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J. Biol. Chem., Vol. 276, Issue 26, 23689-23699, June 29, 2001
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From the Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York 11724
Received for publication, February 28, 2001, and in revised form, April 24, 2001
DNA replication of papillomavirus requires the
viral initiator E1 and the transcription factor E2. Bovine
papillomavirus, type 1 (BPV-1), E1, and E2 bind cooperatively as dimers
to proximal sites in the viral replicator generating a
sequence-specific E1E2-ori complex. This complex is
critical for replication and can be converted to a multimeric
E1-ori initiator complex by displacement of E2 in the
presence of hydrolyzable ATP. However, E2 can function over extended
distances, and E2 at a distal position 33 base pairs upstream of the
E1-binding site also loads an E1 dimer onto ori. Under
these conditions, neither displacement of E2 nor ATP hydrolysis are
required for E1-ori formation, consistent with a need for ATP hydrolysis in E2 displacement from E1E2-ori. However,
ATP is required for stabilization of the resulting E1-ori
complex. These results indicate that BPV (with a proximal E2-binding
site) and human papillomaviruses (with distal E2-binding sites) utilize the same general mechanism for E1 loading but suggest that
E1E2-ori, which forms preferentially on ori,
may perform an additional role in BPV replication.
The papillomaviruses are an extensive family of closely related
DNA viruses. Infection with some human papillomaviruses
(HPV)1 is associated with a
risk of developing certain cancers. For example, HPV-16 and -18 are
strongly implicated in the etiology of cervical cancer. Many other HPV
types infect the mucosal and cutaneous epithelia causing benign
papillomas or warts (1, 2). The fact that papillomaviruses can exist in
a latent state imposes an additional degree of complexity to the
clinical infection. The papillomaviruses are also important models for
eukaryotic gene expression and replication. An understanding of genome
regulation in bovine papillomavirus type 1 (BPV-1), the prototype of
the group, is eclipsed perhaps only by bacteriophage Replication of BPV-1 requires only two viral proteins, E1 and E2, that
perform the early steps in replication initiation. E1 has all the
activities of an initiator protein including DNA binding, origin
melting, and perhaps the ability to act as a regulatory component of
the initiation reaction (4-9). It is also a helicase, responsible for
processive DNA unwinding (10-12). The E2 protein is a prototypic
eukaryotic transcription factor and the major transactivator and
regulator of BPV-1 transcription (reviewed in Ref. 13). The role of
transcription factors in the initiation of DNA replication is a
recurrent, yet poorly understood, theme in eukaryotes. Possible
functions of transcription factors include chromatin remodeling to
expose the DNA sequence determinant of origin specificity, recruitment
of general replication factors, a role in origin melting by DNA looping
or bending, and targeting and assembly of the initiator complex
(reviewed in Ref. 14). For BPV-1, the latter is the focus of much
investigation, since it appears to explain the absolute and specific
requirement of E2 for papillomavirus replication in vivo. It
is also intimately linked to the intriguing ability of E1 to perform
multiple biochemical functions that can be assigned to different
assembly states of E1. From these studies the concept of an assembly
pathway for the E1 helicase, governed by transcription factor E2, has
emerged (9, 15-17).
The minimal BPV-1 origin of replication (ori) consists of
binding sites for E1 and E2 and an A/T-rich region (7, 18-21). We have
characterized extensively a replication pre-initiation complex that
forms on the origin with the E1- and E2-binding sites separated by 3 base pairs (16, 22-24). This configuration is found only in a subgroup
of papillomaviruses that generate lesions with both a fibroblastic and
epithelial cell component. The complex, E1E2-ori, is most
likely a dimer of E1 bound cooperatively with a dimer of E2 (25). The
physical interactions between the two proteins, which are critical for
cooperativity, map to two distinct regions. An interaction between the
DNA binding domains of each protein appears to facilitate a second
interaction between the E2 activation domain and E1 helicase domain
(25-32). Formation of this complex, which forms with high specificity
and affinity, may be critical for the initiation of DNA replication
since E1 is expressed at low levels in infected cells and binds DNA
with low specificity and affinity (22). In vitro,
E1E2-ori can be converted to a higher order
E1-ori complex. Conversion is ATP-dependent and
results in the displacement of E2 and the incorporation of additional
E1 molecules (16). The function of E1-ori appears to
be stable and specific E1 binding, and it also forms the core of a
higher order origin melting complex. E2 bound to distal binding sites
assists formation of this complex (17). Thus, E2 appears to have two
distinct but linked roles in initiator complex assembly, acting as a
specificity factor for E1 binding and a general assembly factor.
A second E2-binding site of intermediate affinity (BS11) exists in
close proximity to the BPV-1 minimal origin of replication, 33 bp
upstream of the E1 site (Fig. 1).
Constructs with only E2 BS11 replicate efficiently in vivo
(15),2 and binding of E2 to
this site readily stimulates formation of E1-ori and the
origin melting complex in vitro (17). Here, we have explored
how E2 bound to a distal E2 site recruits E1 and assembles
E1-ori. The results reveal that E2 serves similar roles at
proximal and distal sites and initially recruits an E1 dimer to
ori. However, E1E2-ori formation at a proximal E2
site may have an additional role in replication, possibly as a
regulatory complex. BPV-1 origins with distal E2-binding sites resemble
the replication origins of HPVs, including those in the oncogenic group, which lack an E2 site proximal to the E1-binding site (3). Like
BPV-1, HPV replication is E2-dependent, and an
E1E2-ori-like complex appears to form on HPV origins
(32-34). The results presented here describe further the early events
in E1 complex assembly and may also be useful to model the early events
in HPV replication, for which there is currently little available
data.
Viral Proteins, Antibodies, and Origin Probes--
E1 and E2
proteins and anti-E2 and HA epitope antibodies have been described
previously (16, 24). Origin probes were constructed from BPV-1
nucleotides 7894 to 27 cloned into pUC19. Nomenclature is based on the
wild type template with E2-binding sites (BS) 11 and 12, denoted 11/12.
Templates were modified to alter the affinity of E2-binding sites as
described previously (17). E2 BS9 is a high affinity site; E2 BS11 is
an intermediate affinity site, and E2 BS12 is a low affinity site (36);
"X" indicates mutation of the E2 site to abolish binding. For
example, a template with high affinity E2 BS9 at the distal position
only would be called 9/X.
DNA Binding Reactions--
Probes were generated by polymerase
chain reaction using pUC19 primers, one 32P-end-labeled
(upstream 5'-GTAAAACGACGGCCAGT, and downstream
5'-GGATAACAATTTCACACAGG), and purified by polyacrylamide gel
electrophoresis and soak elution. Binding buffer was 20 mM
sodium phosphate (pH 7.2), 100 mM NaCl, 1 mM
EDTA, 0.1% Nonidet P-40, 1 mM dithiothreitol, 10% (v/v)
glycerol, 0.1 mg/ml bovine serum albumin, 125 pg/µl
poly(dA-dT)n. When ATP/MgCl2 was added it was at 5 mM. The probe concentration was 0.025 to 0.1 nM
as indicated, and reactions were incubated for 30 min at 20-22 °C.
To cross-link complexes, glutaraldehyde was added to 0.08% w/v, and
the reaction quenched with Tris-HCl (pH 7.5, 80 mM) after 5 min. For the assembly experiment described in Fig. 7,
E2HAE1-ori was pre-formed with probe at 0.4 nM with HAE1 at 20 nM and E2 at 3 nM. After 20 min of incubation, a 40-base pair
oligonucleotide with two high affinity E2-binding sites was added to
800 nM, and the products were immediately diluted 20-fold and mixed simultaneously into a reaction with a 100-fold excess of
untagged E1, poly(dA-dT)n competitor (0.2 ng/µl) and ATP/Mg2+, as indicated. Products were sampled, cross-linked
with glutaraldehyde, and analyzed in agarose gels after treatment with
ethidium bromide.
Gel Shift Analysis--
Cross-linked DNA-protein complexes were
analyzed in 1% agarose gels as described previously (16). Binding
reactions were also analyzed without cross-linking in polyacrylamide
gels (80:1 acrylamide/bisacrylamide, 0.25× Tris borate/EDTA). When
complexes were analyzed in the presence of ethidium bromide (EtBr), it
was added to 25 µg/ml.
Ortho-Phenanthroline-Copper (OP-Cu) Footprinting--
OP-Cu
footprinting was performed according to the general guidelines of
Sigman et al. (37). Binding reactions (0.05 nM
probe, 200 µl) were treated with 0.6 mM
1,10-phenanthroline, 0.135 mM cupric sulfate, and 0.29 mM 3-mercaptopropionic acid for 1 min and quenched with
2,9-dimethyl-1,10-phenanthroline to 0.84 mM. Carrier DNA
was added, and reactions were extracted twice with phenol/chloroform,
before ethanol precipitation of the DNA. Reaction products were
analyzed on 8% urea-polyacrylamide gels.
Hydroxyl Radical Footprinting--
Hydroxyl radical footprinting
in solution was performed according to the general guidelines of Dixon
et al. (38). The binding conditions were as described above,
except that glycerol was omitted. The hydroxyl radical was generated by
addition of 1 mM sodium ascorbate, 0.075% (w/v)
H2O2, 4 mM
((NH4)2Fe(SO4)2·6H2O),
8 mM EDTA for 2 min. Reactions were quenched with 0.5 volume of 200 mM thiourea, 25% v/v glycerol, 2 mM EDTA, 2% SDS, and 0.15 µg/µl carrier DNA. Cleavage
products were recovered and analyzed as described above for OP-Cu
footprinting. To footprint E1 complexes immobilized on Sepharose beads,
DNA-protein complexes were formed in standard binding buffer with
glycerol using hemagglutinin (HA) epitope-tagged E1 (16). After 30 min
of incubation, excess monoclonal anti-HA antibody (12CA5) was added,
and the reaction was incubated for a further 10 min.
HAE1-containing protein DNA complexes were then recovered
on protein G-Sepharose micro-columns (20 µl of beads per ml of
reaction). Sepharose beads were recovered in a micro-tube, washed twice
rapidly with reaction buffer (1 ml, no glycerol), and treated with the hydroxyl radical in binding buffer without glycerol (20 µl buffer per
µl of beads). Reactions were quenched with 0.5 volume of 200 mM thiourea, 25% v/v glycerol, 2 mM EDTA, and
the beads were recovered by centrifugation. DNA was eluted from the
beads in 0.5 M NH4 acetate, 1% w/v SDS, 1 mM EDTA, 15 µg of carrier DNA (37 °C, 30 min), the
solution phenol/chloroform extracted, and precipitated with ethanol.
DEPC Interference--
End-labeled probes were modified with
diethyl pyrocarbonate (39). Approximately 1 µg of end-labeled DNA was
denatured by boiling in 100 µl of water for 3 min. The solution was
snap-cooled on ice and 100 µl of cacodylate buffer (100 mM sodium cacodylate, 2 mM EDTA (pH 7.0)), and
4 µl of DEPC was added. The reaction was incubated at 37 °C for 20 min, and the DNA ethanol was precipitated twice. The DNA was denatured
and reannealed in a small volume of TE-100 mM NaCl before
repurification on a polyacrylamide gel. Binding reactions were
assembled as described above, and complexes resolved by polyacrylamide
gel shift. The desired complexes and free DNA were located by
autoradiography, and the DNA was recovered by electroelution,
phenol/chloroform extraction, and ethanol precipitation. Modified bases were cleaved with piperidine, and the DNA was recovered by butanol extraction and precipitation. Products were analyzed as
described above.
Recruitment of E1 to ori by E2 at Distal Sites--
BPV
ori constructs with a distal E2-binding site replicate
in vivo, and E2 stimulates E1 origin complex formation on
those probes in vitro. To investigate how E2 promotes
E1-ori complex formation from distal binding sites,
protein-DNA complex formation at low E1 and E2 concentrations was
assessed by polyacrylamide gel electrophoresis without protein
cross-linking (Fig. 2). A single
prominent complex, which was not detected with E1 alone, formed with E2
on the probe 11/X with distal E2 BS11 (lanes 2-5). This
complex, now termed E2E1-ori, migrated slower than
E2-ori (lane 6). Curiously, less probe was
shifted in the presence of E1 for reactions that contained the same
concentration of E2 (compare lanes 5 and 6).
Anti-E2 polyclonal antibodies supershifted E2E1-ori and
E2-ori (lanes 4 and 6 compared with
lanes 7 and 8), but only E2E1-ori was
supershifted with anti-E1 antibody (lanes 9 and
10). A similar complex, which migrated slightly slower than
E2E1-ori and was supershifted with anti-E1 and -E2
antibodies, formed with E1 and E2 on the probe with proximal E2 BS12
(X/12, lanes 12-20). This most likely corresponds to the
E1E2-ori complex described previously (22). At high E1
concentrations, in the absence of E2, a series of protein-DNA complexes
formed that bind anti-E1 but not anti-E2 antibodies (lanes
21-23). Six bands can be discerned, the largest of which is the
most prominent form.
We also replaced the distal E2 BS11 with high and low affinity
E2-binding sites (36). With the high affinity E2 BS9 in the distal
position (probe 9/X, lanes 27-30), the E2E1-ori
complex formed more efficiently than with probe 11/X (lane
25 compared with 30). The low affinity E2 BS12 at the
distal position supported E2E1-ori formation to a lesser
extent (lanes 33-36), whereas a probe lacking an E2-binding
site failed to form an E2E1-ori complex (lanes
39-42). As noted above for the probe 11/X, in the presence of E1
less probe was shifted than by E2 alone (compare lanes 36 and 37). On probe 11/12, which corresponds to the
arrangement in the wild type ori, a complex that co-migrated
with E1E2-ori formed at low E2 concentration (lanes
46 and 47 compared with 24). Upon addition
of more E2 (lane 48) a larger complex of unknown composition
was formed. We conclude, based on these results, that E2 bound to a
distal E2 site can recruit E1 to ori. The complex that
forms, E2E1-ori, resembles E1E2-ori, but complex
formation from the distal E2-binding site is less efficient than from a proximal E2-binding site with similar affinity.
E1E2-ori, but not the multimeric E1-ori
complexes, is unstable in the presence of low concentrations of
ethidium bromide (EtBr, 25 µg/ml) (16, 17). The reactions with probes
X/12, 9/X, and 11/X, examined above, were also examined by agarose
gel-shift following glutaraldehyde cross-linking, with and without EtBr treatment. In Fig. 2B, top panel, lane 1 shows
E1-ori formation. The extent of E1E2-ori
formation on probe X/12, is similar to that seen by polyacrylamide
gel-shift (compare lanes 2-7 with Fig. 2A, lanes
13-15). For probe 9/X, E2E1-ori was detected but only
at 50% that observed by polyacrylamide gel electrophoresis (compare
lanes 8-12 with Fig. 2A, lanes 28-30). E2
binding was not detected after cross-linking, as observed previously
(lane 13). For probe 11/X, E2E1-ori was not
detectable under these assay conditions. In the lower panel,
EtBr (25 µg/ml) was added to samples 1-19. Like the
E1E2-ori complex, E2E1-ori is unstable in the
presence of EtBr (lanes 4-6 and 10-12
upper and lower panels). Thus, sensitivity to
EtBr can be used to differentiate the E2E1-ori complex from the multimeric E1-ori complexes and suggests that E1 is
bound to DNA in a similar low oligomeric form as in
E1E2-ori.
High Resolution Footprinting Shows Occupancy of the E1- and
E2-binding Site in E1E2-ori--
To investigate the disposition of
proteins on the DNA in E2E1-ori, complexes were footprinted
with OP-Cu. By using probes with E2-binding sites of varying affinity
and position, E2 was titrated at low concentration of E1. Reactions
were treated with OP-Cu, quenched, and the DNA recovered for analysis
on a sequencing gel (Fig.3, top
strand). On probe 11/X (lanes 1-7), E2 completely protected the 12-base E2 palindrome and one or two more bases up- and
downstream (lane 7). A similar protection was observed on
the bottom strand and for all E2 sites tested. With E1 and E2, the
18-base E1 palindrome and an additional 2 bases up- and downstream were
also protected (lanes 3-6, compared with 2 and 7 as indicated in the annotations to the right).
Cleavage of the DNA between the two binding sites was also slightly
reduced. On probe 11/12 (lanes 8-14), E2 protected BS11,
and there was a similar weak protection over BS12 (lane 14).
With E1 and E2 (lanes 9-13), the E1-binding site was
occupied at a lower concentration of E2 compared with probe 11/X, and
E2 BS12 was completely protected (compare lanes 3-7 with
9-13). With probe 9/X, the E1-binding site was clearly
protected in the presence of E1 and E2 at the lowest concentrations of
E2 where BS9 was fully occupied (lanes 17-20). Cleavage of
all DNA between the two binding sites was reduced 15-30% depending on
the position. For probe X/12 (lanes 22-28), the E1 and E2
sites were completely protected at low concentrations of E1 and E2. The
E1E2-ori protection over E2 BS12 and the E1-binding site was
identical to that observed for probe 11/12. Solution footprints and
in-gel footprints obtained with OP-Cu were identical (data not shown).
Therefore, the complexes characterized in polyacrylamide gels are
likely to be identical to those characterized in solution, and the weak
protection between the E1- and E2-binding sites in E2E1-ori
is unlikely to be due to the binding of additional protein molecules in
a sub-set of the complexes.
In summary, E2 bound to a distal site recruits E1 to the
E1-binding site to form E2E1-ori, but E1E2-ori
forms preferentially on the wild type sequence. E2 bound to BS11 does
not impair E1E2-ori formation but may assist it (compare
lanes 10-13 with 24-27). The protections
suggest that E1 is bound to DNA in the same low oligomeric form in both
E1E2-ori and E2E1-ori. Therefore, initiator complex assembly begins with recruitment of a low oligomeric form of E1
to ori. The E1-binding site is the ultimate determinant of
where assembly begins, which is not altered by the position of the
E2-binding site.
DEPC Interference Analysis of E2E1-ori--
Diethyl pyrocarbonate
carboxyethylates A and G residues and can be used to assess the
importance of specific base contacts in DNA-protein complex formation
(39). DEPC interference data was generated for E2 alone and E1 and E2
bound on probes 11/X and X/12 (Fig. 4).
Modified probe was incubated with protein, and complexes were resolved
on and recovered from polyacrylamide gels. Modified residues were
cleaved with piperidine, and the DNA was analyzed on a sequencing gel.
For E2 bound to BS11 or BS12, many bases in the core E2 palindromes
show strong interference or enhance E2 binding when modified
(lanes 4 and 6, bottom and top strands). Other
positions both up- and downstream of the palindrome also affect binding
when modified (summarized in Fig. 4B). For E1 and E2 bound
to probes 11/X and X/12 (lanes 3 and 5, bottom and top strands), there is a similar interference associated
with each E2-binding site. Within the E1 palindrome, modification of every A and G residue interferes with DNA binding. For both
E1E2-ori and E2E1-ori, a number of bases in the
A/T-rich region affect complex formation when modified, most notably on
the bottom strand where there are more informative A and G residues.
Also, modification of a single G residue on the bottom strand between
the E1- and E2-binding sites affects DNA binding in E1E2-ori
but not when the proteins bind alone or in E2E1-ori
(lane 5, Figs. 4A). Therefore, the principal
sequence determinants for E1 DNA binding are the same in
E1E2-ori and E2E1-ori, although minor qualitative
and quantitative differences are apparent in the DEPC interference assay. These results and those of the protection analysis suggest that
E1 is bound similarly in both E1E2-ori and
E2E1-ori.
Two Monomers of Full-length E1 Bind the E1 Palindrome Along with E2
at a Distal Site--
Full-length E1 is a monomer in dilute solutions
(12). The stoichiometry of E1 binding in E2E1-ori was
determined with a mixing experiment using full-length E1 and E1 tagged
at its N terminus with a hemagglutinin epitope (HAE1) (16).
If E1 binds to ori as a monomer, only two types of E2E1-ori complex would form when E1 and the variant are
mixed, one containing E1 and one containing HAE1. However,
if full-length E1 binds as a dimer, we would expect a novel complex to
form containing tagged and untagged E1, as well as those with the
respective E1 homodimers. Any higher order binding configuration would
result in a more complex mixture of species.
Binding reactions were assembled with probe 9/X, E2, and a mixture of
E1 and HAE1 at ratios from 100% E1 to 100%
HAE1 for polyacrylamide gel-shift analysis (Fig.
5). To resolve clearly the various
complexes, excess anti-HA antibody (12CA5) was added. Fig. 5,
lane 1, is free probe, and lanes 2-5 demonstrate that without E2 neither form of E1 binds the probe, with or without antibody. E2 binds the probe (lane 6) and is not recognized
by the anti-HA antibody (lane 7). E2E1-ori forms
with untagged E1 (lane 8) and is not supershifted with the
anti-HA antibody (lane 9). The E2E1-ori complex
also forms with epitope-tagged HAE1
(E2HAE1-ori) and is supershifted with excess
anti-HA antibody (lanes 14 compared with 15).
When an increasing proportion of tagged HAE1 was mixed with
E1 (lanes 10-13), a single complex of intermediate mobility
formed along with the tagged and untagged complexes. A similar result
was obtained without using anti-HA antibody (data not shown), since the
mobility difference between all the complexes that form is just
sufficient to allow resolution of all complexes (lanes
8 and 15). The complex that migrates between the
E2-containing complex and E2E1-ori is most likely E2-bound
with a monomer of E1, since the complex that forms with
HAE1 can be supershifted with anti-HA antibody (compare
lanes 14 and 15). These results are consistent
with the binding of full-length E1 as a dimer to its binding site in
E2E1-ori.
Hydroxyl Radical Footprints of the E1 Dimer and E2 Bound to
DNA--
E2E1-ori formation offers a unique opportunity to
study the E1 dimer-DNA interaction when E1 and E2 are not bound in
close proximity. Since discrete hydroxyl radical footprints of
E2E1-ori proved difficult to obtain in reactions without
glycerol (a prerequisite for the hydroxyl radical cutting reaction),
the following protocol was adopted. Complexes were formed using
HA-tagged E1 and E2 on probe 9/X, in the standard reaction buffer
containing glycerol. After 30 min of incubation, anti-HA antibody was
added, the reaction incubated for a further 10 min, and the
E2HAE1-ori (or
HAE1E2-ori) complex recovered by passage over
protein G-Sepharose. The Sepharose beads were then washed in reaction
buffer without glycerol, and the bound complex was treated with the
hydroxyl radical. Footprints were then revealed on a sequencing gel
(Fig. 6).
Fig. 6A shows the hydroxyl radical footprints for
E1E2-ori (probe X/9) and E2E1-ori (probe 9/X).
Lane 1 is a G ladder. Lane 2 is the hydroxyl
radical cleavage ladder for probe X/9. Lanes 3 and
4 are the solution footprints for E2 and
HAE1E2-ori, respectively. As indicated in the
annotations to the left, three sets of protections are
observed over and beyond proximal E2 BS9 when E2 is bound. The
footprints of HAE1E2-ori in solution and bound
to protein G-Sepharose beads (lanes 4 and 5) are
identical. There is a general protection over a region encompassing the
E1- and E2-binding sites, within which a periodic set of specific
protections can be discerned. As indicated in the annotations, the
three 5'-most sets of protections are similar to those for E2 and were
previously assigned to E2 (17). Over and beyond the E1-binding site
there are two sets of strong protections and one weak 3'-set
(dashed box) that defines the upstream boundary of E1 bound
to DNA (upper arrow). For the
E2HAE1-ori complex (lane 6), there is
an extended set of periodic protections and a stronger general
protection over and beyond the E1-binding site compared with
E1E2-ori. Three sets of periodic protections, indicated
between the arrows, are similar to a subset in
HAE1E2-ori. Significant E1 protection is
observed in the region corresponding to the proximal E2-binding site
that is protected by E2 alone (lane 3). Three sets of
protections (dark boxes) are clearly observed for E2 bound
to distal E2 BS9 (lane 7), but one set (indicated with an *)
has no counterpart in HAE1E2-ori or E2 bound to
DNA. A similar but weaker protection is found at a related position
downstream of the E1-binding site (dashed box).
The protections on the top strand (lanes 9-16, right) are
similar to those on the bottom strand.
HAE1E2-ori and E2HAE1-ori
share some related protections, indicated between the
arrows. The two 3'-protections in
E2HAE1-ori overlap with protections observed
with E2 alone (lane 10). The E2 protections over distal E2
BS9 (lane 14) are clearly recognizable in
E2HAE1-ori, and one group of protections (*) is
not seen in HAE1E2-ori or E2 bound to DNA (as
above). The hydroxyl radical protections of the E1 dimer bound to DNA
are shown in Fig. 6B. The protein-DNA contacts flanking the
central pair of hydroxyl radical protections are qualitatively
different and are not clearly revealed with OP-Cu (Fig. 3) or DNase I
(boundaries indicated with the arrows; see Ref.
16).3 Although it is formally
possible that the stronger upstream hydroxyl radical protections (*)
are generated by E2, we consider this unlikely since a similar set is
found at a related position downstream of the E1-binding site and at a
related position in E1-ori.
E2E1-ori Is the Precursor of a Higher Order E1-ori Complex--
E2
could load a pre-formed initiator complex on the DNA or assemble
E1-ori in a stepwise fashion from monomers. Consistent with
the latter, we demonstrated that a pre-formed E1E2-ori
complex (formed on probe X/12) was a preferred substrate for
E1-ori formation. ATP (ATP/Mg2+) was required
for the transition to displace E2 from its binding site as additional
E1 molecules were incorporated. The experimental protocol was based on
molecule tagging (16). Briefly, HA-tagged E1 was used to form
HAE1E2-ori in the absence of ATP, conditions
inhibitory for E1-ori formation. This substrate was then
diluted into a reaction containing ATP, a 100-fold excess of untagged
E1, nonspecific competitor DNA, and an oligonucleotide E2-binding site
competitor (the "assembly reaction"). Product was sampled for up to
25 min, cross-linked, and analyzed by gel-shift with and without
specific antibodies (anti-HA and anti-E2). Under these conditions, if
E1-ori is a hexamer, we would expect only 6% of the
E1-ori complexes that form to contain HAE1, if
E1-ori formed by random assortment of E1 molecules. However, the observed value deviates considerably from the one predicted for
random assortment, indicating that E1E2-ori was a direct
precursor for E1-ori. To test whether E2E1-ori is
a substrate for formation of a higher order E1-ori complex,
a similar experiment was performed with
E2HAE1-ori formed on probe 9/X (Fig.
7).
Fig. 7A shows the assembly of a multimeric
E1-ori complex from a pre-formed
E2HAE1-ori precursor. Reaction products were
sampled for up to 25 min after dilution and addition of ATP, untagged
E1 (100-fold excess), and competitor DNAs, cross-linked, and analyzed
in agarose gels after addition of EtBr to 25 µg/ml. Lane 1 is free probe, and lane 2 demonstrates that no
EtBr-resistant products form in the pre-incubation
(E2HAE1-ori is unstable in the presence of EtBr,
Fig. 2B, but was detected by polyacrylamide gel-shift).
Lanes 3 and 4 show E1-ori formed at
high concentrations of HAE1 and E1, and only
HAE1-ori can be supershifted with anti-HA
antibody (lanes 5 and 6). E1-ori is
not supershifted with anti-E2 antibodies (lanes 7 and
8), but some of the probe is retained in the wells.
Lanes 9-13 show rapid formation of an EtBr-resistant
multimeric E1-ori complex upon dilution and addition of
untagged E1 and ATP. Approximately 80% of the complex that forms at
early time in the reaction (<5 min) can be supershifted with anti-HA
antibody and therefore contains epitope-tagged HAE1. This
proportion decreased slightly with time, possibly as a result of
complex formation directly from free or E2-bound probe. The value
deviates considerably from the value predicted for random assortment,
even if E1-ori is composed of up to 12 E1 molecules (12%).
Therefore, like HAE1E2-ori,
E2HAE1-ori is a substrate for formation of
the multimeric E1-ori complex. E2 is a component of the
final E1-ori complex (lanes 19-23),
demonstrating that, from a distal site, E2 remains bound to its site
(16).
In a parallel control reaction (Fig. 7B), E2 was omitted
from the pre-incubation with HAE1 but was added upon
dilution into the assembly reaction, as indicated below Fig.
7B. In Fig. 7B, lanes 1-8 are identical controls to those shown in Fig. 7A, lanes 1-8. E1-ori
formation from free probe was clearly lower (8.6-fold) than for the
reactions described in Fig. 6A, and none of the complex
could be supershifted with anti-HA antibody (lanes 14-18).
Therefore, the final ratio of tagged to untagged E1 is sufficiently
high to allow proper interpretation of the results. Finally, none of
the complex contains E2 (lanes 19-23, Fig. 7B),
so all E2 is effectively bound by the E2-binding site competitor in the
assembly reaction, and the extent of complex formation is likely to be
the maximum that can occur in the absence of E2. Therefore, the
E2HAE1-ori complex is a better substrate for
formation of the multimeric E1-ori complex than free probe.
In Fig. 7C, the ATP/Mg2+ requirement for
formation of E1-ori from E2HAE1-ori
was assessed. Lanes 1-8 are the controls described above. Lanes 9-13 are a time course after dilution of substrate
E2HAE1-ori and addition of untagged E1 and
competitor DNA. Surprisingly, formation of a multimeric
E1-ori complex was observed at early times in the reaction
(2.5 min, lane 9), albeit at a lower extent (2-fold)
compared with the reaction with ATP/Mg2+ (Fig. 7A,
lane 9). However, the complex that formed was unstable with a
half-life of ~11.5 min, which is close to the dissociation rate of
E1-ori that forms without ATP/Mg2+ (16). The
majority of the complex that formed could be supershifted with
anti-HA antibodies, indicating that it was derived from the E2HAE1-ori precursor. Finally, lanes
19-23 demonstrate that E2 does not dissociate from the multimeric
E1-ori complex during its formation. Therefore, additional
E1 molecules can be recruited to E2HAE1-ori in
the absence of ATP/Mg2+, but a stable E1-ori
complex does not form.
In summary, like the E1E2-ori complex, E2E1-ori
is a substrate for formation of a multimeric E1-ori complex,
which appears to form by stepwise binding of E1 to the ori.
However, displacement of E2 is not required for formation of the
multimeric E1-ori complex. Unlike E1E2-ori,
additional E1 molecules are readily recruited to E2E1-ori in
the absence of ATP/Mg2+, consistent with a role for ATP in
the displacement of E2 during E1-ori formation from
E1E2-ori. However, the resulting complex is unstable
relative to the complex that formed in the presence of ATP. Therefore,
the requirement for ATP/Mg2+ in E1-ori complex
formation is manifested at two levels. First, ATP/Mg2+ is
required to displace E2 bound to a proximal E2-binding site; and
second, it is required to stabilize the complex of E1 molecules recruited to the DNA.
BPV-1 is an important model for the initiation of DNA
replication in eukaryotic cells. Although the viral components required for initiation are relatively simple (E1, E2, and a replicator with the
cognate binding sites), a series of initiator complexes and a network
of interactions of surprising complexity have been revealed. We have
been intrigued by the difference in ori structure between
BPV and HPVs, and we undertook these studies to determine how an
initiator complex (E1-ori) is assembled by E2 from a distal site and how this assembly compares to E1-ori formation when
E1 and E2 are bound to juxtaposed sites. The results are gratifying, indicating that the basic mechanism for stepwise assembly that we
observe with the proximal site occurs also with a distal site. The
results presented here indicate that E1-ori formation in HPV may proceed in a similar manner to that in BPV. Thus, the E1-binding site is the ultimate determinant of where complex assembly begins. The
precision of the initial binding event (that of the E1 dimer) is
therefore critical for replication. The E1 site may be required to
correctly position, with respect to each other, the first E1 molecules
that bind ori so that an active multimeric E1 initiator complex can successfully form. Alternatively, the positional
relationship between the E1 site and other DNA sequences necessary for
initiation may be critical.
Binding of the Full-length E1 Dimer to ori--
Binding of an E1
dimer to the E1-binding site is stabilized by E2 bound at proximal or
distal sites. By using a number of footprinting reagents (hydroxyl
radical, OP-Cu, and DNase I), we have compared how full-length E1
interacts with DNA in the two complexes E1E2-ori (E1 and E2
bound to adjacent sites) and E2E1-ori (E2 and E1 bound to
distal sites). From this comparison, we can deduce the extent to which
an E1 dimer can interact with DNA, a form that is not readily detected
in solution. The E1 dimer appears to be capable of more extensive
interactions with the DNA than determined previously. An extended set
of periodic hydroxyl radical protections is observed in
E2E1-ori, and the entire region where E1 interacts is more
resistant to hydroxyl radical cleavage. The DNA between the E1 and
distal E2 sites in E2E1-ori is also generally less reactive
to OP-Cu and DNase I cleavage. However, no discrete
protections are seen with these reagent compared with the hydroxyl
radical. It therefore appears that the E1 interactions in this region
are qualitatively different from those over the E1 palindrome, which
appears to be the principal determinant of sequence-specific DNA
recognition by the E1 DNA binding domain (25). It is possible that
regions of E1, other than the DNA binding domain (DBD), can interact
over this region. Models for helicase-catalyzed DNA unwinding predict
that a minimum of two DNA-binding motifs, one each for single- and
double-stranded DNA (40), are required for helical motor activity. It
is possible that their existence in E1 is being revealed, even in the
initial E1 dimer-DNA interaction.
The second interesting observation to emerge is that E2 clearly can
modulate the way in which an E1 dimer interacts with DNA, depending on
its positional relationship to E1. This also has functional
consequences for the bound E1 molecules. First, the E1-DNA interactions
are more extensive in E2E1-ori, compared with E1E2-ori. Second, hydroxyl radical protections by E1 and E2
alone overlap extensively at proximal sites, indicating that in
E1E2-ori changes in the way the proteins interact with DNA
may occur. It is possible that the proximal interaction of the two
proteins can modulate individual protein domains so that either a new
composite interaction surface is generated or binding of one protein
precludes a DNA interaction made by the other. At this point it is
unclear which of these possibilities prevails. However, the protections in this region in E1E2-ori resemble more closely those
generated by E2, and in the absence of E2, E1 interacts extensively
over the whole proximal E2 site. The concept of structural flexibility in DNA-binding proteins is not without precedent. The Oct-1 POU domain
contains two separate DNA-binding modules joined by a flexible linker,
and the relative position of these domains on DNA can vary depending on
the DNA target (41).
Cooperative binding of E1 and E2 to juxtaposed sites involves two
separate interactions between the individual E1 and E2 DNA binding
domains and the activation domain of E2 and the helicase domain of E1
(25-32). The latter interaction can be readily detected in solution
and on DNA. Indeed, it is the sole determinant of cooperative binding
between E1 and E2 bound to distal sites, and a significant component of
the forces that stabilize E1E2-ori (32).3 The
DBD interaction, however, does not allow DNA replication by itself and
is significant only when E1 and E2 are bound in close proximity (27,
42). Cooperative binding of the E1 and E2 DBDs in E1E2-ori
generates a sharp bend in the DNA (29). E1 and E2 alone can bend DNA,
but the combined bend in the E1E2-ori complex is much
greater than the sum of the individual bends. It is possible that
either the remodeling of binding domains or the exclusion of one
specific interaction at the expense of another is a critical component
of "bending cooperativity." DNA bending is required for the second
interaction between the E2 activation domain and E1 helicase domain to
occur. In E1E2-ori that forms in the absence of ATP, this
interaction may limit (in addition to mutual exclusion by E2 downstream
of the E1 site) E1-DNA interactions, possibly by drawing an interaction
surface away from the DNA. It is enticing to speculate that this could
explain why the E1E2-ori complex, in the absence of ATP, is
a poor substrate for binding of additional E1 molecules (16). This is
clearly not the case when E1 interacts more extensively with DNA in
E2E1-ori, which readily recruits E1 even in the absence of
ATP (Fig. 7). Thus, E1 bound in a specific extended conformation along
the DNA may be the template necessary for further E1 binding and
assembly of a higher order E1-ori complex.
Regulation of E1-ori Complex Formation--
The occurrence of an
E2 site immediately adjacent to the E1 site, and as a consequence the
E1-E2 DBD interaction, is unique to BPV-1 and other
fibropapillomaviruses. Most papillomaviruses, including the HPVs, have
an E2-binding site at a more distal position. An important question is
what role this interaction plays in the BPV-1 life cycle. One distinct
possibility is that it governs a regulatory switch critical for the
regulation of BPV replication. Our data indicate that
E1E2-ori forms preferentially on the BPV ori
sequence with E2 BS11 and 12, regardless of E2 binding to BS11. In the
absence of the nucleotide cofactor ATP, E1E2-ori hinders
E1-ori formation and may negatively regulate its formation. The energy of ATP hydrolysis may induce a conformational change in E1,
which alters the quality of the interaction between the E2 activation
domain and E1 helicase domain, and may also be transmitted to the
cooperative binding surface between the E1 and E2 DBDs destabilizing
the DBD component of cooperativity that facilitated it. The
consequences would then be 2-fold as follows: E2 would freely
dissociate from its low affinity binding site, and the E1 dimer bound
to DNA may be primed to recruit more E1 upon forming more extended
interactions with DNA. Conceivably, E2 bound to BS11 could stabilize
binding of the E1 dimer during the transition, promote the extended
interactions with DNA, as well as recruit additional E1 molecules. The
latter is critical for formation of a stable E1-ori complex
that cannot form in the absence of ATP. The critical evidence required
in support of this hypothesis would be the demonstration that E1 ATP
binding is regulated in vivo. Why this type of negative
regulation may be unique to the fibropapillomaviruses is unclear. The
regulation of E1-ATP binding could also control replication without
E1E2-ori formation, since stable E1-ori formation
and the catalytic activities of the initiator are
ATP-dependent. HPV replication may be controlled in such a way, or alternatively, the proposed regulatory role of E2 at proximal BS 12 may be performed by a cellular DNA-binding protein.
Cooperative interactions are common in processes that involve assembly
of specific multiprotein complexes on DNA. They serve to stabilize
large protein DNA complexes through multiple weaker protein-protein
interactions and can provide regulatory opportunities through
allosteric control. A network of homo- and heterotypic interactions
between the BPV E1 and E2 proteins are now well documented. E1 can
assemble into higher order structures on DNA that are stabilized by
ATP, presumably through a cofactor-driven conformational change. The
heterotypic interactions between E1 and E2 are of two kinds. One
(between the E2 activation domain and the E1 helicase domain) serves
generally in recruitment and assembly of E1 at the origin of
replication. A second, dependent on the proximity of the E1- and
E2-binding sites, recruits E1 and may also cause a substantial re-modeling of the E1-DNA interaction. This has not been described before for cooperating DNA-binding interactions. We suggest that this
phenomenon may be part of a sensitive switch governed by an ATP-induced
conformational change that serves to regulate E1-ori formation.
*
This work was supported by National Institutes of Health
Grant CA 13106 (to A. S.) and in part by a Wellcome International Prize Traveling Fellowship (to C. M. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, April 25, 2001, DOI 10.1074/jbc.M101861200
2
A. Stenlund, unpublished observations.
3
C. M. Sanders, unpublished data.
The abbreviations used are:
BPV-1, bovine
papillomavirus type 1;
HPV, human papillomavirus;
BS, binding site;
DBD, DNA binding domain;
OP-Cu, ortho-phenanthroline-copper;
DEPC, diethyl pyrocarbonate;
EtBr, ethidium bromide;
HA, hemagglutinin.
Mechanism and Requirements for Bovine Papillomavirus, Type 1, E1
Initiator Complex Assembly Promoted by the E2 Transcription Factor
Bound to Distal Sites*
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in prokaryotes and simian virus 40 in eukaryotes. However, BPV-1 offers several distinct advantages over other eukaryotic viral models in regard to
these studies. In the latent state, the viral chromosome is maintained
at a constant low copy number, directs a low level of gene expression,
and replicates in synchrony with the host cell chromosome (3). Thus, in
several important aspects, the viral genome behaves much like the host genome.
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Fig. 1.
Arrangement of E2-binding sites in BPV-1 and
HPV origins of replication. In BPV two E2-binding sites flank the
E1-binding site and A/T-rich region. The low affinity E2 BS12 is
located 3 base pairs downstream of the E1-binding site; the
intermediate affinity E2 BS11 is located 33 base pairs upstream of the
E1-binding site; either E2 site alone supports replication in
vivo. In HPV, the E1-binding site is positioned between two distal
E2 sites, one of which is duplicated. In HPV-18, the up- and downstream
sites are 26 and 23 base pairs, respectively, from the boundaries of
the E1 palindrome. Only one E2 site is required for replication of
chimeric HPV-18 origins, but replication efficiency improves with
multiple sites (35). The nomenclature for the BPV-1 origins used in
this study is as follows. The wild type probe with E2 BS11 and BS12 is
referred to as ori 11/12. Origins with BS11 or BS12 only are
referred to as 11/X and X/12, where X is a mutated E2 site. Origins
with high affinity E2 BS9 at either position are referred to as 9/X
(distal) and X/9 (proximal).
MATERIALS AND METHODS
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Fig. 2.
Complex formation with E1 and E2 bound to
distal sites. A, binding reactions with the indicated
probes were analyzed on polyacrylamide gels. The concentration of E1
used was 20 nM except lanes 21-23 (100 nM). The concentrations of E2 were from 1.25 to 3.75 nM and 3.75 nM without E1. For probes 11/X
(distal E2 site) and X/12 (proximal E2 site), selected reactions were
examined with anti-E1 and -E2 antibodies (lanes 7-10 and
16-19). Lanes 21-23 show complex formation at
high E1 concentration without E2; six bands were observed. Lanes
24 and 25 indicate a small mobility difference between
the E1-E2 origin complexes formed on probes 11/X or X/12. Lanes
26-43 demonstrate the dependence of E2E1-ori complex
formation on E2 site affinity. With the probe 11/12 (proximal and
distal E2 sites, lanes 46-49), the complex that formed at
low E2 concentrations co-migrated with E1E2-ori (lane
24), and formed more efficiently than the complex on probe 11/X
(lanes 3-5). B, E1E2-ori and
E2E1-ori formation assessed by glutaraldehyde cross-linking
and agarose gel electrophoresis, without (top panel) and
with ethidium bromide (25 µg/ml, lower panel). The same
reactions as in A were analyzed. Only E2E1-ori
formed on probes with a high affinity E2 site were detected
(lanes 10-12, BS9, compared with 16-18, BS 11).
Instability due to cross-linking appears to account for this in part.
All complexes were unstable in the presence of EtBr (lower
panel).
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Fig. 3.
E2 bound to a distal site recruits E1 to the
E1 palindrome. Parallel binding reactions were assembled with each
probe (E1, 35 nM; E2, 2.5-20 nM; 20 nM in reactions with E2 only). E2 bound to a distal binding
site stabilizes E1 binding to its palindrome (lanes 1-7 and
15-21), and E1E2-ori forms more readily than
E2E1-ori on probes with the wild type E2 sites (lanes
1-7 compared with 8-14). Footprints for E1 and E2
bound to DNA can be clearly discerned and suggest that E1 is bound in
the same form in each complex. The proportion of unbound probe did not
appear to be lower in reactions with E1 and the same concentration of
E2, as observed by polyacrylamide gel electrophoresis. The former
observation is likely to be an artifact of gel
electrophoresis.
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Fig. 4.
DEPC interference analysis.
A, DEPC interference was performed with probes 11/X and X/12
(both strands) for E2 and the E2E1-ori and
E1E2-ori complexes resolved in acrylamide gels. Probes were
recovered from the gel, cleaved with piperidine, and the products
analyzed on a sequencing gel. B, the results of several
experiments were analyzed using a PhosphorImager. The size of the
circles or triangles is an approximate relative
measure of the changes in band intensity by interference or enhancement
of DNA binding upon DEPC modification (large symbol, >70%
change; intermediate size symbol 40-70% change; less than
40% discernible change, small symbol. No base positions
gave greater than 70% change in band intensity as a result of
enhancement of DNA binding). The positions of the DNA-binding sites are
shown and the BPV-1 nucleotide coordinates are given.
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Fig. 5.
E2 at a distal site recruits a dimer of E1 to
the E1 palindrome. To determine the number of E1 molecules bound
in E2E1-ori, a mixing experiment was performed with probe
9/X, E2 (3.75 nM), and E1 and epitope-tagged
HAE1 mixed at different ratios (15 nM total).
Lane 1, free probe. Lanes 2-5, E1 does not binds
DNA without E2 at the given concentration or with or without anti-HA
antibody. Lane 6 is E2-bound to DNA, which does not react
with the anti-HA antibody (lane 7). Lane 8 is
100% untagged E1, which is not recognized by the anti-HA antibody
(lane 9). Lanes 10-14 are E1:HAE1
ratios of 80:20, 60:40, 40:60, 20:80 and 100% HAE1,
respectively, analyzed in the presence of anti-HA antibody. Lane
15 shows the mobility of E2HAE1-ori in the
absence of antibody. The mobility of the homodimeric complexes and the
mixed form are indicated on the right.
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Fig. 6.
Hydroxyl radical footprinting of E2 and the
E1 dimer bound to DNA. A, footprints of E2 bound to
proximal and distal sites (lanes 3 and 7, bottom
strand, and 10 and 14, top strand) and
HAE1E2-ori (lane 4, top strand, and
lane 11, bottom strand) were generated in solution without
glycerol and compared with footprints of
HAE1E2-ori and E2HAE1-ori
generated under standard reaction conditions and purified on protein
G-Sepharose beads (lanes 5 and 6, bottom strand,
and 12 and 13, top strand). Without
glycerol, discrete E2HAE1-ori footprints proved
difficult to obtain due to nonspecific binding of E1 along the DNA.
Under the reaction conditions used, binding site occupancy in solution
is low, and the reaction products bound to beads dissociate rapidly in
the presence of EtBr (25 µg/ml; see Fig. 2B), indicating
that the minimal E2E1-ori complex is the predominant form
bound to the beads. The footprints are annotated on the left
and right, and E2 protections are indicated with dark
boxes. The region of general hydroxyl radical protection is also
delineated. One protection on each strand, indicated with a *, has no
counterpart in E1E2-ori or E2 bound to DNA. E2 bound to
proximal BS9 and the E1 dimer both extensively protect a similar
region. B, footprint of an E1 dimer bound to DNA. The
periodic hydroxyl radical protections are indicated immediately
adjacent to the origin DNA sequence, and the size of each
box is a relative measure of the degree of protection. The
upstream protections (indicated * in A) are most likely due
to E1 since a similar protection is seen at a related position
downstream of the E1-binding site and in E1-ori. The results
of the OP-Cu protections over the E1-binding site are indicated by the
bars, and the hatched regions are partial
protections. The arrows indicate the boundaries of DNase I
footprints.
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Fig. 7.
Conversion of epitope-tagged
E2HAE1-ori to a multimeric E1
complex. Probe 9/X was used in the preincubation reactions, and
the assembly reactions with a 100-fold excess of untagged E1 were
performed in parallel as described below each panel. Under
these condition, only 6% of the E1 origin complexes that form would be
expected to contain a tagged E1 molecule if E1-ori is a
hexamer and forms by random assortment of E1 molecules. The control
reactions (lanes 1-8) indicate the mobility of free probe
(lane 1), the absence of EtBr stable complex formation in
the preincubation (lane 2), the mobility of E1 origin
complexes formed at high E1 concentrations (lanes 3 and
4), and their specific reactivity to the antibodies used
(lanes 5-8). Reactions were sampled from 2.5 to 25 min,
cross-linked with glutaraldehyde, and analyzed with and without
specific antibodies as indicated (lanes 9-13, no
antibody (No Ab); lanes 14-18, anti-HA antibody;
lanes 19-23, anti-E2 antibody). A,
HAE1 and E2 were preincubated before dilution into an
assembly reaction containing ATP/Mg2+. B, E2 was
omitted from the preincubation before dilution into an assembly
reaction containing ATP/Mg2+ and E2. C,
HAE1 and E2 were preincubated before dilution into an
assembly reaction without ATP/Mg2+.
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MATERIALS AND METHODS
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FOOTNOTES
To whom correspondence should be addressed: Cold Spring Harbor
Laboratory, P. O. Box 100, Cold Spring Harbor, NY 11724. Tel.: 516-367-8407; Fax: 516-367-8454; E-mail: Stenlund@cshl.org.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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