the sample was heated
to 100°C, all the labeled oligonucleotide
was released and migrated
at the bottom of the gel (Fig.
3A, lane
7). When purified E1 protein
was incubated in the presence of
ATP, the oligonucleotide was also
displaced (Fig.
3A, lanes 8
to 10), while under the same conditions in
the absence of ATP,
no displacement was observed (Fig.
3A, lanes 11 to
13), indicating
that, as expected, the displacement of the
oligonucleotide by
E1 was ATP dependent. The gradient fractions 20 to
25 had detectable
helicase activity. These fractions coincided exactly
with the
E1 oligomer peak, as measured by Western blotting with a
monoclonal
E1 antibody (compare Fig.
3A, lanes 1 to 5 to Fig.
3B, lanes
1
to 5). Whether the helicase activity resides in the oligomer or
possibly results from the rearrangement of the oligomer upon incubation
is unclear; however, the stable nature of the E1 oligomer, which
allows
sedimentation, may indicate that the preformed oligomer
is indeed the
active helicase.
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FIG. 3.
Fractions containing the oligomeric E1 complex have DNA
helicase activity. (A) Glycerol gradient fractions containing the peak
of tracer oligonucleotide were analyzed for DNA helicase activity by an
oligonucleotide displacement assay (lanes 1 to 5). Purified E1 (300, 30, or 3 ng) was used in the same assay as a positive control with the
same substrate in the presence (lanes 8 to 10) and absence (lanes 11 to
13) of ATP. (B) Western blot of the same gradient fractions that were
used in the helicase assay probed with a monoclonal antibody directed
against E1.
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|
The multimeric E1 complex has a molecular mass consistent with that
of an E1 hexamer.
To estimate the relative molecular mass of the
oligomeric form of E1, we performed glycerol gradient centrifugation
experiments to determine the sedimentation rate of the oligomeric E1
complex relative to those of marker proteins under the conditions
described above. The sedimentation values relative to those of the
marker proteins were determined for four separate gradient runs and
averaged. We also determined the Stokes' radius of the complex
relative to those of marker proteins by gel filtration. Gel filtration chromatography was performed on a low-pressure 1-cm2 by
50-cm Sephacryl S-300 column equilibrated with buffer A. The sample was
generated in 200 µl of buffer A containing 4 mM ATP and 0.1 mg of
BSA/ml with 12 µg of E1 protein and 2 µg of 28-mer oligonucleotide.
The elution of the marker proteins was determined by Bradford protein
assays. The thyroglobulin peak fraction corresponded to the void volume
(18 ml). The mixture was incubated for 10 min at room temperature
before it was loaded onto the column. The Stokes' radius of the
complex relative to those of marker proteins was determined from six
different gel filtration experiments and averaged. An example of these
analyses is shown in Fig. 4. The results
from these experiments demonstrated that the oligomeric E1 complex
sedimented at 12.7S ± 0.5S with a Stokes' radius of 82 ± 5 Å. Using these values, we could calculate the relative molecular mass
of the oligomeric E1 complex to be 420 ± 40 kDa. This would
correspond well to a stoichiometry of six E1 molecules (68 kDa each) in
the oligomeric E1 complex. This would be a far from surprising result;
several helicases characterized to date form hexameric complexes. These
results also indicate that the stoichiometry of binding between E1 and
the oligonucleotide, which we have determined to be approximately 6 to
1, most likely means that the E1 oligomer consists of a hexamer of E1
bound to one molecule of oligonucleotide.
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FIG. 4.
The relative molecular mass of the oligomeric E1 complex
is consistent with that of a hexamer of E1. The relative molecular mass
of the oligomeric E1 complex was estimated by using a combination of
glycerol gradient centrifugation (A) and gel filtration analysis (B) to
determine the S value and Stokes' radius for the oligomeric complex
relative to those of marker proteins. The Stokes' radius for the
oligomeric E1 complex was determined to be 82 ± 5 Å, and the
sedimentation rate was 12.7S ± 0.5S, resulting in an estimated
molecular mass of 420 ± 40 kDa. The marker proteins were BSA (35 Å; 4.2S), aldolase (ALD) (46 Å; 8.3S), catalase (CAT) (52 Å; 11.3S),
and thyroglobulin (THR) (85 Å; 18.5S).
|
|
Under conditions where ATPase activity of E1 can be detected, purified
monomeric E1 protein is quantitatively converted into
a large
oligomeric form with a molecular mass consistent with
that of a
hexamer. Fractions containing this complex show both
ATPase and DNA
helicase activities, suggesting that both of these
activities reside in
the hexameric form of E1. This is consistent
with results obtained for
SV40 large T antigen, where similar
experiments have indicated that a
hexamer has helicase activity
(
15,
29). It is difficult,
however, to completely rule out
the possibility that a rearrangement of
the isolated hexamers
can occur after isolation and that this
rearrangement results
in the active enzyme. A stimulatory effect of
oligonucleotide
on the formation of hexameric helicases is not
unprecedented;
it has been demonstrated, for the T7 gene 4 protein as
well as
for DnaB, that single-stranded oligonucleotides are strongly
stimulatory
for hexamer formation and that the stoichiometry of binding
is
very close to one DNA molecule per hexamer (
4,
5,
10,
19). A likely possibility is that the helicase assembles on
its
cognate substrate. Like SV40 large T antigen, the E1 protein
is capable
of inducing KMnO
4 sensitivity in the sequences flanking
its
binding site (
2,
9,
18,
20). A simple model for
the loading
of the DNA helicase is a situation where a region
of single-stranded
DNA in a structurally distorted
ori could serve
as a
specific inducer and site of formation for the hexamer. If
the E1
hexamer forms a ring-like structure, as has been suggested
for
hexameric helicases, one implication of this model would be
that E1
encircles one DNA strand (
7,
24).
| |
ACKNOWLEDGMENTS |
This work was supported by National Institutes of Health grant CA
13106 to A.S.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Cold Spring
Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Phone: (516) 367-8407. Fax: (516) 367-8454. E-mail:
stenlund{at}cshl.org.
| |
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J Virol, August 1998, p. 6893-6897, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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