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Allosteric control of simian virus 40 T-antigen binding to viral origin DNA.

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Copyright

©

1986, American

Society

forMicrobiology

Allosteric

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

is

regulated

primarily by

the gene A

product,

large

T (tumor)

antigen

(38, 45). T

antigen is

a

multi-functional

protein required for

regulation of early and late

viral

gene

expression and initiation of

viral

DNA

replication.

In

addition, it influences

patterns of cellular transcription

and

DNA

synthesis

and may

well affect cellular

metabolism

in

more

subtle

ways.

The known

biochemical

properties of

T

antigen

are

diverse, including

sequence-specific binding

to two

major sites in the SV40 control

region,

nonspecific

binding

to

native and denatured

DNA,

binding

toa

cellular

phosphoprotein, p53, nucleotide

binding,

and

cleavage of

ATP

and dATP.

Sequence-specific

binding of

T

antigen

to

the

SV40 control

region mediates

autoregulation of early

transcription (14, 22, 39), initiation

of viral

DNA

replication

(13, 32,

43),

and

stimulation of

late

transcription (6, 7, 26, 27)

in a

temporally

controlled sequence.

Viral

DNA

replication

appears to

require

ATPase

activity in addition (10). Studies

with

polyomavirus suggest that

nucleotide-binding activity

may

be distinct from ATPase-substrate

binding

and may

also

be essential

for viral

DNA

replication (12).

T

antigen

appears to

be

composed

of

several

domains,

some

of which

can

be

clearly correlated with

a

subset

of its

biological

or

biochemical activities.

For

example, studies

with SV40

mutants

and hybrid virus

proteins have localized

the

DNA-binding domain

of

T

antigen

to an

amino-terminal

region (10,

33,

35). Studies with

monoclonal antibodies (9)

and

mutant T

antigens (10)

have

implicated

the

carboxy-terminal

half

of

T

antigen

in ATPase

activity.

The

ATP-binding site of

T

antigen

is

also located

at

the

carboxy-terminal

end

of

the

protein

(12).

Thus, it

is

conceivable

that

these biochemical

activities, being localized

at

different

ends

of

a

large molecule,

may be

independent of

each other. As a

first approach

to

this

question,

we have

investigated

the

sequence-specific DNA-binding

activity of

T

antigen in

the presence and absence of ATP. The results demonstrate that DNA

binding

and

nucleotide

binding

are not

independent

activities

of

T

antigen

and suggest that nucleotide

binding

allosterically inhibits

T

antigen

binding

to the viral

origin

region. Possible biological implications of this

newproperty

of

T

antigen

are

discussed.

*Correspondingauthor.

(A

preliminary

account

of this work

was

presented

at

the

Cold

Spring Harbor Conference

on

Cancer

Cells, Cold

Spring Harbor, N.Y.,

4to8

September, 1985.)

MATERIALS ANDMETHODS

Cells

and antibodies. The culture

of COS1 (20) cells

has

been

described

previously (8). Hybridoma cells obtained

from E. Harlow

(23),

R.

Ball

(4), and

Gurney

et al.

(21a;

PablO8)

were

cultured in

Dulbecco-modified Eagle medium

(GIBCO Laboratories, Grand Island, N.Y.) supplemented

with

15% fetal calf

serum

(GIBCO

or

Biochrom KG, Berlin,

Federal

Republic

of Germany)

and

antibiotics.

Immunoglob-ulin

G

(IgG)

was

purified

as

described

previously (16). Pab2O4

and

Pab2O5

IgG (9)

were a

gift

from David Lane,

London,

England.

SV40 DNAbinding. T

antigen

was extracted

from

COS1

monkey

cells

(20)

in

lysis buffer (50

mM

Tris

[pH

9]-120

mM

NaCl-0.5% Nonidet P-40) for

30min at

0°C

(107

cells

per

ml).

Samples of

100

,ul (50

to 100 ng

of

T

antigen)

were

im-munoprecipitated with

5 ,ug

of

purified

monoclonal

PablO8

IgG and fixed

Staphylococcus

aureus

(28)

as

described

previously (17). PablO8 recognizes

a

denaturation-resistant

epitope

located between 0.65 and 0.62

SV40

map

units

(21a).

It

binds

essentially all soluble

T

antigen and has

no

detect-able

effect

onT

antigen binding

to

the

SV40

origin

regioui,

as

shown

by

DNase

footprinting

(24, 41). ATPase

activity and

ATP

affinity

labeling of

T

antigen

were

also

readily

detect-able

in

the presence

of

PablO8

(24).

Immune

complexes

were washed

with

50 mM

Tris

(pH

7.5)-150

mM

NaCl-5

mM

EDTA-0.05% Nonidet

P-40

(NET)

with or

without

0.5 M

LiCl and

then

with

NET.

Immunopurified

T

antigen complexes

were taken up in 0.15 ml

of

binding

buffer

(10

mM HEPES

[N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid]

[pH

7.8]-80

mM

KCl-0.5 mM

MgCl2-1

mM dithiothreitol-1 mM

phenylmethylsulfonyl fluoride-0.2

mg

of

glycogen

per ml -1 mg

of

bovine serum albumin per ml-50 ,ug of

sonicated

Escherichia coli DNA per

ml).

The DNA

templates

used were

pSV-wt (18), wild-type

SV40 DNA

cloned

in the

BamHI site of pAT153 (47),

and

p1097

(24),

the

SV40

deletion

mutant cs1097

(14), which

lacks

T-antigen-binding

site I

(nucleotides

5178 to

5208),

cloned in the

opposite

orientation

in the BamHI site

of

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

of

immunopurified

T

antigen.

Duplicate samples

ofCOSiT

antigen

were

immunopurified

oneach of the

purified

monoclonal antibodies

(Pab)

indicated. One of thetwo

samples

was

preincubated

with 20 mM ATP for 30 mmnatO0C(+).

Binding

toend-labeled

fragments

of

pSV-wt

DNAwas

assayed

withoutE. coli DNAasdescribed in Materials and Methods. Marker DNA (M)was12.5 ng(5%) of the

input

DNA.SV40 Hindlll

fragments

are indicatedonthe left.

pLg

per

assay)

was presentin excess and incubated with T

antigen

to

equilibrium,

unless stated otherwise. In some

experiments,

E. coli DNA was

omitted

from the

binding

buffer,

as stated in the

figure legends.

Bound DNA was

dissociated from

immune

complexes

and

analyzed

by agarose

gel

electrophoresis

and

autoradiography

as

de-scribed

previously

(18).

Gel retardationDNA-binding assay.

Plasmid

pON-wt

car-rying

a

synthetic

19-base-pair

DNA sequence

from

T-antigen-binding site

I was

shown

tobind T

antigen with

an

affinity comparable

to that

of

intact

origin

DNA

(41).

The

EcoRI-Sall fragment of pON-wt carrying

this sequence was

cloned

into the EcoRI-SalI sites of

vector

pSDL13 (30) and

designated pSDL13-wt.

A

TaqI-NaeI

fragment of

89

base

pairs carrying

the

binding site

was5' end labeled and

purified

by

polyacrylamide gel electrophoresis. Biochemically

puri-fied

lytic

T

antigen

(44), a

gift from

P. Tegtmeyer, was

incubated

with4 ngof the

purified fragment

and 1.75 ,ugof unlabeled E. coli DNA for 1 h at

0°C

in 10 mM Tris

hydrochloride

(pH

7.4)-i

mM EDTA-20 mM NaCl. The

reaction

mixwasthenanalyzed

by

electrophoresis

(100 V

for

2

h)

in

6%

polyacrylamide gels

in 10 mM Tris acetate

(pH

7.8)-i

mM EDTA as described

previously (19)

and

autora-diography.

RESULTS

Inhibition ofT-antigen-DNA binding by ATP. The SV40-transformed cell line

COS1,

which

constitutively

expresses wild-type SV40Tantigen (20),wasselectedas a sourceofT

antigen.

T

antigen

was

immunopurified

with

eight

different monoclonal

antibodies which

bind to

epitopes

distributed throughout the

protein.

Pab419 and PablO8 bindtothe amino terminus ofTantigen (21a, 23).

Pab416,

Pab1630, Pab204,

Pab2O5,

and

Pab4O2

bind to determinants

mapped

to the

middle portion,

and

Pab4O5

binds to a

determinant

at the

carboxy

terminus of the

protein

(4, 9, 23).

Specific binding

of

immunopurified

T

antigen

to SV40 DNAwasassayed with 5'-end-labeled restriction fragments

of cloned

wild-type DNA,

which carries

T-antigen-binding

sites

I

and

II in the

HindIll C fragment.

DNA

binding

was measured in the presence and

absence

of ATP

(Fig.

1).

Specific

DNA

binding of

T

antigen

was detected with all

eight

antibodies, though

the

efficiency of binding differed

between

antibodies.

For

example,

T

antigen immunopurified

onPablO8

and

Pab416 bound considerably

more

origin

DNA

fragment

than the subclass

of

T

antigen purified

on

Pab4O5

(Fig. 1).

The amount

of

origin fragment

bound in the pres-ence

of

ATP was

markedly reduced, regardless of

the

antibody

used

for

immunopurification (Fig.

1).

The

experimental conditions

were then

varied

to investi-gate the factors involved in the observed

inhibition.

Since

PablO8

was

previously

shown to

bind

T

antigen

as well as

did

hamstertumor serum

(24)

and

did

not

affect

origin

DNA

binding

as

assayed by

DNase

footprinting

(41), it was selected foruse in

subsequent experiments.

When

immuno-precipitation

was

carried

out with

PablO8

in

combination

with each of the otherantibodies,

DNA-binding

experiments with ATP showed similar inhibition of

origin

binding (data

notshown). Moreover, several other variations in the assay conditions had little or no effect.

Stringent

washing

of T

antigen

immune

complexes

with buffer

containing

0.5 M

LiCl

prior

to

DNA-binding

tests did not affect the ATP inhibition

(Fig. 2A). Furthermore,

the presenceofa20-fold excessof

nonspecific

DNAand the timeof

preincubation

of T

antigen

with ATP did not

greatly

affect the extent of inhibition

(Fig.

2B).

Finally,

ATP inhibited

origin

DNA

binding

at 20 and

37°C

aswellas at

0°C (Fig.

2C).

Atrivial

explanation

for these results would be thatATP causes T

antigen-antibody complexes

to dissociate. How-J. VIROL.

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[image:2.612.154.471.71.317.2]
(3)

A

+LiCI

L_ Ct

M ATP

B

30

30

0

15

M - - + + + +

.m

*:..

"':

ATP

min

DNA

M + - + - + - A

D -+ +

--ATP

+ - + - -

pSV-wt

T T T N

T

W#*

4

* T

Om 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, at

three

different

tempera-tures, on twodifferent DNA templates,

with

lytic

and

COS!

T

antigens,

andin the presence andabsence of monoclonal

antibodies,

cellular

proteins,

and excess

nonspecific

DNA.

Nucleotide

specificity and concentration requirements for inhibition. The

inhibitory activity of several adenine

ribonucleotides

on

T-antigen-DNA binding

was

measured

with end-labeled

fragments of

two

different

templates,

pSV-wt and

p1097

DNA. Since plasmid

p1097

carries a

31-base-pair deletion

encompassing

all

of

T-antigen-binding

site

I

(14), this

template

may be used to measure

binding

tosite II. Specific DNA binding of T antigen to both templates was

markedly reduced

in

the

presence

of

ATP

and,

to a

lesser

extent, ADP and

adenylyl-imidodiphosphate (AMP-PNP)

(Fig. 4), in

agreement

with the results with crude

cell

extracts

(Fig. 3A).

AMP

reduced

origin

DNA

binding only

slightly. Thus, binding

tosite II alone

(p1097),

as well as to the stronger site I

(15, 25),

is

reduced

in

the

presence

of

nucleotide.

The

inhibitory activity

of adenine

rbonucleotides

on

T-antigen-DNA binding

was then

measured

as a

function

of

nucleotide concentration

(Fig. 5).

The amount

of

origin

DNA

fragment bound

at

equilibrium by

T

antigen

was

already

markedly reduced

in the presence

of

20 ,uM

nucleotide and

reached

a minimum between 2

and

20 mM

nucleotide.

Although

the level

of maximum inhibition varied somewhat

from one preparation of T antigen to another, ATP was

consistently

more effective than ADP and AMP-PNP. A

semilog plot

of bound

origin

DNA as a

function of

nucleotide concentration

(Fig. 5;

inset)

shows that

50% inhibition of

binding

occurredat anATP

concentration of about

10,uM or atADPorAMP-PNP

concentrations of

about 1 mM.

The

nucleotide

specificity

of

this

inhibitory effect

was further

investigated by assaying binding of immunopurified

T

antigen

to

pSV-wt

DNAinthe presence

of various

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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 most

effective 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,

and

5),

while

pyrimidine

nucleoside

triphosphates,

nucleoside

monophosphates,

nucleosides,

cy-clic

AMP,

and

NAD+

had littleorno

inhibitory activity

(Fig.

4

through

6; datanot

shown).

Is enzymatic activity required for ATP inhibition of DNA

binding?

T antigen is modified

posttranslationally by

phos-phorylation

at

multiple

sites (42,

48), by

adenylylation

(5), and

by

ADP

ribosylation (21). Since

divalent metal

cations

are

generally

required

for

enzymatic modification reactions

involving ATP,

we

assayed

T-antigen-DNA

binding

with ATPin the presence and absence

of

divalent

cations

(Fig.

i).

ATPinhibited

origin

DNA

binding

in the presence

of

excess EDTA

(Fig. 7A,

lane

1)

or EGTA

[ethylene

glycol-bis(p-aminoethyl

ether)-N,N,N',N'-tetraacetic

acid] (Fig.

7B,

lane 1)

just

as

effectively

asin the presenceof 0.5 mM

Mg2+

or

Ca2+

ions

(Fig.

7A and

B,

lanes

2). Origin

DNA

binding

in

the presenceof

high concentrations

of metal ionswasmuch weaker, but a

slight

reduction in the presence

of

ATPwas

h. 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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(5)

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 8

9

10

.1...

still detectable

(Fig.

7Aand

B,

lanes

3). Thus,

divalent metal cations do not appear to berequired for ATP inhibition of DNA

binding.

To furtherinvestigate whether a covalent modification of T

antigen

in the presence of ATP might cause inhibition of DNA

binding, immunopurified

T antigen was

preincubated

with

or

without

ATP in the usual way. T antigen immune

A

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

w

A2*

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]
(6)

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,and

taken

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

were

then

washed and

again taken

up

in

binding

buffer in the

presence or

absence

of ATP (Fig.

8A). A

comparison of

DNA

binding in lane

1(pretreated

with

ATP

and then suspended without ATP)

and

lane

3

(control

not

treated with

ATP) indicates that

ATP

inhibition is

readily

reversible

upon

removal of the nucleotide.

ATP

inhibition in

this

experiment

was atthe usual

level (compare lanes

2and

3).

Thus,

we

conclude that

simple binding of

ATP to T

antigen

and

not

covalent modification of

T

antigen is

prob-ably responsible

for

inhibition

of DNA

binding.

Further-more,

the results

confirm that

ATP

does

not cause

dissocia-tion of

T

antigen

from the immune complexes (Fig. 2D).

The

reversibility of ATP inhibition raised the

question

whether

dissociation of

DNA

bound

to T

antigen might be

facilitated

by

exposure to ATP. To

address

this

question,

T

antigen

was

incubated with

pSV-wt

DNA

for

only

30

min,

a

time

period

too

short

for the

binding

reaction

to

reach

equilibrium

at

0°C,

or

for

2 h as usual

(Fig.

8B). Continued

incubation of these

samples in the

presence

of

ATP

revealed

that

the

amount

of bound

origin

fragment

was arrested or even

slightly reduced, despite

the presence

of

a

large

excess

of unbound

origin

DNA

(Fig.

8B). Thus, it

appears

likely

that ATP

binds

to

free

T

antigen, interfering somehow with

its

ability

to

bind

origin

DNA.

In

a

second series of

experiments,

T

antigen

was

incu-bated with excess labeled

pSV-wt

DNA

to

equilibrium,

but

unbound DNA was washed away

prior

to

addition

of ATP in excess.

Parallel

samples which

still

contained

excess DNA were

incubated

with and

without

ATP

(Fig. 8C).

Bothwith

and

without

excess

DNA, the

amount of DNA

which

re-mained

bound after addition of

ATP was

slightly

reduced

after

1 h

(lanes

1 and

3)

and

dramatically

reduced

after

16 h

(lane 5).

Dissociation of

T

antigen-DNA complexes

was

also

detected

after

16 h in the presence

of

unlabeled

pSV-wt

DNA,

but

a

significant

fraction of

the DNA remained

bound

(lane 7).

The

results

suggest that in

addition

to

binding

to

those

T-antigen molecules which dissociate from origin

DNA

spontaneously, thereby

preventing

theirreassociation with the

DNA,

ATP may also induce the dissociation of T

antigen

from

origin

DNA.

DISCUSSION

T-antigen binding

to

regions

I

and

II

in

the

SV40

origin of

DNA

replication and

to a

19-base-pair

sequencefrom

site

I

is

diminished

in

the

presence

of low concentrations of purine

nucleoside triphosphates (Fig.

1

through

6).

Nucleotide

inhibition of

origin

DNA

binding

is observed in three

dif-ferent

DNA-binding

assays with

immunopurified COS1

T

antigen, biochemically purified lytic

T

antigen,

and

crude

COSi

cell

extract

(Fig.

1

and

3).

ATP

inhibits

DNA

binding

of

T

antigens

expressed by

10

other

SV40-transformed

mouse, rat,

and

hamster

cell

lines,

aswell as

lytic

T

antigen

subclasses

(B.

Vogt and E.

Fanning, manuscript in

prepara-tion).

ATP

inhibition is

independent of

the presence

of

nonspecific DNA, the

temperature

of

the assay,

and

the

antibody used for immunopurification (Fig.

1

and

2).

Thus,

we suggest that ATP

inhibition of origin

DNA

binding is

a new,

intrinsic biochemical

property

of

T

antigen.

Several different mechanisms for the observed

ATP

inhi-bition

of DNA

binding

are

conceivable.

The

interaction

between

T

antigen

and ATPmay

modify

the

template

in such away that T

antigen

can no

longer

bind to

origin

sequences.

Although it is difficult

to

exclude this

possibility completely,

we

feel

it

is

unlikely because inhibition is observed with

three

different linear

DNA

templates,

one

of which carries

a

minimal

binding

signal

of

only

19

base

pairs. Moreover,

inhibition

is observed with noncleavable ATPanalogs and in the absence

of

divalent metal

cations, which

would

presum-ably

be

required for enzymatic alteration of

the

template.

Alternatively,

ATP may be used as a

substrate for

a

modification of

T

antigen. However,

theeasy

reversibility of

inhibition

upon removal of ATP suggests that

modification

of

T

antigen

is

unlikely

to cause

inhibition

of DNA

binding (Fig.

8A).

In support of this

interpretation,

ATP

inhibition

does not

require

Mg2+

or

Ca2+

cations

(Fig. 7). Furthermore,

noncleavable

ATP

analogs

and ADP can also inhibit

T

antigen-DNA binding, albeit

less

effectively

than ATP

(Fig.

3A and

4).

The data

would, however,

be

consistent with

the

interpretation

that

simple binding

of ATP to T

antigen

inhibits

DNA

binding.

J. VIROL.

.,.4-OF-,

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(7)

Assuming

this

to

be the

case, ATP

could either

compete

directly

with

origin

DNA

for

T

antigen

or

allosterically

induce

a

conformational

change

in T

antigen, thereby

ren-dering

itunableto bind

origin

DNA

efficiently.

If ATP and

origin

DNA were

competing

for

the same

site,

we

would

expect DNA

binding

to

be

favored

even in

the

presence

of

ATP, since the

affinity

of

T

antigen for origin

DNA

is

great

enough

to

withstand

vigorous washing

of

DNA

immune

complexes,

while ATP

dissociates

completely

under

the

same

conditions

(Fig. 8A).

Yeteven

low concentrations of

ATP

inhibit

origin binding

when added

prior

to

addition of

DNA

(Fig.

1

through 6).

Therefore,

we

feel it is

unlikely

that

ATP

and

origin

DNA

bind

tothesame

site. On

the contrary,

the

data

are

entirely

consistent with the idea

that T

antigen

is

an

allosteric

protein.

We suggest

that it

exists in

two

different

but

related stable

conformations,

one

(N) which

binds nucleotide but

not

origin

DNA and

another

(D) which

binds

origin

DNA

but

notATP

(Fig. 9).

These

conformations

are

postulated

to

exist in

equilibrium

in

solution. Which

conformation

T

antigen

assumes

would thus

depend

onthe

relative concentrations of the

two

ligands,

ATP

and

SV40

DNA,

and

the

affinity

of

T

antigen

for each of them.

As

predicted by

this

model,

preformed T-antigen-DNA

com-plexes

are

moderately

stable

despite

the presence

of

ATP

added later

(Fig.

8B and

C).

However,

upon

removal of

excessDNA

and

prolonged

incubation with

excess

ATP,

the

equilibrium

is

displaced

to

the

N

conformation of

T

antigen

(Fig.

8C). In support

of this

interpretation,

wehave

recently

identified

mutantT

antigens

which

bind

specifically

to

SV40

DNA

in

the presence

of

ATPaswellasin

its absence

(Vogt

and

Fanning,

in

preparation).

The

location of the

ATP-binding

site

responsible

for

allosteric

inhibition of

origin binding

has not yet been

determined.

However,

a

nucleotide-binding

site

on

SV40

and

polyomavirus

T

antigens

was

detected

previously by

ATP

affinity labeling

(11,

12).

Recent

evidence

suggests

that

this

nucleotide-binding

site is located

at

the

carboxy-terminal

end

of

the

protein

and

is distinct from

the ATPase

substrate-binding

site

(12).

The

affinity-labeled

nucleotide-binding

site

has

several

properties

in

common with the

binding

site which mediates

the

allosteric inhibition of

origin

DNA

binding;

both have

a

fairly

broad

specificity

for

the

ligand

bound

(11;

Fig.

3

through 6),

and the Km

for

ATP

affinity labeling

is

approximately

the same as the ATP

concentration which

causes

50% inhibition of

origin

DNA

binding (12; Fig. 5). Alternatively,

either the

ATPase

sub-strate-binding

site

or a

hitherto undetected

ATP-binding

site

could

mediate

the

inhibition of

DNA

binding. Thus,

local-ization of

the ATP

allosteric

effector-binding

site

requires

further work.

ATP

binding

hasbeen

reported

tomodulate the

activity

of

several

proteins

involved

in DNA

replication

and

recombi-nation.

Binding

of

E.

coli dnaB

protein

to

ATP, for example,

allosterically

activates

binding

to

single-stranded

DNA

(3,

34).

Sequence-specific binding

of

Tn3 transposase to the

inverted

repeats

of

the transposon

requires

ATP but not metal

ions, suggesting possible

activation

by

ATP

binding

(49).

On the other

hand,

the

activity

of

purified

topoisomerase

I

from

HeLa

cells

and

Ustilago maydis

is

negatively regulated by physiological

concentrations

of

ATP

orATP

analogs (31, 40).

The

biological

consequences of

negative

regulation by

ATP remain

puzzling.

Both

T-antigen-DNA binding

and

topoisomerase

I

relaxation activities

are

presumed

to be

active in the

cell,

andyet the intracellular and intranuclear concentrationsof

ATP,

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 severe

inhibition

of these activities. This apparent

paradox could be explained

in severalways for T

antigen.

Cellsmay contain factors which protect intracellular T

antigen against

the

inhibitory

effect of ATP.

However,

the

fact that

ATP

inhibits

DNA

binding of

T

antigen

in crude cell extracts, as well as

immunopurified

T

antigen (Fig.

1 and

3),

argues

against

this explanation. Another

possibility

is that the

actually

available ATP con-centration in the cell isquite different from that calculated by

isolating

ATP

from

the cell

(1, 36, 37). Given the multitude

of

proteins and

enzymes

which bind and

utilize

ATP

and

the

possible sequestration

of ATP in subcellularcompartments, this

possibility

is difficult to evaluate. Yetanother possibility is that ATP inhibition of DNA

binding

is a transient event

associated

withatemporary

change

in

T-antigen

conforma-tion,

for

example,

in

initiation

or

elongation of viral

DNA

replication.

Afinal

possibility

is that

T-antigen

accumulation orATPase

activity

orboth overcome the ATP

inhibition

of

origin binding.

This

alternative

is attractive because it

pro-vides

a

plausible explanation

as to

why

T

antigen

synthe-sized

early

ininfection does not lead toimmediate repression of

early

transcription.

ATPinhibition of DNA

binding

could thus act as a

molecular

switch sensitive

to the

metabolic

stateof the infectedcellaswell as tothe number

of

infecting

SV40

genomes and their

expression

activity. ACKNOWLEDGMENTS

Wethank 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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49. Wishart, W., J. Broach, and E. Ohtsubo. 1985. ATP-dependent specific binding of Tn3 transposase to Tn3 inverted repeats. Nature (London)314:556-558.

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

Figure

FIG.1.ofBinding(M) the ATP inhibits SV40 DNA binding of immunopurified T antigen. Duplicate samples of COSi T antigen were immunopurified on each purified monoclonal antibodies (Pab) indicated
FIG. 2.Thepolyacrylamide(T)20minweretemperaturesbiotin-labeledcontainingantigen(+DNA)(20°C), mM Parameters of ATP inhibition of T-antigen-DNA binding
FIG. 3.pSDL13forDNA-proteinlyticwithdiphosphonate;autoradiography.(pH ATP inhibits SV40 DNA binding of T antigen in crude cell extract
FIG. 7.enceassayedcontainingMgCl2(B)CaC12 ATP inhibition of T-antigen-DNA binding in the pres- of chelating agents
+2

References

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