Copyright
©
1986, AmericanSociety
forMicrobiologyAllosteric
Control of Simian Virus 40 T-Antigen Binding
to
Viral
Origin
DNA
BRIGITTE VOGT, EVANGELIA VAKALOPOULOU, ANDELLEN FANNING*
Institutefor Biochemistry, 8000 Munchen 2, Federal Republic of Germany
Received 11December 1985/Accepted 6 March 1986
Simian virus 40(SV40) large tumorantigen (T antigen)possessesseveral biochemical activities localized in different domains of theprotein.These activities includesequence-specific bindingtotwomajor sites,IandII,
intheSV40 control region, ATPase,andnucleotide-binding activity.In the presentcommunication,wepresent
evidence that specific binding of immunopurified T antigen to SV40 DNA is markedly inhibited by low concentrations ofATP,dATP, GTP,and dGTP. The inhibition is reversible after removal of thenucleotide, suggesting that simple nucleotide bindingrather thanacovalent modification of Tantigeninthepresenceof ATP isresponsible for the inhibition. The results suggest that Tantigenmay assumetwoconformations, one active and one inactive in binding to the SV40 origin of replication. In the presence of purine nucleoside triphosphates,the inactive conformation is favored.
The
simian virus
40(SV40)
infectious
cycle in cultured
monkey cells
isregulated
primarily by
the gene Aproduct,
large
T (tumor)antigen
(38, 45). Tantigen is
amulti-functional
protein required for
regulation of early and late
viral
geneexpression and initiation of
viral
DNAreplication.
In
addition, it influences
patterns of cellular transcription
and
DNAsynthesis
and maywell affect cellular
metabolism
in
moresubtle
ways.The known
biochemical
properties of
Tantigen
arediverse, including
sequence-specific binding
to twomajor sites in the SV40 control
region,
nonspecific
binding
tonative and denatured
DNA,
binding
toacellular
phosphoprotein, p53, nucleotide
binding,
andcleavage of
ATP
and dATP.
Sequence-specific
binding of
Tantigen
tothe
SV40 control
region mediates
autoregulation of early
transcription (14, 22, 39), initiation
of viral
DNAreplication
(13, 32,
43),and
stimulation of
latetranscription (6, 7, 26, 27)
in a
temporally
controlled sequence.Viral
DNAreplication
appears to
require
ATPaseactivity in addition (10). Studies
with
polyomavirus suggest thatnucleotide-binding activity
may
be distinct from ATPase-substrate
binding
and mayalso
be essential
for viral
DNAreplication (12).
T
antigen
appears tobe
composed
of
severaldomains,
some
of which
canbe
clearly correlated with
asubset
of its
biological
orbiochemical activities.
Forexample, studies
with SV40
mutantsand hybrid virus
proteins have localized
the
DNA-binding domain
of
Tantigen
to anamino-terminal
region (10,
33,35). Studies with
monoclonal antibodies (9)
and
mutant Tantigens (10)
haveimplicated
thecarboxy-terminal
halfof
Tantigen
in ATPaseactivity.
TheATP-binding site of
Tantigen
isalso located
atthe
carboxy-terminal
endof
theprotein
(12).Thus, it
isconceivable
thatthese biochemical
activities, being localized
atdifferent
endsof
alarge molecule,
may beindependent of
each other. As afirst approach
tothis
question,
we haveinvestigated
thesequence-specific DNA-binding
activity of
Tantigen in
the presence and absence of ATP. The results demonstrate that DNAbinding
andnucleotide
binding
are notindependent
activities
of
Tantigen
and suggest that nucleotidebinding
allosterically inhibits
Tantigen
binding
to the viralorigin
region. Possible biological implications of this
newpropertyof
Tantigen
arediscussed.
*Correspondingauthor.
(A
preliminary
accountof this work
waspresented
atthe
Cold
Spring Harbor Conference
onCancer
Cells, Cold
Spring Harbor, N.Y.,
4to8September, 1985.)
MATERIALS ANDMETHODS
Cells
and antibodies. The cultureof COS1 (20) cells
hasbeen
described
previously (8). Hybridoma cells obtained
from E. Harlow
(23),
R.Ball
(4), and
Gurney
et al.(21a;
PablO8)
werecultured in
Dulbecco-modified Eagle medium
(GIBCO Laboratories, Grand Island, N.Y.) supplemented
with
15% fetal calf
serum(GIBCO
orBiochrom KG, Berlin,
Federal
Republic
of Germany)
andantibiotics.
Immunoglob-ulin
G
(IgG)
waspurified
asdescribed
previously (16). Pab2O4
and
Pab2O5
IgG (9)
were agift
from David Lane,
London,
England.
SV40 DNAbinding. T
antigen
was extractedfrom
COS1
monkey
cells(20)
inlysis buffer (50
mMTris
[pH
9]-120
mMNaCl-0.5% Nonidet P-40) for
30min at0°C
(107
cells
perml).
Samples of
100,ul (50
to 100 ngof
Tantigen)
wereim-munoprecipitated with
5 ,ugof
purified
monoclonal
PablO8IgG and fixed
Staphylococcus
aureus(28)
asdescribed
previously (17). PablO8 recognizes
adenaturation-resistant
epitope
located between 0.65 and 0.62
SV40
mapunits
(21a).
It
binds
essentially all soluble
Tantigen and has
nodetect-able
effect
onTantigen binding
tothe
SV40
origin
regioui,
asshown
by
DNasefootprinting
(24, 41). ATPase
activity and
ATP
affinity
labeling of
Tantigen
werealso
readily
detect-able
in
the presenceof
PablO8(24).
Immunecomplexes
were washedwith
50 mMTris
(pH
7.5)-150
mMNaCl-5
mMEDTA-0.05% Nonidet
P-40(NET)
with orwithout
0.5 MLiCl and
thenwith
NET.Immunopurified
Tantigen complexes
were taken up in 0.15 mlof
binding
buffer
(10
mM HEPES[N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid]
[pH
7.8]-80
mMKCl-0.5 mM
MgCl2-1
mM dithiothreitol-1 mMphenylmethylsulfonyl fluoride-0.2
mgof
glycogen
per ml -1 mgof
bovine serum albumin per ml-50 ,ug ofsonicated
Escherichia coli DNA per
ml).
The DNAtemplates
used werepSV-wt (18), wild-type
SV40 DNAcloned
in theBamHI site of pAT153 (47),
andp1097
(24),
theSV40
deletion
mutant cs1097(14), which
lacksT-antigen-binding
site I
(nucleotides
5178 to5208),
cloned in theopposite
orientation
in the BamHI siteof
pAT153.
Specific
DNA(0.25
765
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766 VOGT ET AL.
108
204
205
1630
402
405
416
419
Pcib
M
+ - + - + -+
- + -
+
-
M+
- +
-
ATP
Al
FIG. 1. ATPinhibitsSV40DNA
binding
ofimmunopurified
Tantigen.
Duplicate samples
ofCOSiTantigen
wereimmunopurified
oneach of thepurified
monoclonal antibodies(Pab)
indicated. One of thetwosamples
waspreincubated
with 20 mM ATP for 30 mmnatO0C(+).Binding
toend-labeledfragments
ofpSV-wt
DNAwasassayed
withoutE. coli DNAasdescribed in Materials and Methods. Marker DNA (M)was12.5 ng(5%) of theinput
DNA.SV40 Hindlllfragments
are indicatedonthe left.pLg
perassay)
was presentin excess and incubated with Tantigen
toequilibrium,
unless stated otherwise. In someexperiments,
E. coli DNA wasomitted
from thebinding
buffer,
as stated in thefigure legends.
Bound DNA wasdissociated from
immunecomplexes
andanalyzed
by agarosegel
electrophoresis
andautoradiography
asde-scribed
previously
(18).Gel retardationDNA-binding assay.
Plasmid
pON-wt
car-rying
asynthetic
19-base-pair
DNA sequencefrom
T-antigen-binding site
I wasshown
tobind Tantigen with
anaffinity comparable
to thatof
intactorigin
DNA(41).
TheEcoRI-Sall fragment of pON-wt carrying
this sequence wascloned
into the EcoRI-SalI sites of
vectorpSDL13 (30) and
designated pSDL13-wt.
ATaqI-NaeI
fragment of
89base
pairs carrying
thebinding site
was5' end labeled andpurified
by
polyacrylamide gel electrophoresis. Biochemically
puri-fied
lytic
Tantigen
(44), agift from
P. Tegtmeyer, wasincubated
with4 ngof thepurified fragment
and 1.75 ,ugof unlabeled E. coli DNA for 1 h at0°C
in 10 mM Trishydrochloride
(pH7.4)-i
mM EDTA-20 mM NaCl. Thereaction
mixwasthenanalyzedby
electrophoresis
(100 Vfor
2
h)
in6%
polyacrylamide gels
in 10 mM Tris acetate(pH
7.8)-i
mM EDTA as describedpreviously (19)
andautora-diography.
RESULTS
Inhibition ofT-antigen-DNA binding by ATP. The SV40-transformed cell line
COS1,
whichconstitutively
expresses wild-type SV40Tantigen (20),wasselectedas a sourceofTantigen.
Tantigen
wasimmunopurified
witheight
different monoclonalantibodies which
bind toepitopes
distributed throughout theprotein.
Pab419 and PablO8 bindtothe amino terminus ofTantigen (21a, 23).Pab416,
Pab1630, Pab204,
Pab2O5,
andPab4O2
bind to determinantsmapped
to themiddle portion,
andPab4O5
binds to adeterminant
at thecarboxy
terminus of theprotein
(4, 9, 23).Specific binding
ofimmunopurified
Tantigen
to SV40 DNAwasassayed with 5'-end-labeled restriction fragmentsof cloned
wild-type DNA,
which carriesT-antigen-binding
sites
Iand
II in theHindIll C fragment.
DNAbinding
was measured in the presence andabsence
of ATP(Fig.
1).Specific
DNAbinding of
Tantigen
was detected with alleight
antibodies, though
theefficiency of binding differed
between
antibodies.
Forexample,
Tantigen immunopurified
onPablO8
and
Pab416 bound considerably
moreorigin
DNAfragment
than the subclassof
Tantigen purified
onPab4O5
(Fig. 1).
The amountof
origin fragment
bound in the pres-enceof
ATP wasmarkedly reduced, regardless of
theantibody
usedfor
immunopurification (Fig.
1).The
experimental conditions
were thenvaried
to investi-gate the factors involved in the observedinhibition.
SincePablO8
waspreviously
shown tobind
Tantigen
as well asdid
hamstertumor serum(24)
anddid
notaffect
origin
DNAbinding
asassayed by
DNasefootprinting
(41), it was selected foruse insubsequent experiments.
Whenimmuno-precipitation
wascarried
out withPablO8
incombination
with each of the otherantibodies,
DNA-binding
experiments with ATP showed similar inhibition oforigin
binding (data
notshown). Moreover, several other variations in the assay conditions had little or no effect.
Stringent
washing
of Tantigen
immunecomplexes
with buffercontaining
0.5 MLiCl
prior
toDNA-binding
tests did not affect the ATP inhibition(Fig. 2A). Furthermore,
the presenceofa20-fold excessofnonspecific
DNAand the timeofpreincubation
of Tantigen
with ATP did notgreatly
affect the extent of inhibition(Fig.
2B).Finally,
ATP inhibitedorigin
DNAbinding
at 20 and37°C
aswellas at0°C (Fig.
2C).Atrivial
explanation
for these results would be thatATP causes Tantigen-antibody complexes
to dissociate. How-J. VIROL.on November 10, 2019 by guest
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[image:2.612.154.471.71.317.2]A
+LiCI
L_ Ct
M ATP
B
30
30
0
15
M - - + + + +
.m
*:..
"':
ATP
min
DNA
M + - + - + - AD -+ +
--ATP
+ - + - -
pSV-wt
T T T N
T
W#*
4
* TOm an1
*iwf§
IgH..IgL
FIG. 2. ParametersofATPinhibition ofT-antigen-DNA binding. (A) COS1 T antigen immunopurified onPablO8was washed with NET containing 0.5 M LiCl or with NET alone and then assayed for binding to pSV-wt DNA fragments in the presence (+) and absence (-) of 20mMATP. M,Marker DNA. (B) ImmunopurifiedCOS1 T antigen was preincubated with (+) or without (-) 20 mM ATP for 30, 15, or0 min before the addition of end-labeled pSV-wt DNA fragments alone (-DNA) or mixed with 50 ,ug of unlabeled sonicated E. coli DNA (+DNA) per ml. M, Marker DNA. (C) Immunopurified COS1 T antigen was preincubated with (+) or without (-) 20 mM ATP at the temperatures(in°C)indicated before the addition of end-labeled pSV-wt DNA fragments. Bound DNA was determined after 2 h(0°C),1 h (20°C),or30min(37°C). M, Marker DNA. (D) COS1Tantigen was immunopurified onPabl08 (T) or nonimmune mouse IgG (N). Pabl08-T antigen complexes were preincubated for 30 min in binding buffer with (+) or without (-) 20mM ATP.End-labeled fragments of pSV-wt DNA were added as indicated (+) and incubated for 1 h. Immune Complexes were dissociated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (29) andWesternblotting (46). The first antibody was PablO8(1.7,ug/ml), and the second antibody was biotin-labeledanti-mouse IgG (1:500, Amersham), followed by preformed streptavidin-horseradish peroxidase complexes (1:400, Amersham). The blotwasdeveloped with 0.5 mg of diaminobenzidine perml-O.02%hydrogen peroxide-0.03% NiCl2for 20 min (2). Bands of T antigen (T) andimmunoglobulin heavy (IgH) and light(IgL)chains are indicated on the right.
ever, since dissociation of T antigen from immune
com-plexes in thepresenceof ATP, DNA,orATP and DNAwas
not observed (Fig. 2D), we conclude that it is the
DNA-binding activity of T antigen which is inhibited in the presence of ATP. Another possibility would be that the monoclonalantibodies induceaconformationalchange in T antigen upon binding, causing its inability to bind origin DNA efficiently. The fact that T antigen associated with eight different antibodies is subjecttoATP inhibitionargues against this possibility.
However, to confirm these results, weexposed COS1 T antigen in crude cellextract toATPand several ATPanalogs inthe absence ofantibody. SV40 DNA fragmentswerethen incubatedtoequilibrium, and protein-DNA complexeswere immunoprecipitated with PablO8 (Fig. 3A). Inhibition of originDNAbindingwasobserved with all of thenucleotides,
although inhibitionwasgreatestwith ATP and ATP-S [aden-osine-5'-O-(3-thiotriphosphate)]. Thus, exposure to ATP in the absence ofanantibody also results in inhibition of origin DNAbinding, although wecannotcompletely rule outthat the antibody could not influence T antigen behavior when added atthe end of the assay.
Thus, DNA-bindingassayswereperformed with
biochem-ically purified lytic T antigen (44) and a purified labeled restrictionfragment containingasynthetic T-antigen-binding
site I (41) (Fig. 3B). Specifically bound DNAwas detected by reduced electrophoretic migration of the restriction
frag-mentwhenboundtoTantigen (Fig. 3B, firsttwolanes). The
amount ofspecifically bound fragment was reduced in the
presence of excess unlabeled site I DNA but not in the presence of control vector DNA (Fig. 3B, last twolanes). Preincubation of purified T antigen with ATP or ATP-S resulted inmarked reduction ofspecific DNA binding (Fig. 3B, middle two lanes). These results, taken together,
dem-onstratethat ATPrapidly inhibits T-antigen-DNA binding in
three
different
assay systems, atthree
different
tempera-tures, on twodifferent DNA templates,with
lytic
andCOS!
T
antigens,
andin the presence andabsence of monoclonalantibodies,
cellularproteins,
and excessnonspecific
DNA.Nucleotide
specificity and concentration requirements for inhibition. Theinhibitory activity of several adenine
ribonucleotides
onT-antigen-DNA binding
wasmeasured
with end-labeled
fragments of
twodifferent
templates,pSV-wt and
p1097
DNA. Since plasmidp1097
carries a31-base-pair deletion
encompassing
all
of
T-antigen-binding
site
I(14), this
template
may be used to measurebinding
tosite II. Specific DNA binding of T antigen to both templates wasmarkedly reduced
inthe
presenceof
ATPand,
to alesser
extent, ADP and
adenylyl-imidodiphosphate (AMP-PNP)
(Fig. 4), in
agreementwith the results with crude
cell
extracts
(Fig. 3A).
AMPreduced
origin
DNAbinding only
slightly. Thus, binding
tosite II alone(p1097),
as well as to the stronger site I(15, 25),
isreduced
inthe
presenceof
nucleotide.
The
inhibitory activity
of adeninerbonucleotides
onT-antigen-DNA binding
was thenmeasured
as afunction
of
nucleotide concentration
(Fig. 5).
The amountof
origin
DNAfragment bound
atequilibrium by
Tantigen
wasalready
markedly reduced
in the presenceof
20 ,uMnucleotide and
reached
a minimum between 2and
20 mMnucleotide.
Although
the levelof maximum inhibition varied somewhat
from one preparation of T antigen to another, ATP was
consistently
more effective than ADP and AMP-PNP. Asemilog plot
of boundorigin
DNA as afunction of
nucleotide concentration(Fig. 5;
inset)
shows that50% inhibition of
binding
occurredat anATPconcentration of about
10,uM or atADPorAMP-PNPconcentrations of
about 1 mM.The
nucleotide
specificity
ofthis
inhibitory effect
was furtherinvestigated by assaying binding of immunopurified
Tantigen
topSV-wt
DNAinthe presenceof various
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[image:3.612.73.546.74.241.2]768 VOGT ET AL.
A
E I < < <
B
OL CL
(-) Z
a-
0-OL
L
:2
2-
--
ATP-S
...
._
.f.
ATP
-NUCLEOTIDE
S
NS COMPETITOR
+
+
TAg
f.. A
FIG. 3. ATPinhibits SV40DNAbinding ofTantigen in crude cell extract. (A) Crude lysate of COS1 cells was adjusted to25 mMTris (pH7.8)-75mMNaCl4.2mg of glycogen per ml-1 mg of bovine serum albumin perml-0.25% NonidetP-40andincubated without (-) and with 10 mM nucleotide for 30 min at0°C, as indicated. ATP-S, adenosine-5'-O-(3-thiotriphosphate);AMP-PCP, adenylyl (P-y-methylene)-diphosphonate; AMP-PNP, adenylyl-imidodiphosphate. End-labeled fragments of pSV-wt DNA were added and incubated for 1 h. DNA-protein complexes werethenimmunoprecipitated with PablO8 and fixed S. aureusandanalyzed by agarosegelelectrophoresis and autoradiography. (B)Apurified end-labeledDNAfragment containing SV40 siteIsequenceswasincubatedwith(+) or without (-)purified lytic Tantigen (44). Competition assays were performed by adding a sixfoldexcess of unlabeled specific pSDL13-wt (S) or nonspecific pSDL13(NS) DNA together with the labeled fragment.Tantigen (TAg)waspreincubated without (-)orwith20 mMnucleotide as indicated for 30 min before addition of labeledDNA.
pSV-WT
plO97
MN 1
I*
2 3 4 5
[image:4.612.130.486.69.302.2]F ..
FIG. 4. Adenineribonucleotides inhibitT-antigen bindingtosite I and siteII inSV40 originDNA. ImmunopurifiedCOS1 Tantigen
waspreincubatedfor 30minat0°C with buffer (lane 1)orwith 20 mM ADP (lane 2), AMP (lane 3), ATP (lane 4), or AMP-PNP (Boehringer GmbH, Mannheim, Federal Republic of Germany) (lane 5) inbinding bufferwithout E. coli DNA. T-antigen samples
werethen assayed for specificbinding toend-labeled fragments of pSV-wt or p1097 DNA as described in Materials and Methods.
Autoradiographywasfor 2(gels shown here)or20(gelsnotshown)
tides. Purine nucleoside
triphosphates
with either riboseor deoxyriboseasthe sugar moiety and theATPanalog ATP-S were the mosteffective inhibitors
(Fig. 3A and 6). Purine
nucleoside
diphosphates
and other noncleavable ATP ana-logs(AMP-PNP
andAMP-PCP) inhibited DNA bindingto a lesserextent(Fig. 3A, 4,
and5),
whilepyrimidine
nucleosidetriphosphates,
nucleosidemonophosphates,
nucleosides,cy-clic
AMP,
andNAD+
had littleornoinhibitory activity
(Fig.
4
through
6; datanotshown).
Is enzymatic activity required for ATP inhibition of DNA
binding?
T antigen is modifiedposttranslationally by
phos-phorylation
atmultiple
sites (42,48), by
adenylylation
(5), andby
ADPribosylation (21). Since
divalent metalcations
are
generally
required
for
enzymatic modification reactions
involving ATP,
weassayed
T-antigen-DNA
binding
with ATPin the presence and absenceof
divalent
cations
(Fig.
i).
ATPinhibited
origin
DNAbinding
in the presenceof
excess EDTA(Fig. 7A,
lane1)
or EGTA[ethylene
glycol-bis(p-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid] (Fig.
7B,
lane 1)just
aseffectively
asin the presenceof 0.5 mMMg2+
orCa2+
ions(Fig.
7A andB,
lanes2). Origin
DNAbinding
inthe presenceof
high concentrations
of metal ionswasmuch weaker, but aslight
reduction in the presenceof
ATPwash. Marker DNA(M) was12.5 ngof the labeled DNA used for the bindingassays.SV40HindlIl fragmentsareindicatedonthe left.A
controlsample (N)wasimmunopurifiedonnonimmunemouseIgG.
Note that thenonspecifically bound A2andBfragments ofp1097 DNA comigrate just above the specifically bound C fragment containing T-antigen-bindingsite II.
S
._
_I
I'l
M
N
1
2
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[image:4.612.60.299.393.640.2]02 2 5 10
NUCLEOTIDE CONCENTRATION (mM)
[image:5.612.88.524.77.339.2]15 20
FIG. 5. Inhibition of T-antigen-DNA binding as a function of nucleotide concentration. Immunopurified COS1 T antigen was assayed for bindingtopSV-wt DNA fragments in the presence of ATP (0), ADP (0), or AMP-PNP (x) at the concentrations indicated. Bands of bound HindIII-C DNA were excised from the gel, solubilized in Luma-Solve/Lipoluma (J.T.Baker Chemical Co., Phillipsburg, N.J.) and counted. (Inset)Semilog plot of counts per minute bound as a function of nucleotide concentration.
2
3 4 5 6 7 89
10.1...
still detectable
(Fig.
7AandB,
lanes3). Thus,
divalent metal cations do not appear to berequired for ATP inhibition of DNAbinding.
To furtherinvestigate whether a covalent modification of T
antigen
in the presence of ATP might cause inhibition of DNAbinding, immunopurified
T antigen waspreincubated
with
orwithout
ATP in the usual way. T antigen immuneA
4.;
+ _ + _- L - 4.
s
a d
DE
FIG. 6. Immunopurified COSiTantigenwasassayedfor
binding
topSV-wtDNAfragmentsasdescribed in Materials and Methods in thepresence of various nucleotides, eachataconcentration of20 mM. Lanes: M, MarkerDNA; 1, without nucleotide; 2, ATP; 3, dATP; 4, CTP; 5, dCTP; 6, GTP; 7, dGTP; 8, TTP; 9, UTP; 10, NAD'. SV4O HindIII fragmentsareindicated atthe left.
a
a0
ao
a
FIG. 7. ATP inhibition ofT-antigen-DNA binding in the pres-enceofchelatingagents.(A) Immunopurified COS1Tantigenwas
assayed for binding to pSV-wt DNA fragments in binding buffer containing5mMEDTA(lanes 1),0.5 mMMgCl2(lanes 2),or10 mM
MgCl2(lanes3) in thepresence(+)orabsence(-) of 20 mM ATP.
(B) Theexperimentwasrepeated, substitutingEGTA for EDTA and
CaC12for
MgC92.
4--4 0
IT
CD3C
C
-4 z
2-c
n
0 m
ox
0
x
tx --6
2
~~~~~~~~~~~~~~~0.2
2
20 mM
1
\
E
_
o .
.
.
.
.
/t
t
II
A S
AI
wA2*
2
0+
a a
a
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[image:5.612.69.286.399.677.2] [image:5.612.312.554.487.667.2]770 VOGT ET AL.
A
M
1
2
3
B
M
1
2
3
4
5 6
C
M 1
2
3
4
M
5 6
7
F w-. w
w
-.. B.
#.. .. ...
sF
It
FIG. 8. Reversibilityof nucleotide inhibition of T-antigen binding. (A) Immunopurified COS1 T antigen was preincubated for 30 min at0°C in the presence (lane 1) or absence (lanes 2 and 3) of 20 mM ATP in
binding
buffer. Immune complexes were spun down, washed three times in NET,andtaken
up inbinding buffer. T-antigen samples were preincubated with 20 mM ATP (lane 2) or without ATP (lanes 1 and 3) for 30min at0°C.End-labeled pSV-wt DNA fragments were then added to all samples. Bound DNA in all samples was analyzed after 1 h at0°C. M, Marker DNA. (B) Immunopurified COS1 T antigen was preincubated for 30 min in the presence (lane 1) or absence (lane 2) of 20 mM ATP andthen withpSV-wt DNA fragments for 2 h. The samples in lanes 3 through 6 were incubated with pSV-wt DNA fragments for 30 min. Incubation was continued in the presence of 20 mM ATP for 10 (lane 4), 30 (lane 5), or 60 (lane 6) min. M, Marker DNA. (C)Immunopurified COS1 T antigen was incubated to equilibriumat0°Cwith labeledpSV-wt DNA fragments. Unbound DNA was washed away(lanes
3 through 7), and immune complexes were suspended in binding buffer without (lanes 2, 4, 6, and 7) or with (lanes 1, 3, and 5) 20 mM ATP or with 0.25 ,ugof unlabeled pSV-wt DNA fragments (lane 7). Incubation was continued at0°Cfor 1 (lanes 1 through 4) or 16 (lanes 5 through 7) h.complexes
werethen
washed and
again taken
upin
binding
buffer in the
presence orabsence
of ATP (Fig.
8A). Acomparison of
DNAbinding in lane
1(pretreatedwith
ATPand then suspended without ATP)
andlane
3(control
nottreated with
ATP) indicates that
ATPinhibition is
readily
reversible
uponremoval of the nucleotide.
ATPinhibition in
this
experiment
was atthe usuallevel (compare lanes
2and3).
Thus,
weconclude that
simple binding of
ATP to Tantigen
and
notcovalent modification of
Tantigen is
prob-ably responsible
for
inhibition
of DNA
binding.
Further-more,
the results
confirm that
ATPdoes
not causedissocia-tion of
Tantigen
from the immune complexes (Fig. 2D).
The
reversibility of ATP inhibition raised the
question
whether
dissociation of
DNAbound
to Tantigen might be
facilitated
by
exposure to ATP. Toaddress
this
question,
Tantigen
wasincubated with
pSV-wt
DNAfor
only
30min,
atime
period
tooshort
for the
binding
reaction
toreach
equilibrium
at0°C,
orfor
2 h as usual(Fig.
8B). Continued
incubation of these
samples in the
presenceof
ATPrevealed
that
the
amountof bound
origin
fragment
was arrested or evenslightly reduced, despite
the presenceof
alarge
excessof unbound
origin
DNA(Fig.
8B). Thus, it
appearslikely
that ATP
binds
tofree
Tantigen, interfering somehow with
its
ability
tobind
origin
DNA.In
asecond series of
experiments,
Tantigen
wasincu-bated with excess labeled
pSV-wt
DNA
toequilibrium,
but
unbound DNA was washed away
prior
toaddition
of ATP in excess.Parallel
samples which
stillcontained
excess DNA wereincubated
with andwithout
ATP(Fig. 8C).
Bothwithand
without
excessDNA, the
amount of DNAwhich
re-mained
bound after addition of
ATP wasslightly
reduced
after
1 h(lanes
1 and3)
anddramatically
reducedafter
16 h(lane 5).
Dissociation of
Tantigen-DNA complexes
wasalso
detected
after
16 h in the presenceof
unlabeledpSV-wt
DNA,
but
asignificant
fraction of
the DNA remainedbound
(lane 7).
Theresults
suggest that inaddition
tobinding
tothose
T-antigen molecules which dissociate from origin
DNA
spontaneously, thereby
preventing
theirreassociation with theDNA,
ATP may also induce the dissociation of Tantigen
from
origin
DNA.DISCUSSION
T-antigen binding
toregions
Iand
IIin
theSV40
origin of
DNA
replication and
to a19-base-pair
sequencefromsite
Iis
diminished
inthe
presenceof low concentrations of purine
nucleoside triphosphates (Fig.
1through
6).Nucleotide
inhibition of
origin
DNAbinding
is observed in threedif-ferent
DNA-binding
assays withimmunopurified COS1
Tantigen, biochemically purified lytic
Tantigen,
andcrude
COSi
cell
extract(Fig.
1and
3).
ATPinhibits
DNAbinding
of
Tantigens
expressed by
10other
SV40-transformed
mouse, rat,
and
hamstercell
lines,
aswell aslytic
Tantigen
subclasses
(B.
Vogt and E.Fanning, manuscript in
prepara-tion).
ATPinhibition is
independent of
the presenceof
nonspecific DNA, the
temperatureof
the assay,and
theantibody used for immunopurification (Fig.
1and
2).
Thus,
we suggest that ATP
inhibition of origin
DNAbinding is
a new,intrinsic biochemical
propertyof
Tantigen.
Several different mechanisms for the observed
ATPinhi-bition
of DNAbinding
areconceivable.
Theinteraction
between
Tantigen
and ATPmaymodify
thetemplate
in such away that Tantigen
can nolonger
bind toorigin
sequences.Although it is difficult
toexclude this
possibility completely,
we
feel
itis
unlikely because inhibition is observed with
three
different linear
DNAtemplates,
oneof which carries
aminimal
binding
signal
of
only
19base
pairs. Moreover,
inhibition
is observed with noncleavable ATPanalogs and in the absenceof
divalent metalcations, which
wouldpresum-ably
berequired for enzymatic alteration of
thetemplate.
Alternatively,
ATP may be used as asubstrate for
amodification of
Tantigen. However,
theeasyreversibility of
inhibition
upon removal of ATP suggests thatmodification
of
T
antigen
isunlikely
to causeinhibition
of DNAbinding (Fig.
8A).
In support of thisinterpretation,
ATPinhibition
does notrequire
Mg2+
orCa2+
cations(Fig. 7). Furthermore,
noncleavable
ATPanalogs
and ADP can also inhibitT
antigen-DNA binding, albeit
lesseffectively
than ATP(Fig.
3A and
4).
The datawould, however,
beconsistent with
theinterpretation
thatsimple binding
of ATP to Tantigen
inhibits
DNAbinding.
J. VIROL.
.,.4-OF-,
on November 10, 2019 by guest
http://jvi.asm.org/
[image:6.612.124.508.82.230.2]Assuming
this
tobe the
case, ATPcould either
competedirectly
with
origin
DNAfor
Tantigen
orallosterically
induce
aconformational
change
in Tantigen, thereby
ren-dering
itunableto bindorigin
DNAefficiently.
If ATP andorigin
DNA werecompeting
for
the samesite,
wewould
expect DNA
binding
tobe
favored
even inthe
presenceof
ATP, since the
affinity
of
Tantigen for origin
DNAis
greatenough
towithstand
vigorous washing
of
DNAimmune
complexes,
while ATPdissociates
completely
under
thesame
conditions
(Fig. 8A).
Yetevenlow concentrations of
ATP
inhibit
origin binding
when added
prior
toaddition of
DNA
(Fig.
1through 6).
Therefore,
wefeel it is
unlikely
that
ATP
and
origin
DNAbind
tothesamesite. On
the contrary,the
data
areentirely
consistent with the idea
that Tantigen
is
an
allosteric
protein.
We suggestthat it
exists in
twodifferent
butrelated stable
conformations,
one(N) which
binds nucleotide but
notorigin
DNA andanother
(D) which
binds
origin
DNAbut
notATP(Fig. 9).
These
conformations
are
postulated
toexist in
equilibrium
in
solution. Which
conformation
Tantigen
assumeswould thus
depend
ontherelative concentrations of the
twoligands,
ATPand
SV40
DNA,
andthe
affinity
of
Tantigen
for each of them.
Aspredicted by
this
model,
preformed T-antigen-DNA
com-plexes
aremoderately
stable
despite
the presenceof
ATPadded later
(Fig.
8B andC).
However,
uponremoval of
excessDNA
and
prolonged
incubation with
excessATP,
the
equilibrium
is
displaced
tothe
Nconformation of
Tantigen
(Fig.
8C). In supportof this
interpretation,
wehaverecently
identified
mutantTantigens
which
bindspecifically
toSV40
DNA
in
the presenceof
ATPaswellasinits absence
(Vogt
and
Fanning,
inpreparation).
The
location of the
ATP-binding
site
responsible
for
allosteric
inhibition of
origin binding
has not yet beendetermined.
However,
anucleotide-binding
site
onSV40
and
polyomavirus
Tantigens
wasdetected
previously by
ATP
affinity labeling
(11,
12).
Recentevidence
suggeststhat
this
nucleotide-binding
site is located
atthe
carboxy-terminal
endof
theprotein
and
is distinct from
the ATPasesubstrate-binding
site
(12).
The
affinity-labeled
nucleotide-binding
site
hasseveral
properties
in
common with thebinding
site which mediates
theallosteric inhibition of
origin
DNA
binding;
both have
afairly
broad
specificity
for
theligand
bound
(11;
Fig.
3through 6),
and the Kmfor
ATPaffinity labeling
isapproximately
the same as the ATPconcentration which
causes50% inhibition of
origin
DNAbinding (12; Fig. 5). Alternatively,
either the
ATPasesub-strate-binding
site
or ahitherto undetected
ATP-binding
site
could
mediate
theinhibition of
DNAbinding. Thus,
local-ization of
the ATPallosteric
effector-binding
site
requires
further work.
ATP
binding
hasbeenreported
tomodulate theactivity
of
several
proteins
involved
in DNAreplication
and
recombi-nation.
Binding
of
E.coli dnaB
protein
toATP, for example,
allosterically
activates
binding
tosingle-stranded
DNA(3,
34).
Sequence-specific binding
of
Tn3 transposase to theinverted
repeatsof
the transposonrequires
ATP but not metalions, suggesting possible
activation
by
ATPbinding
(49).
On the otherhand,
theactivity
of
purified
topoisomerase
Ifrom
HeLacells
andUstilago maydis
isnegatively regulated by physiological
concentrationsof
ATPorATP
analogs (31, 40).
The
biological
consequences ofnegative
regulation by
ATP remain
puzzling.
BothT-antigen-DNA binding
andtopoisomerase
Irelaxation activities
arepresumed
to beactive in the
cell,
andyet the intracellular and intranuclear concentrationsofATP,
estimated
atabout4 to5mM(1,
36,
+ATP
TN-ATP
~-A
TN
-ATP
+
ORI-DNA
w
TD
z TD ORI
-ORI-DNA
FIG. 9. Model forallosteric inhibition of T-antigen-DNA binding activity by purine nucleotides. See the text for details. TN, N conformation of T antigen; TD, D conformation of T antigen; ORI-DNA and ORI, originDNA.
37), would be
sufficient
to cause severeinhibition
of these activities. This apparentparadox could be explained
in severalways for Tantigen.
Cellsmay contain factors which protect intracellular Tantigen against
theinhibitory
effect of ATP.However,
thefact that
ATPinhibits
DNAbinding of
Tantigen
in crude cell extracts, as well asimmunopurified
Tantigen (Fig.
1 and3),
arguesagainst
this explanation. Anotherpossibility
is that theactually
available ATP con-centration in the cell isquite different from that calculated byisolating
ATPfrom
the cell(1, 36, 37). Given the multitude
ofproteins and
enzymeswhich bind and
utilize
ATPand
thepossible sequestration
of ATP in subcellularcompartments, thispossibility
is difficult to evaluate. Yetanother possibility is that ATP inhibition of DNAbinding
is a transient eventassociated
withatemporarychange
inT-antigen
conforma-tion,
forexample,
ininitiation
orelongation of viral
DNAreplication.
Afinalpossibility
is thatT-antigen
accumulation orATPaseactivity
orboth overcome the ATPinhibition
oforigin binding.
Thisalternative
is attractive because itpro-vides
aplausible explanation
as towhy
Tantigen
synthe-sizedearly
ininfection does not lead toimmediate repression ofearly
transcription.
ATPinhibition of DNAbinding
could thus act as amolecular
switch sensitive
to themetabolic
stateof the infectedcellaswell as tothe number
of
infectingSV40
genomes and theirexpression
activity. ACKNOWLEDGMENTSWethank AndreaSchmid, UrsulaMarkau, and Silke Dehde for excellent technical assistance, Ed Harlow, Elisabeth Gurney, Wolfgang Deppert, Roland Ball, and DavidLaneforhybridomas and monoclonalantibodies,PeterTegtmeyer for
purified
lytic Tantigen, WolfgangDeppert for communication of resultspriortopublication, and GuidoHartmannfor critical review of themanuscript.The financial support of the Deutsche Forschungsgemeinschaft (Fa 138-1/1 and 1/2), Fonds der Chemischen Industrie, and the Konrad-Adenauer-Stiftung(to E.V.) isgratefully acknowledged.
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