From the
We present a preliminary biochemical characterization of two
simian virus 40 mutants that affect different T antigen replication
functions. SV40 T antigen mutants dl1135 (
Simian virus 40 (SV40) has been a useful model for the study of
DNA replication and transcription as well as for better understanding
the mechanisms of tumorigenesis. The 708-amino-acid large tumor (T)
antigen is the major regulator of productive and transforming
infections(1, 2, 3, 4, 5, 6, 7) .
Many of the biochemical functions of T antigen have been mapped to
specific domains of the polypeptide (Fig. 1)(1, 8) . These include: DNA binding
(residues 131-259), ATPase/DNA helicase activity(302-627),
and DNA polymerase
A
monomer of T antigen can bind to one pentameric sequence in the absence
of ATP, but ATP not only increases T antigen binding severalfold but it
also induces a conformational change in T antigen such that 12 monomers
of T antigen become organized into a two-lobed structure spanning the
entire core ori(10, 15, 16, 17, 18) .
The T antigen dodecamer complex then melts the origin. The ability of T
antigen to bind to and melt site II is regulated by phosphorylation
(19-21). An intact zinc finger region, which maps between amino
acid residues 302 and 320, is required for hexamer assembly on the
origin and for melting of the origin DNA(22) . The presynthesis
complex consists of T antigen assembled as a double hexamer in the
presence of ATP onto a site II duplex subsequently bound by replication
protein A (RP-A), a mammalian single-stranded DNA binding
protein(7, 16, 17, 23) . With the
association of DNA polymerase
SV40
mutants dl1135 and 5080 have been studied extensively
with regard to their ability to transform cells in culture and induce
tumors in transgenic
mice(28, 29, 30, 31, 32) . The
mutant 5080 carries an amino acid substitution of P584L
located within a stretch of hydrophobic residues near the
carboxyl-terminal boundary of the ATPase/p53 binding domain. Previous
studies indicated that the 5080 T antigen expressed in
transformed murine cell lines was defective for ATPase activity and
showed an aberrant pattern of oligomerization and
phosphorylation(33, 34, 35, 36) .
The dl1135 mutant carries an in-frame deletion that results in the
expression of a T antigen missing amino acids 17-27(29) .
This mutant was of interest since it had been reported to be defective
for both ATPase and sequence-specific DNA binding, activities thought
not to require the amino-terminal domain(37) . Additionally, dl1135 is defective for transformation of some cell types
although it retains the ability to complex with pRb and p53. The
mutation appears to abolish a third transforming activity of T antigen
that can functionally replace the adenovirus E1A 300K binding function
(38).
To investigate in more detail the molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed both of the mutant proteins using the
baculovirus system and purified them by immunoaffinity
chromatography(39, 40, 41) . Purified
baculovirus T antigen has been shown to be comparable to T antigen
purified from infected monkey cells in all assays tested(42) .
Using the purified mutant proteins, we examined some of the biochemical
activities of T antigen required for replication.
We then examined the ability of the purified
mutant T antigens expressed in the baculovirus system for
sequence-specific DNA binding activity using an immunoprecipitation
assay. A modified McKay assay was done using plasmids containing sites
I or II in either a HEPES/EDTA buffer (McKay buffer) on ice or a
HEPES/MgCl
We next wished
to determine whether dl1135 and 5080 were capable of
ATP-dependent assembly on the minimal origin (site II) of SV40 DNA. The
ATP-dependent assembly of T antigen on the origin as a double hexamer
is required for the presynthesis complex (for a review, see Ref. 7).
Virshup et al.(55) showed that in using a plasmid
containing the core ori in a DNA mobility shift assay two
DNA
The SV40 mutants dl1135 and 5080 are both
replication-defective in vivo. Previous studies have suggested
that, in both cases, the mutations result in effects on activities
thought to reside in different domains. Thus, these mutants might be
useful for understanding interdomain communication. The mutant 5080, for instance, which carries a substitution of residue
P584L located within the ATPase/p53 domain of T antigen, does not bind
to pRb even though the mutation lies outside of the Rb binding region
(34). The single amino acid change also renders the protein defective
for ATPase and p53 binding(33) . Similarly, dl1135,
which carries an in-frame deletion that results in the expression of a
T antigen missing amino acids 17-27, appeared to be defective for
DNA binding and ATPase activity(37) . Both of these biochemical
activities map to a different region of the molecule than the dl1135 mutation. In this paper, we report the characterization
of the purified baculovirus T antigen encoded by those mutants.
Tack et al.(33) studied the 5080 T antigen
expressed in transformed C3H10T1/2 cells and found that it was
defective for ATPase. As expected, we also found that the purified
baculovirus-expressed 5080 was defective for ATPase, although
it showed some activity, retaining approximately one-sixth of the
wild-type activity. Since T antigen ATPase activity is required for its
helicase and unwinding functions, the greatly reduced ATPase activity
is sufficient to explain the replication defect of 5080. Tack et
al.(33) also examined the oligomeric state of 5080 T antigen in C3H10T1/2 cells by glycerol gradient sedimentation.
Also Ludlow et al.(34) studied oligomerization using
extracts of CV-1P cells transfected with 5080. In both
instances, 5080 was found to exist in 5-10 S forms only
compared with wild-type 20 S and 14 S forms. Thus, 5080 appeared to be defective for forming higher oligomers.
By
electrophoresis in nondenaturing gels, we found that the purified 5080 T antigen was in a high molecular weight form that barely
penetrated the gel. Lin et al.(36) reported a similar
result with a T antigen harboring the same mutation. The large
oligomers which barely enter the gel are reminiscent of zinc finger
mutants described by Loeber et al.(22) . Two possible
explanations for the discrepancy in the oligomerization results from
the different techniques are the following: 1) the higher molecular
weight forms represent aggregated T antigen resulting from the
purification process and are not reflective of the in vivo situation or a real property of 5080; 2) the lower
molecular weight forms reported by Tack et al.(33) are
an artifact and a consequence of the aggregated T antigen being
discarded in the clarification step of the immunoprecipitation.
We
found that the dl1135 T antigen purified from
baculovirus-infected insect cells was able to stimulate SV40 ori-dependent DNA replication in vitro. Although less
active than wild-type, 1135 exhibited significant levels of
ATPase, helicase, and ori unwinding activity. Gel-shift
experiments indicated that 1135 could efficiently bind to and
assemble double hexamers on the ori. On the other hand, 1135 showed very reduced binding to site I and site II in
immunoprecipitation DNA binding assays. One explanation for the
difference in the binding of site II by 1135 in the modified
McKay assay versus the gel-shift assay may lie in a decreased
affinity or a quick on/off rate by the mutant compared with wild-type.
The cross-linking agent used in the gel-shift assay may compensate for
a quick release of the core ori by ``freezing'' the
complex so that it is detectable, whereas without a cross-linking
agent, the immunoprecipitation assay does not allow the complex to be
detected. This may also account for the results of Clark et
al.(37) , who reported little or no origin binding activity
by dl1135 as measured by a method similar to the McKay assay.
The dl1135 mutant is one of several mutants in the
amino-terminal domain which can replicate DNA in vitro but not in vivo. For example, mutants 3213 (E107K), 5110 (D44N), and
GMSV40 (
We thank Robert Lanford for the recombinant
baculovirus 941T. We are especially grateful to Tom Kelly and his
laboratory for sharing their expertise, protocols, and HeLa cell
extract. We thank all members of the Pipas laboratory, especially Tim
Kierstead, Ashok Srinivasan, and Alex Castellino, for their advice and
comments. Critical reading of the manuscript by Roger Hendrix and Keith
Peden is greatly appreciated.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
17-27
amino acids) and 5080 (P-L) have been studied extensively with
regard to their ability to transform cells in culture and induce tumors
in transgenic mice. Both mutants are defective for viral DNA
replication in vivo. In order to assess in more detail the
molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed the mutant proteins
using the baculovirus system and purified them by immunoaffinity
chromatography. With each of the purified proteins, we examined some of
the biochemical activities of T antigen required for replication, viz. ATPase, binding to the origin of replication (ori) and assembly on ori, DNA helicase and
unwinding, and replication in in vitro assays. Consistent with
previous studies, we found that the 5080 protein is defective
for multiple biochemical activities including ATPase, helicase, ori-specific unwinding, and ATP-induced hexamerization.
However, this mutant retains some sequence-specific DNA binding
activity. In contrast, the dl1135 protein exhibited
significant levels of activity in all assays, including the ability to
drive SV40 DNA replication in vitro. Thus, dl1135 is
one of several mutants with an altered amino-terminal domain which can
replicate DNA in vitro, but not in vivo. Thus, while
the 5080 mutation affects a T antigen enzymatic function
directly required for viral DNA synthesis, dl1135 may alter an
activity required to prepare the cell for viral replication.
-primase binding (1-82;
260-517)(3, 9) .
Figure 1:
Sites of mutations in SV40 large T
antigen, functional domains, and regions recognized by relevant
monoclonal antibodies. The primary sequence of T antigen is linearly
diagrammed and numbered. The vertical lines with a P at the ends indicate positions of clustered phosphorylated serine
and threonine residues. Mapped regions of large T antigen indicated
include binding sites for products of the retinoblastoma (Rb)
and p53 tumor suppressor genes, DNA binding domain, ATPase/helicase
domain, and putative zinc (Zn) finger region. Arrows show the location of mutants dl1135 (17-27) and 5080 (P584>L). The regions recognized by monoclonal
antibodies specific for SV40 T antigen, PAb419 and PAb108, detect
epitopes at the amino terminus, and PAb101 and KT3, specific to
epitopes within the carboxyl terminus. HR, host range domain. cr1 and cr2, conserved regions 1 and 2,
respectively.
The first step in viral DNA
synthesis is the sequence-specific binding of T antigen within the
origin of replication. The region of the origin of replication contains
three T antigen binding sites (site I, site II, and site III) in
decreasing order of affinity(1, 10) . Site II, the core ori, is part of a 64-base pair sequence that is necessary and
sufficient for viral DNA replication; site I and site III can stimulate
viral replication(10, 11) . Both site I and site II have
pentameric 5`-GAGGC-3` sequences, but in different arrangements.
Additionally, site II has two other regions, a 15-base pair inverted
repeat on the early side and a 17-base pair A/T-rich region on the late
side, which are involved in ori melting and untwisting of the
DNA helix, respectively(12, 13, 14) .
-primase, hexameric T antigen in
complex with RPA unwinds the DNA bidirectionally in a reaction driven
by the hydrolysis of
ATP(7, 24, 25, 26, 27) .
Recombinant Baculovirus Vectors That Express Wild-type
and Mutant T Antigens
Recombinant baculovirus 941T which
expresses wild-type SV40 large T antigen was kindly provided by Robert
Lanford(39) . Both wild-type (wt) and mutant T antigens were
expressed in BTI-TN-5B1-4 (``High 5'') insect cells
(Invitrogen). The T antigen mutants dl1135, which has a
deletion of amino acids 17-27, and 5080 which has an
amino acid substitution of P584L have been
described(28, 29) . The plasmids pdl1135 and p5080 contain the complete mutant viral genomes inserted at
the BamHI site of pBR322 (28) or a
derivative(29) . Recombinant baculovirus containing these
mutants were constructed by digesting the pdl1135 plasmid or
the p5080 plasmid with BamHI and StuI and
ligating the purified SV40 fragment (2657 base pairs) into the BamHI and SmaI sites of the baculoviral transfer
vector pVL1393. Subsequently, the recombinant transfer vectors
containing the cDNAs of the mutants were made in an exchange reaction
with 941T following digestion of both the wild-type and mutant
constructs with EcoNI. The new recombinant transfer DNA was
cotransfected with purified wild-type baculovirus DNA into insect cells
followed by identification and purification of the recombinant virus by
three rounds of dot-blot hybridization.
T Antigen Purification
High 5 insect cells were
infected with the above recombinant baculoviruses, and the infected
cells were lysed 36-42 h later using a buffer containing 0.2 M LiCl, 20 mM Tris, pH 8.0, 1 mM EDTA, 0.5%
Nonidet P-40, 0.2 mM dithiothreitol (DTT),(
)and a protease inhibitor mixture. The T antigens were
immunoaffinity purified from the lysates using a protein A-Sepharose
(Pharmacia) column cross-linked to either PAb416 (43) or PAb101 (44) as described previously(45, 46) . After high
pH elution (pH 11.4), the proteins were dialyzed overnight against a
buffer containing 10 mM HEPES, pH 8.0, 1 mM EDTA, 0.1 M NaCl, 50% glycerol, 1 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride.
Plasmid DNAs
pUC.HSO contains SV40 nucleotides
5171 to 128 (47). The pDV.XH plasmid carries the SV40 minimal origin of
replication corresponding to 64 base pairs of sequence between SV40
nucleotides 5211 to 32(48) . pTBS1 contains T antigen binding
site I within SV40 nucleotides 5171 to 5228(49) .
In Vitro Replication of SV40 DNA
The reactions
were performed according to the methods previously
described(47, 50) . HeLa cell extracts, kindly provided
by Dr. Thomas J. Kelly (Johns Hopkins University), were prepared as
described previously(51) . The DNA replication products were
ethanol-precipitated, subjected to agarose gel electrophoresis, and
visualized by autoradiography.
Immunoprecipitation DNA Binding
Modified
McKay(
)assays were done with site I and site II
DNAs using two different buffers and conditions: McKay buffer and
conditions and replication buffer with conditions appropriate for in vitro replication(52) . The McKay reaction buffer
contained 10 mM HEPES, pH 7.3, 0.1 mM EDTA, 0.05%
Nonidet P-40, 1 mg/ml bovine serum albumin, 2 mM DTT, and
radiolabeled DNA. T antigen was then added, and the reactions were
incubated on ice for 45 min to 1 h. Two hundred microliters of a 1:1
mixture of KT3/PAb101 hybridoma supernatant was added and the mixture
was incubated for 30 min on ice, followed by the addition of 50 µl
of protein A-Sepharose. The precipitated protein
DNA complex was
washed three times with 10 mM Tris-HCl, pH 8.0, 150
mM NaCl, 0.5% Nonidet P-40. The replication buffer contained
30 mM HEPES, pH 7.5, 7 mM MgCl
, 1 mM DTT, 40 mM creatine phosphate, 10 mg/ml bovine serum
albumin, plus radiolabeled DNA. These reactions were started by first
adding T antigen and then 1 mM AMP-PNP (Sigma). The reactions
were incubated at 37 °C for 30 min, followed by the addition of 200
µl of KT3/PAb101 (44, 53) and incubation for 20 min
at 37 °C. The protein A-Sepharose was then added and incubated for
15-20 min. The immunocomplex was washed with 1 M
Tris-HCl, pH 7.5, 0.5 M EDTA, and 10% Nonidet P-40. In both
instances, the final pellet was resuspended in 10 mM Tris-HCl,
pH 7.5, 7.5 mM EDTA, 0.5% SDS, 10% glycerol and heated to 65
°C for 2 min. The protein A-Sepharose beads were pelleted for 2
min, and the supernatant was analyzed by electrophoresis on a 1% or
1.4% agarose gel followed by autoradiography.
Nondenaturing Polyacrylamide Gradient Gel
Electrophoresis
The procedure has been described by Loeber et al.(22) . T antigen was incubated at 37 °C for
20-30 min in a reaction buffer consisting of 30 mM
HEPES, pH 7.5, 7 mM MgCl, 1 mM DTT, and 4
mM ATP. The protein was then cross-linked for 10-15 min
at 37 °C with 0.1% glutaraldehyde. Sample buffer containing 15% 0.5 M Tris-HCl, pH 6.8, 50% glycerol, and 0.0025% bromphenol blue
at pH 6.8 was added, and the oligomers were analyzed on a 4-22%
nondenaturing polyacrylamide gradient gel in Tris glycine buffer for 11
h at 4 °C at 35 mA.
ori-specific Band Shift
The assay was performed as
described by Virshup et al.(48) . A 67-base pair
fragment containing the ori (site II) was obtained by
digesting the pDV.XH plasmid with ApaLI and SnaBI,
purified and radiolabeled. T antigen was added to an assay mixture
containing HEPES, pH 7.8, 7 mM MgCl, 40 mM creatine phosphate, 1 mM DTT with 4 mM AMP-PNP
(Sigma), and 3 fmol (10,000-15,000 cpm/reaction) of
P-labeled DNA fragment and the reactions were incubated at
37 °C for 30 min after which the cross-linker glutaraldehyde was
added to a final concentration of 0.05% and the reactions were
incubated at 37 °C for 5 more min. The DNA
protein complexes
were electrophoresed on 0.5% agarose, 2.4% acrylamide gel in 89 mM Tris borate, 2 mM EDTA (TBE) for approximately 1 h at
room temperature at 200 V. The gels were dried on DE81 Whatman paper
and then exposed to XAR film at -70 °C with an intensifying
screen.
ATPase Assays
The assay buffer consisted of 25
mM HEPES, pH 7.0, 5 mM MgCl, 0.1 mM EDTA, 0.05% Nonidet P-40, 1 mM DTT, and 1.3 µM ATP including [
-
P]ATP. Each reaction
mixture contained 10% glycerol. The reaction was started by the
addition of T antigen and incubated at 37 °C. At timed intervals,
1-8 µl of reaction mixture was pipetted into tubes containing
an equal volume of 0.75 M KH
PO
, pH
3.5. Samples of 1-4 µl were subsequently spotted onto
polyethyleneimine-cellulose plates and developed in 0.75 M
KH
PO
at room temperature for approximately 2 h.
The plates were then exposed to XAR film, and the corresponding ATP and
ADP spots were cut out and quantitated in a liquid scintillation
counter, or the appropriate spots were quantitated by the AMBIS
radioanalytic imaging system.
DNA Helicase Assay
This assay is based upon the
method of Stahl et al.(54) . The annealed substrate
consisting of M13mp18 (Life Technologies, Inc.) with a 17-nucleotide
forward-sequencing primer (U. S. Biochemical Corp.) was extended from
the primer using Klenow DNA polymerase in the presence of
[-
P]dATP, dGTP, and dTTP as described by
Stahl et al.(54) . The assay mixture consisted of 0.3
fmol of annealed primer, 25 mM Tris, pH 7.5, 5 mM MgCl
, 2 mM ATP, 1 mM DTT, 40
mM phosphocreatine, 0.5 µg of creatine phosphokinase, 0.5
µg of poly(dI-dC) (Boehringer Mannheim). The reaction was started
with the addition of 100 to 500 ng of T antigen followed by incubation
at 37 °C for 1 h. The reaction was stopped by the addition of an
equal volume of 1.5% SDS, 0.2 M EDTA. Subsequently, 5 µl
of sample buffer was added to each reaction, and the reactions were
electrophoresed on a 12% acrylamide gel in TBE buffer for 2 h at 200 V.
ori-dependent Unwinding Assays
The unwinding
assays were performed as described by Virshup et
al.(55) . The plasmid pUC.HSO, containing the minimal
origin of replication, was linearized with HindIII, labeled
using the Klenow fragment and [-
P]dCTP, and
digested with BamHI and HinfI. This results in two
labeled fragments, an ori
and an ori
fragment. The ori
fragment served as an internal control for the unwinding
reactions. T antigen was incubated with 0.5 fmol of labeled substrate
DNA in a buffer containing 30 mM HEPES, pH 7.8, 7 mM MgCl
, 4 mM ATP, 40 mM creatine
phosphate, and single-stranded DNA binding protein (United States
Biochemical Corp.). The reactions were incubated for 1 h at 37 °C
and then stopped with 0.5% SDS, 10 mM Tris, pH 7.5, 5 mM EDTA, and proteinase K (10 mg/ml). The incubation was continued
for 1 h at 37 °C. DNA sample buffer was then added, and the
reactions were heated at 65 °C for 2 min. The reactions were then
analyzed on an 8% acrylamide gel, dried on DE81 Whatman paper, and
autoradiographed with XAR film.
Purification of T Antigens
Recombinant
baculoviruses expressing 5080 and dl1135 were
constructed as described under ``Experimental Procedures,''
and the T antigens were subsequently immunoaffinity-purified from
infected insect cells. Samples of each protein were assessed for purity
and integrity by silver staining and Western blot analysis (Fig. 2). The silver stain shows a predominant band of
approximately 97 kDa for both mutant and wild-type T antigens. Based on
a visual assessment of this gel, we estimate that 5080 and dl1135 T antigens are greater than 90% pure. Fig. 2B shows a Western blot of purified dl1135 protein, and
wild-type T antigen demonstrates that the monoclonal antibodies PAb101
and KT3, which recognize epitopes within the carboxyl-terminal region
of T antigen, detect both mutant and wild-type proteins. However, the dl1135 deletion results in a loss of reactivity for PAb419 and
PAb108. Since both PAb419 and PAb108 recognize denaturation-resistant
epitopes, it seems likely that the deletion of residues 17-27
encompasses the actual epitopes for these monoclonal antibodies. The
mutant protein 5080, like wild-type T antigen, is detected by
antibodies PAb101 and KT3 as well as PAb419 and PAb108 (not shown).
Figure 2:
Immunoaffinity-purified
baculovirus-expressed wild-type and mutant T antigens. A,
silver-stained SDS-PAGE with electrophoresed proteins (1 µg). The
molecular masses of marker proteins (Bio-Rad) are shown at the left in kilodaltons. B, Western blot of denaturing
polyacrylamide gel with electrophoresed wild-type and mutant
baculovirus T antigens. Antibodies used for detection are indicated
below the immunoblot. In the lanes labeled KT3/PAb101, a
mixture of these two antibodies was used. Immunoblot detection employed
enhanced chemiluminescence (ECL; see ``Experimental
Procedures'').
Oligomerization and DNA Binding
When
electrophoresed through a nondenaturing gradient gel, T antigen
resolves as multiple oligomeric forms that appear as a ladder extending
from the monomer to dodecamer and higher. At 37 °C in the presence
of ATP and magnesium, T antigen undergoes a conformational change such
that the predominant species is the hexamer(15, 56) .
This reaction can occur in the presence or absence of DNA; however, DNA
cooperatively enhances protein to protein
association(18, 56) . T antigen assembles on the origin
of replication (ori) as a double hexamer in the presence of
ATP and participates in localized melting of the
origin(7, 12, 13) . Therefore, we wished to see
if the mutant proteins formed the normal pattern of oligomeric forms
and if they formed hexamers in response to ATP. The dl1135 protein resolved into an oligomeric ladder in the absence of ATP
at 4 °C without cross-linker, but unlike wild-type extended only
from monomer to apparently hexamer with clear resolution. Some higher
oligomeric forms were present, but these appeared fainter than with the
wild-type T antigen (Fig. 3). There also seems to be a
non-integer band with 1135 which is not present with
wild-type, which we have seen in multiple experiments and for which we
cannot account. When cross-linker was added at 4 °C, the oligomeric
forms appeared to be indistinguishable from wild-type. With the
addition of 4 mM ATP at 37 °C, some of the dl1135 protein shifted to the hexameric form, although most of it
appeared to be in the monomeric form, suggesting that the response was
less efficient than wild-type.
Figure 3:
Nondenaturing gradient gel for resolution
of oligomeric forms of T antigen. Immunoaffinity-purified wild-type and
mutant T antigens were electrophoresed through a 4-22% acrylamide
gradient gel following incubation for 20-30 min at 37 °C in a
HEPES/Mg buffer with or without 4 mM ATP.
The cross-linker glutaraldehyde was added to a final concentration of
0.1% except for a control with T antigens at 4 °C in the same
buffer (see ``Experimental Procedures''). The final glycerol
concentration in all reactions was 15%. The gel was fixed and
silver-stained.
Most of 5080 protein seems
to be a smear in the gel with a only few lower molecular weight bands
migrating near the level of dimer in the wild-type lane at 4 °C
without cross-linker (Fig. 3). With cross-linker, the few lower
molecular weight bands are no longer visible, and there appears to be
more smearing toward the top of the gel, suggesting more higher
oligomeric or aggregated forms. The 5080 mutant also did not
show any monomer as did both the wild-type and 1135 proteins.
At 37 °C with cross-linker in the presence and absence of ATP, the 5080 protein did not appear to enter the gel but remained at
the top of the gel, suggesting that perhaps the protein forms a greater
aggregate at the higher temperature or that the smaller oligomers are
unstable at the higher temperature. This property of the 5080 T antigen has been reported previously by Lin et
al.(36) .
buffer with ATP and without EDTA (replication
buffer) at 37 °C. The wild-type T antigen appeared to bind to site
I and site II slightly better in McKay buffer (on ice) than in
replication buffer at 37 °C (Fig. 4). The 5080 T
antigen bound to site I nearly as well as wild-type in replication
buffer. In contrast, 5080 bound to site I very inefficiently
in McKay buffer, although it retained some specific DNA binding
activity (Fig. 4) The 1135 protein also could
specifically bind to site I, but the efficiency of the binding was much
less than with wild-type.
Figure 4:
Immunoprecipitation DNA binding assay.
Affinity-purified T antigen was added to 0.3 ml of reaction buffer in
the concentrations as indicated. A, the reaction buffer
contained labeled restriction enzyme-digested (HincII) plasmid
harboring site I. B, the reaction buffer contained labeled
plasmid harboring the minimal origin (site II) digested with
restriction enzymes ApaLI and NaeI. HEPES/EDTA (on
the right) or HEPES/Mg with AMP-PNP
incubated with the T antigen on ice or at 37 °C, respectively, for
30-45 min. The DNA-protein complex was precipitated with a
mixture of monoclonal antibodies KT3/PAb101 followed by the addition of
protein A-Sepharose. The pellets were washed and resuspended in sample
buffer (see ``Experimental Procedures''), then heated for 2
min. The supernatants were electrophoresed through a 1.4% agarose gel,
and the gel was subsequently prepared for autoradiography. Arrows indicate the specific band.
The 5080 T antigen bound very
inefficiently to site II under both reaction conditions (Fig. 4).
Site II binding by the 1135 protein was barely detectable when
assayed in McKay buffer (Fig. 4). Clark et al.(37) also reported detecting no ori binding for
the 1135 protein purified from infected CV1 monkey cells in a
similar immunoprecipitation DNA binding assay. However, 1135 did bind to site II in replication buffer, but, in addition, it
bound two other fragments. This binding is not entirely nonspecific
since the fastest migrating band of 458 base pairs was not bound by 1135. This pattern of binding was seen in several independent
experiments and with different protein preparations.
protein complexes are retarded, which correspond to a
faster-migrating T antigen hexamer bound to the origin and a
slower-migrating T antigen dodecamer bound to the origin. In the
gel-shift assay, the mutant 5080 gave no distinct band shifts,
only a diffuse smear not seen in the control lane (data not shown). The
smear suggests some ability of 5080 to bind the core ori; however, it fails to assemble hexamers in the presence of
ATP. When the mutant T antigen dl1135 was examined in a
gel-shift assay, it bound to site II both as a dodecamer and as a
hexamer similar to wild-type (Fig. 5). This is consistent with
the results of the nondenaturing gradient gel experiment which showed
that dl1135 is able to hexamerize in response to ATP. Thus,
despite the different McKay site II result, this assay indicates that 1135 binding to ori is normal although this assay
would not detect additional ``nonspecific binding.''
Figure 5:
DNA mobility shift assay. The probe was a
minimal origin (site II)-containing fragment. T antigen was added to an
assay mixture containing the replication buffer with 1.5 mM AMP-PNP and 3 fmol (10,000-15,000 cpm/reaction) of P-labeled DNA fragment. The reactions were incubated at 37
°C for 30 min followed by the addition of the cross-linker
glutaraldehyde to a final concentration of 0.05%. The DNA-protein
complexes were electrophoresed on an agarose-acrylamide gel and
prepared for autoradiography.
In
the course of our studies, we noted a disconcerting variability in the
biochemical activity profile of different 1135 T antigen
preparations. A gel-shift assay using the ori fragment with a
preparation different from the one used in Fig. 5showed more
hexamer than dodecamer, suggesting a loss of cooperativity between
hexamers. This preparation was defective for replication and ori-specific unwinding, but retained ATPase and helicase
activity and the same immunological reactivity as the
replication-positive preparations of dl1135. Apparently, it is
possible to disrupt specific T antigen activities by purification. This
emphasizes the need to assay multiple preparations of each mutant
protein for each activity.
ATPase Activity of Mutant T Antigens
Wild-type T
antigen carries an ATPase activity which along with the helicase
activity is required for unwinding DNA strands for
replication(54, 58) . The mutant 5080 was very
defective for ATPase activity as reported by Tack et
al.(33) , retaining only about one-sixth of the ATPase
activity of wild-type T antigen (Fig. 6). Increasing the amount
of T antigen used in the assay also increases the amount of ATP
hydrolysis for both dl1135 and wild-type, although less so for 5080 (Fig. 6). As expected, the dl1137 protein
showed no measurable ATPase activity (Fig. 6). The 1135 T antigen exhibited a significant level of ATPase activity ranging
from 50-70% of wild-type with different preparations. This is in
contrast to the report of Clark et al.(37) , which
reported that 1135 was defective of ATPase activity. Possible
explanations of the discrepancy are provided under
``Discussion.''
Figure 6:
ATPase assays. The reactions were started
by the addition of T antigen, 941T (wild-type), 1135, 5080, 1137, and were incubated at 37 °C. The
assay buffer contained both cold and
[-
P]ATP (see ``Experimental
Procedures''). A, samples of each reaction mixture were
taken at different time points, and a portion was spotted onto
polyethyleneimine plates for thin layer chromatography. B, ATP
hydrolysis determined for different concentrations of the mutant and
wild-type T antigens; the time interval used was 15 min. The spots
corresponding to ATP and ADP were cut out of the polyethyleneimine
plates and quantitated in a liquid scintillation counter or the ATP and
ADP spots were quantitated by AMBIS radioanalytic imaging.
, 941T;
, dl1135; &cjs2110;, 5080; dl1137 values were not above background and were not
shown.
DNA Strand Displacement Assay
The DNA helicase
activity of T antigen was assessed using M13mp18 annealed to a
complementary synthetic oligonucleotide (17-mer) as a substrate.
Different amounts of T antigen were incubated with labeled annealed
substrate in replication buffer at 37 °C for 1 h. The products were
then electrophoresed through a 12% polyacrylamide gel in TBE buffer and
then prepared for autoradiography. The results are shown in Fig. 7. The dl1135 mutant was active for helicase
activity, exhibiting about 40% the level of wild-type. As expected, no
helicase activity was detected for the ATPase-defective 5080 protein.
Figure 7:
DNA
helicase assay. Varying amounts of mutant and wild-type T antigens were
incubated under replication conditions with end-labeled oligonucleotide
annealed to single-stranded M13 DNA. The products were electrophoresed
through a 12% native polyacrylamide gel and prepared for
autoradiography.
Origin-specific Unwinding
In order to assess the
ability of the dl1135 protein to unwind the origin, a plasmid
containing minimal ori was linearized and radiolabeled. The
labeled fragment was then digested with two restriction enzymes to
generate ori and ori
fragments, thus allowing an internal negative control. T antigen
was then incubated with the labeled substrate for 1 h, and the products
were electrophoresed through an 8% TBE gel. Fig. 8shows that dl1135 unwinds the origin, although less efficiently than
wild-type T antigen.
Figure 8:
Origin (ori)-specific unwinding
assay. T antigen at the amounts indicated was incubated with
origin-specific and non-origin DNA in replication buffer containing E. coli SSB at 37 °C for 1 h. The reaction products were
electrophoresed in an 8% polyacrylamide gel which was subsequently
dried and exposed to film.
In Vitro DNA Replication of Wild-type and Mutant T
Antigens
Both dl1135 and 5080 are defective
for replication of viral DNA in monkey cells(28, 29) .
We next evaluated the ability of purified 5080 and dl1135 to activate DNA replication in an in vitro system. The dl1135 and 5080 proteins were incubated for 2 h in
replication buffer with HeLa cell extract at 37 °C. The products
were resolved by electrophoresis through a 1% agarose gel and
visualized by autoradiography. Incorporation of radiolabel into DNA was
measured by the acid-precipitable counts to quantify the amount of
replication. The results are shown in Fig. 9. As expected, the 5080 T antigen was completely defective in this in vitro assay. Surprisingly, the dl1135 protein showed
significant activity, approximately 40-60% of wild-type.
Figure 9:
In vitro SV40 DNA replication by
wild-type and mutant T antigens, dl1135 and 5080 using human cell extracts. The reaction mixture included the
template, pUC.HSO and the concentrations of T antigen indicated in the
conditions described under ``Experimental Procedures.'' The
reactions were incubated at 37 °C for 2 h. The replication products
were electrophoresed through a 1% agarose gel and then visualized by
autoradiography. The control was the reaction mixture without T
antigen. A, replication activity of wild-type and 5080; B, replication activity of wild-type and dl1135.
67-82) are all defective for in vivo replication but support replication in
vitro(59, 60) . The amino terminus of T antigen
plays a role in several functions including transformation and
transactivation and interacts with numerous cellular proteins, i.e. pRb, p107, etc.(61, 62) . Although the amino
terminus may not be directly involved with the enzymatic/origin binding
activities of T antigen in vivo, it may activate a cellular
target or affect some preliminary step to the initiation of
replication.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.