The Finger
Domain of
Simian Virus 40
Large T
Antigen Controls
DNA-Binding
Specificity
ADOLF HOSS, ISMAIL F. MOAREFI, ELLEN FANNING, ANDAVRIL K. ARTHUR*
Institut fur Biochemie, Karlstrasse 23, 8000 Munich 2, Federal Republic
of
GermanyReceived 24 April 1990/Accepted 21 August 1990
The
specificity
andregulation
of protein-DNA interactions playacrucial role in allaspectsof communicationbetween
genotypeandphenotype
inacell. The large T antigenof
simianvirus 40 bindstoidentical, yetquitedifferently
arranged,
pentanucleotide motifs
in thesimian
virus 40 control region, sites I andII. Wild-type Tantigen preferentially binds
siteI. We demonstrate thatabacterialpeptide
encoding residues 1to259 (T260) includesthe
essential amino acids required for binding
toboth DNAsites
butpredominantly binds
site II.However,
alonger peptide (residues
1to369; T370)
binds almostexclusively
tositeI. Thus, theadditionof
amino
acids 260
to369
totheT260 peptide results
in the lossof
siteIIbinding.
This region includes
asingleputative metal-binding region,
andmutation
of T370
ateither conserved cysteine of
thefinger
results inequal
butinefficient binding
toboth sites.
While
nometal binding
has been showntobedirectly associated
with thissequence,
these
resultssuggestanovel,perhaps
structural,function
for suchafinger
motif,
since thisdomain
of
Tantigen
appearstoplay
acrucial
role inmodulating
theDNA-binding
behavior of T-antigen
peptides.T-antigen origin DNA-binding activity is highly controlled
since only
afraction of the protein
presentin lytically
infected cells has specific DNA-binding activity
(24).In
addition, differential binding
toeither site I
orsite
IIis
influenced
by
numerousparameters,including
phosphoryla-tion,
oligomerization, ATP, and
Mg2+
(reviewed in
refer-ences 8 and
27),
but whatdetermines how T antigen binds
with
different activities
toeither site is still
notunderstood.
Itis known,
however, that T-antigen
binding
tosite
II,which
contains two
pairs of
pentanucleotide
ininverted
orienta-tion, is essential for the initiation of
simian virus 40 (SV40)
DNAreplication,
while theminimal site
I,involved
primar-ily
inautoregulation of early transcription, contains
twoof
the samepentanucleotides
asdirect
repeats(Fig. 1A)
(re-viewedin references 6 and 27). Thus, the
twosites
arenotonly involved
indifferent
functions but
arepresented in
a verydifferent DNA
conformational
context:site II with each
pair
ofinverted repeatsappropriately spaced
to lie on oneside ofanormal B-DNA
helix,
and thesite
Idirect
repeatmotif
presentasbent DNAdue
totheA+T-rich
spacer(21)
(Fig.
1A).
We and others have
previously
located theDNA-binding
domainof Tantigen for bothsites
betweenaminoacids
131 and 259(1, 18, 27,
and referencestherein).
While it isclear
that these sequences areboth
necessary andsufficient for
specific binding
to both sites I and II, dataobtained
withlonger peptides (1, 18)
indicated that additional regions of T
antigen play
arole
inmodifying the DNA-binding activity.
Thisprompted
us toinvestigate
further the effectof
se-quences outside of the
DNA-binding
domain on theDNA-binding
profile
with the aimofcharacterizing features
essen-tial forefficient
binding
toeither site
I orsiteIIintheSV40
control
region.
Site I and site II DNA
binding.
Weanalyzed
theDNA-binding
profile
offull-length
Tantigen
and severaltruncatedT-antigen
peptides
(depicted schematically
inFig.
1B)
*Correspondingauthor.
expressed
in Escherichiacoli,
either as crude extracts orafter
immunopurification
to nearhomogeneity (Fig. 2) (1).
Expression
vectors,bacterial growth conditions,
prepara-tion of
E. coli crude extracts, andpurification of peptides
expressed
in E.coli
wereessentially
aspreviously described
(1, 13).
From500 mlof
cells,
approximately
2 mgof
greater than 90%pureT260
or0.1mgof
pureT370
wasobtained.
Allpurified proteins
wereanalyzed by immunoblotting
with theappropriate monoclonal
antibodies toconfirm
theexpected
T-antigen epitopes (data
notshown). Cell
lysatesuperna-tantsfrominduced E.
coli
cultures(6
mlof
Ti, T302, T305;
2 ml ofallothers)
or250ngofpurified baculovirus
Tantigen
or 200 ng of
purified
bacterial mutantpeptides
were incu-bated withequimolar
amounts(50 fmol) of restricted,
end-labeled
pONwt
andp1097
orpSVwt
DNA(Fig.
1A)
inbinding
buffer(20
mM HEPES[N-2-hydroxyethylpipera-zine-N'-2-ethanesulfonic acid] [pH 7.8,
50 mMNaCl,
2 mMEDTA,
1 mMdithiothreitol,
1 mMphenylmethylsulfonyl
fluoride,
8%glycerol,
0.2 mg ofglycogen
perml,
1 mg ofbovine serumalbumin per
ml,
1,ug
ofsalmon sperm DNAper
ml).
After 1 h at4°C,
boundcomplexes
wereimmuno-precipitated
with monoclonalantibody Pab419
(12)
or, forT131-260, Pab220 (gift
from S. Mole and D.Lane).
The bound DNA wasseparated
on 1.5% agarosegels
and visu-alizedby
autoradiography
asdescribedpreviously (1, 26).
In all cases
tested,
the material from the crude extracts bound the DNAfragments
in a mannerindistinguishable
from that of the
purified
material(Fig. 3,
comparepanels
A andC).
The controlprotein
in theseexperiments
was Tantigen purified
from insect cells(recombinant baculovirus),
theexpression
andpurification
of which have been described elsewhere(13a).
Baculovirus Tantigen
bound both site I-and siteII-containing fragments,
butpreferably
site I, in a manner similarto that of Tantigen
from mammalian cells(Fig. 3A,
lane4)
asreported previously (15, 20). Full-length
T
antigen
expressed
in E.coli, however, only detectably
bound site I(Fig. 3A,
lane 3) aspreviously
described(19).
The failure to bind site II has been ascribed to theunder-phosphorylated
nature ofthisprotein (17, 19).
Thebacterial6291
Copyright © 1990, American
Society
for
Microbiology
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A
I
I I
5171
4-
EP
--p37
WT
--L}o
...
gn
H .4- --No .- N
5171
5177
H
H
5208
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..-..-- . /=
R
B
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F 182831T370
1 82 83
wU
DNA a
Z
BINDING U.
H
1708
-X--~~~~~~
369
1 8 2 33 259
T1
31-2 60-T302/370 1
I 82 83
T305/370
-FPwqiN
1 82 83
7X.
369
3 6 9
q6
FIG. 1. (A) Top (WT): Diagram of the arrangement of the perfect pentanucleotides GAGGC (shaded boxes) and one imperfect pentanucleotide (empty box) found in site I and siteIIof theSV40 controlregion, their directionandside of the DNA helix(indicated by the
arrows), and the early palindrome (EP) located between them. SiteIIcontainstwopairs of perfect invertedpentanucleotides separated by
7 basepairs, while site I contains twoperfectpentanucleotides in directrepeatseparated bya7-base-pair A+T-richspacer(dotted region) which confersabend in the DNA (21). Thesequencebetween the HindIII (H; nucleotide 5171) and NcoI (N; nucleotide 37) siteswasthe substrate for DNase Ifootprinting of the wild-type origin (see Fig. 4). Below: Substrates used in the McKay DNA-bindingassays(see Fig. 3A, C, and D). The siteIIsubstrate(middle)was aHindIII (H; nucleotides 5171to1046) digest ofp1097 which carriesadeletion of 31 base pairsencompassing all of site1(7). The site I substrate(bottom)wasanEcoRI (R)-SalI(S)fragment from pONwt (22) which containsa 19-base-pairoligonucleotide with thetwoshaded pentanucleotides plus spacerregion from siteIcloned into pAT153 (double lines), and which is bound withaffinity equaltowild-type site1(22). The wild-type origin fragment (WT) for the McKayassays(seeFig. 3B) is the 1,117-base-pair HindIll fragment of pSVwt(pAT153 containing the entire SV40 genome) (26). (B) Diagram of the peptides described in thisreportwith T-antigen residues numbered below. The peptideswerecloned and expressed from the lacpromoterof pUC9asdescribed previously (1). N-terminalfusion amino acidsareindicated by dashes. Stop codons, novel restriction sitestoremovethe intron, andamino acid substitutions (Cys-302or-305toSer[X])wereintroduced byoligonucleotide site-directed mutagenesisasdescribedpreviously (1, 26). The DNA-binding
domain andputative finger regionareshown above, and the clusteredphosphorylated residues (P)areshown below(23). T-Ag,Tantigen.
peptide
T370(residues
1 to369) bound the mixture of
fragments in
a manneridentical
to thatof
thefull-length
bacterialprotein (Fig. 3A,
compare lanes 2 and 3). Contraryto
this, T131-260 (residues
131 to259)bound
both sites butpreferred site
II(Fig.
3A, lane 5). Similarly, a peptideincluding the first
259residues
of T antigen, T260, boundpreferentially
tosite
II,although site
Ibinding
was also detected withhigher
amountsof protein (Fig.
2A orC, lane 1).This result is
in direct
contrast topreviously reported data(17) which showed that
analmost identical
peptide boundwell
tosite
Ibut
verypoorlytositeIIunless phosphorylatedin vitro
by
cdc-2 kinase. This difference may stem fromvariations between the
assayconditions, since
weobserved
preferential
site IIbinding with that
peptide (gift from
D.McVey). Therefore,
to confirm the site IIDNA-binding
specificity
of thepeptide T260,
weperformed
alternative assays,including
DNaseIfootprint analyses
of thewild-type
origin region.
Purifiedbaculoviruswild-type
Tantigen,
usedas a
control, protected site
Iwith low
amountsof protein,
whilewithgreater amounts,fullprotection extended through
theearly palindrome
and site II andsometimes
into theA+T-rich
region (Fig.
4Aand
B).
In contrast, T260pro-tected site II with the lowest amount of
protein
and withII
p1097
pON-wt
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T260
M
180
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8 4
-5 8
-
48.5-36.5
-M
B
- 116
- 84
- 58
- 48.5
- 36.5
- 26.6
,itl
26.6M
T-Ag
C
1 1 6 -.;
84
-58
-48.5
-36.5
-26.6
-FIG. 2. Polyacrylamide gelelectrophoretic analysis of T antigen and peptides purified by immunoaffinity chromatography. (A) E. coli-expressedpeptide T260, Coomassieblue-stained gel. (B) E. coli-expressed peptide T370, silver-stained gel. (C) Baculovirus-expressed full-lengthTantigen, silver-stained gel. Lanes M, Prestained protein size markers (Sigma). Sizes are shown by numbers on sides of gels (in kilodaltons).
increasing amounts partially protected site I, but failed to
protect
the early
palindrome
between (Fig. 4B). While this
may
be
because
T260
is a much smaller protein than
full-length
Tantigen, its
failure to protect the early palindrome is
notdue to an
inability
of T260 to form oligomers (A. Hoss
and A. K. Arthur, unpublished data). Filter binding
experi-mentswith
isolated
site I or site
IIDNA restriction
frag-mentsshowed that T260 bound site
IIwith
agreater
affinity
than
site I
(Hoss and Arthur,
unpublished
data). These
results
confirm
the
specific
and
preferential binding of T260
tosite
II.
Influence of finger domain on DNA binding.
The only other
mutations
so
far described
which
modulate the
DNA-binding
activity of
T
antigen, particularly site
II
binding,
arecon-servative
amino acid
changes
of potentially
phosphorylated
serine
orthreonine
residues
clustered
atthe N
and C termini
of the
protein
(8, 26)
(Fig.
1B).
However,
by
expressing
our mutantsin E.
coli,
we areessentially analyzing
unphos-phorylated peptides (A. Arthur,
unpublished
data)
asdem-onstrated
by
their
being
excellent
substrates for various
cellular
kinases in vitro (13a). In
addition,
the
peptide
T131-T260,
which contains
noneof the
potentially
phosphor-ylated
residues
(Fig.
1B), bound site II
efficiently,
in
a mannerequivalent to that
of
the T260
peptide, which does
contain the
N-terminal cluster
of
phosphorylation
sites.
However, the
peptides
T260 and T370 bear
identical
Ntermini
(Fig.
1B)
but bind sites
I
and II
quite differently,
implicating
thesequence between residues 259 and
369 asbeing
responsible for the results reported here.
Located between residues 260 and 370 is
apossible
metal-binding
sequence
(14, 16),
which
is
highly
conserved
among
six related
papovavirus
Tantigens
(J.
M.Pipas,
personal
communication).
Replacement of either
cysteine
residue 302
or305,
which would be involved in
forming
afinger
structure,
by serine reduced
specific origin
DNA-binding activity of full-length
Tantigen
tolevels
which
areundetectable
by
this assay
(Fig.
3B, lanes
4 to6) (1).
These
mutations have also been shown to greatly reduce
T-antigen-mediated SV40 DNA
replication
in
monkey cells and
trans-formation in
ratcells
(16). When these mutations
wereintroduced into the truncated T370
peptide, specific
DNA
binding
wasretained
(T302/370, T305/370; Fig.
3B, lanes
2and
3). However,
it
is
clear that the overall
binding capacity
of these
mutants wasgreatly reduced,
since
they bound
only
5 to
10%
of the
origin fragment
that
wasbound
by the T370
peptide
orfull-length
Tantigen (determined
by densitometry
of the
autoradiogram
in
Fig.
3B; data not
shown),
despite
using appropriate
volumes of each extract such that
equiv-alent
amountsof
Tantigen
and
peptides
werepresent
in all
assays
(as
determined
by
immunoblot;
data
notshown).
When
binding
tothe
individual sites I and II
wastested
with
equivalent amounts of
purified mutant T370 proteins,
binding
tosite
I wasfound
tobe
considerably
reduced
compared
with
wild-type T370,
while
binding
tosite II
wasincreased
(Fig. 3D).
These
mutations, therefore,
neither
confer
the
efficient
site II
binding
which results from the
deletion of the
finger region,
asdemonstrated
by
the T260
mutant,
since detectable site II
binding required
alonger
exposure
of the
autoradiograph (compare Fig.
3C and
D),
nor
do
they
retain the efficient site I
binding
of
their
progenitor,
T370. The
DNA-binding profile
of the
mutantsT302/370
and
T305/370
is thus intermediate
between that of
T260
and T370
and
may
be due
tothe
single
amino
acid
substitution
partially disrupting
the
finger,
which could then
lead
toreduced
DNAbinding,
with
nostrong
preference
for
either site.
There
is still
noconclusive evidence that the
finger
se-quence
of
Tantigen
specifically
binds any metal
ion.
Obvi-ously, vigorous
testing
of
purified
peptides,
both
biochemi-cally and
structurally,
will
be
required
todetermine whether
this sequence is indeed
ametal-binding
finger.
Nevertheless,
our
data show that both
cysteines
302 and 305 appear crucial
for
somestructural
feature of
Tantigen,
since their deletion
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FIG. 3. (Aand B)Specific DNA binding of E. coliextracts containing mutant peptides or full-length Tantigen(Tl). Immunoaffinity-purified recombinant baculovirus Tantigen was tested as a control (T-Ag). Site I-, site II-, or wild-typeorigin-containing fragments are indicated.(B) A longexposure of theautoradiogram (wild-type origin binding) is shown so that binding in lanes 2 and 3 (T302/370, T305/370) is visible.Nospecific bandswere detected in lanes 5 or 6 (full-length bacterial T antigen containing Cys-Ser substitutions at position 302 or 305; seetext), regardless ofthe exposure time. Equivalent amounts of protein expressed by all constructs were used, asdetermined by immunoblot (datanotshown).(Cand D) DNA binding ofimmunoaffinity-purifiedpeptides from E.coli or the wild-type baculovirus T antigen (T-Ag). (C)Lane M,20% oftheinputDNAused in the binding reactions, 6-h film exposure time; lane M*, a shorter exposure of the marker lanetoshow theindividualrestrictionfragments. (D)Overnightexposure of lanes 1 to 3; lane M*, a shorter exposure of the marker lane.
or
substitution has such a profound effect on the
DNA-binding
activity of the protein.
Metal-binding finger
motifs have been implicated, and in
some casesproved to be directly involved in
sequence-specific
protein-DNA interactions of transcription factors
(reviewed
in references 2, 9, and 14). However, it is also
becoming
clear that they may often perform other roles
related to protein conformation, stability, and
protein-pro-tein interactions
(9,
28), for example, being essential for the
activity
but not the sequence specificity of GAL4 DNA
binding
(5) or, in the case of the second finger of the
glucocorticoid-estrogen
receptors, the
protein-protein
inter-actions
between subunits (2, 11). Other functions include
non-sequence-specific
interactions with DNA, as, for
exam-ple,
in
the gene 32
protein
of
bacteriophage T7, which binds
single-stranded
DNA
(10),
oradenovirus
ElA
(4);
double-stranded RNA
binding of
aretroviral
gag
protein (25);
orprimase activity of
the T7 gene 4 (3).
Our results suggest that the
finger
sequence
of
Tantigen
does
notplay
adirect role
in
contacting the
pentanucleotides
of either site
I orII, since the T260
peptide
canbind both
sites
specifically.
Instead, the wild-type
finger sequence
seems tomodulate the
DNA-binding
specificity
of
the
pro-tein,
either
interfering
with site
IIbinding or favoring site I
binding.
While it has yet to be tested whether the
altered
DNA-binding preferences
of T260 and T370
aredue
toprotein-protein
interactions
orto
aprotein
conformational
change
induced
by
the
finger
structure,
the mutants
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FIG. 4. DNase I protection analysis of a wild-type SV40 origin fragment (Fig. 1A, WT) with purified bacterial T260 or recombinant baculovirus wild-typeTantigen.Thesingleend-labeled
(32p)
fragment in panel A was subjected to Maxam-Gilbert sequencing reactions (A+G andC+T)withMerckkit(catalogno. 6889) or digested with DNase I in the absence of protein (lane M) or after incubation with 500 ng (lane 1)or10 mg(lane 2) of wild-type baculovirusTantigen.Reactions were incubated with 20 fmol of the wild-type origin fragment (144 base pairs) for 20min at room temperaturein25 mlof binding bufferwith 500 ngof sonicatedsalmon sperm DNA as the competitor. DNase I (Pharmacia) wasaddedto 50pg/ml ina70-ml
reaction mixture (final volume; 10 mM HEPES [pH 7.8], 10mM MgCl2,
5 mMCaCl2)
for 1 minat room temperature.The reactionwas stopped by adding 10 mM EDTA, 0.3 M sodium acetate, and 50 mg of salmon sperm DNA per ml. The DNA wasphenolextracted, ethanol precipitated, and analyzed on an8%
DNA-sequencing gel. (B) Incubation with increasing amounts of T260 (lane1, 200 ng;lane2, 500 ng; lane 3, 1,250 ng; lane 4, 2.5 ,ug; lane 5, 5 ,ug) or baculovirus T antigen (lane 1, 200 ng; lane 2, 500 ng; lane 3, 2.0,ug; lane 4, 5 ,ug; lane 5, 20p.g)
protein before digestionwith DNase I.scribed here provide a relatively simple system to test
directly
which
residues, modifications,
or
amino acid
se-quences
influence
the
capacity
of
peptides
to
bind
specifi-cally
to
site I or II.
Wethank Ronald Mertz, DorisWeigand, and AndreaSchweizer foroligonucleotide synthesis;Haralabos Zorbas for invaluable tech-nical adviceandinterest;Stephan Glaser for critical reading ofthe manuscript; Silke Dehde and Birgit Posch for supplying media, antibodies, and othermaterials;and Irene Dornreiter for advice on thepurification of baculovirusTantigen.Wegratefullyacknowledge SaraMole and David Lane for kindly supplying monoclonal
anti-bodies,
Duncan McVey for supplying the 259.3 peptide, and all members ofourgroup forhelpful discussions.A.H., I.F.M., and A.K.A. weresupported by the Bundesminis-terium fur Forschung und Technologie (Genzentrum), and addi-tionalfundingfor this work came from the Deutsche Forschungsge-meinschaft and Fonds der Chemischen Industrie.
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[image:5.612.111.502.75.386.2]proteins. Cell 53:675.
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