Formation of a cruciform structure at the simian virus 40 replication origin abolishes T-antigen binding to the origin in vitro.

(1)

Vol. 56, No. 1 JOURNALOF VIROLOGY, OCt. 1985, p.293-297

0022-538X/85/100293-05$02.00/0

NOTES

Formation of

a

Cruciform Structure

at

the Simian Virus

40 Replication Origin

Abolishes T-Antigen Binding

to

the Origin

In

Vitro

DANIEL G. TENEN,lt* LORAL. HAINES,' ULLA M. HANSEN,' ROBERT G. MARTIN,2

AND DAVID M. LIVINGSTON'

Dana-Farber CancerInstituteand the Harvard MedicalSchool, Boston, Massachusetts 02115,1 andLaboratory of

MolecularBiology, National Institute of Arthritis, Diabetes, andDigestiveandKidney Diseases, Bethesda, Maryland

202052

Received 28 March 1985/Accepted 5 June 1985

HeteroduplexDNAmoleculeswereformedby annealinganintact simianvirusreplicationorigin-containing

fragmenttoamutantderivativelacking the indigenous wild-type 27-base-pair (bp) invertedrepeatwithinthis structureandcontaining a nonhomologous 26-bpinverted repeatsequenceinitsplace. Results ofrestriction

enzymeand Si endonucleasecleavage analyses strongly suggestedthata13-bp stem-loopstructureformedat

thesite ofnonhomologybetween thesetwoDNAs. Thisstructurelies within theboundaryofsimian virus40 T-antigen-bindingsite2, and itspresenceinhibited T-antigen bindingtothatsequencebutnot toanadjacent

higher-affinity binding site (site 1). Therefore, the conformation of sequences within an otherwise intact

T-antigen-binding sitecanhave major effectsupon T-antigen bindingthere.

Theorigin of simian virus40(SV40) DNAreplication, like

other procaryotic and eucaryotic origins, contains a large inverted repeat sequence with the potential for forming a

stem-loop structure. This 27-base-pair (bp) sequence (a

perfect 13-bp invertedrepeatseparated by1bp) lieswithin a

37-bp strongbinding site, site 2, for SV40large Tantigen. Site2constitutesthe coreoftheviralreplicationorigin(1, 3,

7). Since bindingto this site isessential forthe initiation of

viral DNA replication, it was of interest to investigate the

effects ofthe formation ofa stem-loop structure atthis site

on T-antigen binding to the origin region in vitro. In this report, we describe the results of such experiments.

Previously, we described the formation of heteroduplex

molecules between replication origin-containing fragments ofwild-type SV40DNAandthose ofadeletionmutantthat

lacked 18 of the 27bp ofthe site2palindrome attheorigin (8). These heteroduplex molecules contained a stem-loop

structure consisting ofa 7-bp stem and a 3-bp loop on the

wild-type strand. The presence of this hairpin structure on

either the E or the L strand inhibited T-antigen binding to

either strand atsite 2. The presenceof this structureonthe Estrand alsoinhibitedT-antigen bindingtothe Ebutnotthe L strand at site 1, a high-affinity binding site abutting the

early sideofthecoreorigin sequence. Inspeculatingonthe reasons forT-antigen failing to bindto the altered site 2 in

these heteroduplexes, we pointed out that the asymmetric deletion within the 27-bp inverted repeat resulted in an

"abnormal" stem-loop (7-bp stem with a 3-bp loop versus

13-bp stemwith a 1-bp loop). It was therefore stillpossible

thatsuccessfulbindingtosite 2 would result ifastem-loopof the same size containing the same number of internal base

*Correspondingauthor.

tPresentaddress: Beth IsraelHospital,HarvardMedicalSchool,

Boston, MA02215.

pairs were formed at this locus in both DNA strands. The

experiments describedbelow eliminate thispossibility.

A mutant viral genome (pRGM29) containing a

substitu-tion of 26 bp of foreign sequence for the entire 27-bp

palindrome at the origin has recently been described (C. Nobileand R. G. Martin, submittedforpublication). The 26

bp substituted for the wild-type sequence also contained a

13-bp invertedrepeat,butthisrepeat wastotallyunrelated to the wild-type repeat. When heteroduplexes were formed between the deletion-substitution mutant and wild-type

DNA, the resulting hybrids were found to contain a

stem-loopstructureoneach DNA strand within site 2 (Nobile and

Martin, submitted), as shown below. The heteroduplex,

wild-type, andpRGM29 homoduplex DNAs were

individu-ally incubatedwithincreasingamountsofpurifiedTantigen, whichwas isolated fromSV40-infected monkey cells.

Bind-ing by the latter to the origin region of each DNA was

measured bythe DNasefootprint technique(4, 8, 9). The mutantand wild-type plasmids both contain pBR322

sequences

extending

from the SphI site clockwise to the

HindIII site and SV40 sequences running clockwisefrom the

HindIll site at nucleotide 5172 to the SV40 SphI site at nucleotide 129. One mutant plasmid (pRGM28) had

sus-tained a 27-bp deletion from nucleotide 5230to nucleotide 13, and anothermutant plasmid (pRGM29) had sustained a

26-bp insertion atthe site of this deletion. The insert

con-tainedtwo Sall and a single EcoRI restriction site (Fig. 1).

These restriction site sequences were oriented sothatthey constituted a

26-bp

inverted repeatstructure

(Fig.

1). Each

recombinant plasmid was digested with MboII, which cleavesatpBR322 nucleotide4346,alocation 46 nucleotides downstream of the above-noted SV40 HindlIl site. The MboII-cleaved DNAwasthen 5' end labeled withT4

poly-nucleotide kinase after

digestion

of the 3'

overhanging

se-quencewith T4 DNA

polymerase.

After

subsequent

cleav-ageofeach

plasmid

withMspI,a

250-bp fragment containing

293

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(2)

SITE

1

11 SITE 2

5180 5190 5200 5210 5220 5230 5243/0 10 20 30 40

WT 5' ...GCAAGCCTAGGCCTCCAAMAAGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAMTTAGTCAGCCATGGGGCGGA...3'

t

HaeIII AluI HaeIII BgII HaeIII

pRGM29 5' ...GCAM...3'GGTCGACCCCGMTTCGGGGTCGACC_...

Wild Tvnp

ICG GC

GC

AT

GC

CG

C G

G C

A T

"I

.u

iy

.,I

G

A

5'. ..CTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTC

3' ...GATCCGGAGGTTTTTTCGGAGGAGTGATGAAGACCTTATCGAc

C

pRGM29 A

C

T

GCATAAATAAAAAATTAGTCAGCCATGGGGCGGA.. .3'

GGTATTTATTTTTTAATCAGTCGGTACCCCGCCT

... 5'

G

T

G C C G T A

G C G C

G C

G C C G A T A T

[image:2.612.89.561.70.160.2]

FIG. 1. Possible cruciformstructure attheSV40originofreplication generated byaheteroduplexbetweenwild-typeDNAand themutant pRGM29. Thetop line shows thewild-type SV40 sequence from nucleotide 5180to46(usingthe SV numberingsystem[10]. Above this sequence are T-antigen-binding sites 1 and 2, as previously defined by DNase footprinting (9). Beloware the locations ofcleavage by

restriction endonucleasesHaeIII, AluI,andBglIinwild-typehomoduplexDNA.Thesecond line denotesthesequence ofthemutantplasmid pRGM29, which is identicalto that ofthewild type exceptthatwild-type bases 5230through 13were deleted and 26bases of restriction enzymelinker sequenceweresubstituted,asindicated.Atthebottom of thefigureisacruciformheteroduplexmolecule that could formas aresultofmixing wild-type andpRGM29 origin fragments(Nobile andMartin, submitted). Thearrowsdenotetwoofthefourpentanucleotide

sequencesfelttobeimportanttoT-antigenbinding atsites 1 and2(2,5).

the SV40 origin sequences and labeled onthe early side of

theoriginwaspurifiedbyelectrophoresis through

polyacryl-amide gels and identified by autoradiography. These

wild-type and deletion-substitution mutant DNA segments were

usedin subsequent experiments.

Thefirst questionwas whether Tantigen canbindto site

2from thedeletion-substitution mutant (pRGM29), as mea-suredbyDNasefootprint analysis. Previously, it was shown that an 18-bp deletion at this locus decreased, but did not

abolish, binding to this site (8). In the pRGM29 site 2 sequence,allfouroftheGAGGC pentanucleotide

T-antigen-bindingsequenceswithin the 27-bp inverted repeat (2; Fig. 1) had been deleted and substituted by linker DNA, and no

bindingtosite2 wasobserved at the highest T-antigen input, inkeeping with the results of others (2; Fig. 2). By contrast,

bindingto the wild-type sequence was clearly noted. More-over,T-antigenbinding to site 1 proceeded normally in both the5'-end-labeled mutantand wild-type DNAs (Fig. 2) and in their 3'-end-labeled equivalents (data not shown).

Al-though there was a suggestion of a small difference in the

amount ofT antigen needed to protect wild-type/wild-type

versuswild-type/mutant site1 sequences(see Fig.4),itwas notreproducible in repeated titrations.

Next, 250-bp heteroduplex DNA molecules were formed in vitro between the above-noted wild-type and deletion-substitutionmutant250-bpMboIIlMspI fragments,andtheir

structure was analyzed. Specifically, end-labeled wild-type

orpRGM29 DNA fragments were mixed withan excess of

thecorresponding,unlabeled mutantorwild-type fragment.

After denaturation and reannealing, each reaction mixture

was electrophoresed through a nondenaturing

polyacryl-amidegel. Repeatedly,wild-type/mutantheteroduplex

struc-tures migrated more slowly than did homoduplexes of the

same length, as was noted previously for DNA molecules

containinganotherstem-loopstructureinthis region (8) and for these molecules (Nobileand Martin, submitted).

There-fore, it was possible to purify the desired heteroduplex molecules away from the parental homoduplex molecules

(datanotshown). Analysisof the products ofSi

endonucle-ase cleavage ofsuch gel-purified heteroduplexes showed a

single Si-sensitivesite oneach strand resultingfrom cleav-age of1 to 2 bp centered about nucleotide 5243/0, i.e., the

I

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NOTES 295

WT

29 7r

-r

---

-

A

B

C

D

E

F

G

T(pg)

0 1.2 7.2 7.2 3.6 1.2

0 "'pplnqI

# _

_~~NA.W

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/29

A B

29*

/WT

C

D E

A

G

-20

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.--

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_mp

-

5204

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am

-5191

-. ...

18onee-

10

[image:3.612.55.290.70.504.2]

. _

FIG. 2. SV40 T-antigen footprint protection of wild-type and pRGM29 homoduplex DNAs. The 250-bp wild-type (WT) and 249-bp mutant pRGM29 (29) MboIIlMspI fragments, each 5' end labeledatthe MboIIsiteatpBR322nucleotide 4346,weresubjected

toDNase footprint analysisafter the addition of T antigen purified fromlytically infected monkey (CV-1P) cells and preheatedat30°C for 5 min, as previously described (9). The amount of T antigen added isindicatedatthetopof each lane, and theDNA

concentra-tion ineach reaction mixture wasapproximately 3 x 10-10 M. A similar concentration was employed in all experiments. Bands

correspondingtobinding sites 1 and 2 (9)areindicatedby brackets

along the left side of theautoradiograph.

center of the axis of symmetry of each inverted repeat

sequence (Fig. 1 and 3). No such cleavage was observed

whenthe heteroduplexes were incubated in the absence of the enzyme. Moreover, no Si nuclease cleavage of the

wild-type ormutant homoduplex DNA was observed (data not shown). Similar resultswereobtained with these mole-culesbyNobile and Martin(submitted). Given theabnormal

electrophoreticbehaviorof theheteroduplex molecules,the inverted repeat nature of the wild-type and inserted linker

FIG. 3. Electrophoretic analysisofthe productsof Si nuclease cleavage of heteroduplex molecules. Heteroduplexes formed

be-tween 250-bp 5'-end-labeled (*) MboIIlMspI wild-type (WT) and unlabeled pRGM29 (29) fragments (WT*/29, lanes A and B) and 5'-end labeled pRGM29 and unlabeled wild-type fragments (29*/WT, lanes C and D)werepurifiedbynondenaturing

polyacryl-amide gel electrophoresis and subjected to partial Si nuclease cleavage as previously described (8). Markers included a partial

DNase digest ofa wild-type homoduplex fragment (lane E) and

products of Maxam-Gilbert chemical sequencing reactions (6) of 250-bpwild-type homoduplexMboIIlMspIfragments 5' endlabeled

at the MboII site (lanes G and G + A). The arrow on the left

indicates aband(s) at nucleotide 5243/0 produced by Si nuclease cleavage. The numbersonthe right referto specific nucleotides in the SV40sequence(10).

sequences, and the identification of a single Si nuclease-sensitive siteinananalogous placeoneachstrand,itseemed

likelythat themajorityofheteroduplexmoleculescontained the cruciformstructureshown inFig. 1. Inkeepingwiththis possibility, two restriction enzymes (BglI and HaeIII),

which normally cleave at sites in the wild-type sequences

thatconstitute partof the proposedheteroduplex cruciform

structure, failed to cleave the wild-type strand in the

--ffi~~~~-a

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(4)

296 NOTES

WT7WT

WT%29

29 /29 29 /WT

--~~-- r -- ...--- --..

A

B

C

D

E

F G

H

I

J

K

L

M N

O

0

3 6

12

0

3

6

12

0 3 6

12

3

6

12

7p

qj __::_:

T( 9)

r

'

2

WT

*/WT

WT /29

A

B

C

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E F

G

H

0 3

6 12 0 3

6

12

1

.. _._f

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-

Wn

:-S g- "mow'4'

-0

FIG. 4. T-antigen footprintprotection ofhomoduplex wild-type (WT*/WT), homoduplexmutant(29*/29),andtwoheteroduplex (WT*/29 and29*/WT) 250-bpMboIIlMspIDNAfragments,5'end labeledattheMboIIsite(*).Leftpanel,DNAwas5' end labeledattheMboIIsite

asindicated in the text and subjected to DNasefootprint analysis after addition of the indicated amountof purified T-antigen protein. Fragments analyzed included WT*/WT and 29*/29 homoduplexes (lanes A to D and I to L, respectively), a heteroduplex between

5'-end-labeled wild-type and unlabeled pRGM29 DNA fragments (WT*/29, lanes E to H), and a heteroduplex between 5'-end-labeled pRGM29 and wild-type DNA fragments (29*/WT, lanes M to 0). Bands corresponding to nucleotides within binding sites 1 and 2, respectively,areindicated bybracketsalongtheleft border. Right panel,Anoverexposureof lanes Athrough Hof theexperiment shown in theleft panel,demonstratingthe bandspresentin site 2 of thewild-typehomoduplexDNA and ofWT*/29DNA. Densitometerscanning of these bands in the WT*/29 heteroduplex lanes revealed nodecrease in intensity of bands within site 2 on T-antigen addition when

normalizedfor the totalintegralof theareasunder thepeaksineach lane. Densitometrywasperformedwith aJoyce-LoeblChromoscan3

densitometer.

heteroduplex molecules (data not shown). BglI resistance

was also observed by Nobile and Martin (submitted). By

contrast,anenzyme(AluI) that normally recognizesa

neigh-boring sequence believed to lie outside of the sequences constituting the cruciform structure cleaved the

hetero-duplex molecules efficiently (data not shown). In short, a

specific sequencethat abuts the proposed hairpin structure wasnotsufficiently perturbed in the heteroduplextobecome immune to restriction enzyme cleavage. Analogous results

were obtained previously with the hairpin arising from the formationofheteroduplexes between wild-type origin DNA

and a restriction origin fragment bearing an 18-bp deletion

withinsite 2(8).

When T antigen was incubated with the above-noted

heteroduplex molecules, 5' end labeled on either the

wild-typeormutantstrand,nobindingtosite2could be detected,

eventhough binding to site 1 was as efficient asbinding to site 1 inwild-type homoduplex DNA (Fig. 4). In particular,

nobindingwasobservedtowild-type site2sequenceswhen

theywerepresentintheabove-noted hairpin structure(Fig.

1), evenafter adding enough T antigentobind nearly all of the available site 2 sequences in wild-type homoduplex

molecules (Fig. 4). Results of two autoradiographic expo-sures are presented to indicate the lack of reduction in intensity of the detectable site 2 bands in the heteroduplex

DNAs. Asanadditionaltestof thiscontention,no

T-antigen-dependent reduction in intensity could be detected in densi-tometric analyses of the 5'-end-labeledwild-type/mutant site 2 bands in this experiment. Included among the

heteroduplex site 2 digestion products were certain rela-tively intense bands that were identical to those of the wild-type/wild-type digest. In thecaseofhomoduplexDNA, thesebandsweremarkedly suppressed bythe addition of12

,g of T antigen. Theywereunaffected, however, whenthe same amount of protein was added to wild-type/mutant

DNA. Similar results were obtained with the same DNA

fragments 3' end labeled on the other strand (data not shown). Moreover,asnotedearlier, with these DNAs there

was noapparent inhibition of site 1 binding.

Taken together, the data presented here show that it is

T(pg)

2

1

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(5)

NOTES 297

possible to generate DNA molecules which, in their native configuration, appear to contain stem-loop structures on

both strands at the viral replication origin. In the case

described here, the data suggest that two 13- by 13-bp hairpins were generated by annealing a molecule bearinga

wild-type inverted repeat at the SV40 origin of replication to another that had sustained a complete deletion of this structure and reinsertion of an artificial structure with the same dyad symmetry. When T antigen and this DNA mole-cule were mixed, binding was observed atsite 1, butnone

was detected within site 2, including to sequences within eitherhairpin structure. By contrast, in wild-type homodu-plex DNA, T antigen is known to bind to a sequence (site 2A)composed of 16 nucleotides flanking the early side of the 27-bp inverted repeat structure describedhere plus approx-imately 9 of 13 bp within thewild-type hairpin noted above (8). These 9 bp containone copy of the pentamer GAGGC believed to play an important role in promoting specific

binding of T antigen to sequences in this region (2, 5). Therefore,failuretobindto the same sequence now,inpart, present as astable hairpin structure could be interpreted in at least three ways, none of which are mutally exclusive. First, if theprotein is capable of binding to site 1 and to the 16nucleotides thatconstitute the segmentof site 2A

imme-diately adjacenttotheabove-noted wild-typestemandloop

in normal duplex DNA, then failure to observe such an

interaction in the case noted here may have resulted from

steric interference with a specific protein-DNA binding

event in this region. A second interpretation is that the

protein cannotbind stably to hairpin structures even when theycontainwild-type duplex sequences that normally

con-stitute a high-affinity T-antigen binding site. Third, the presenceofthe26-bpmutant insertionmay preventbinding

of T antigen to the heteroduplex site 2 sequence. From

earlier work, it is clear that binding to site 2 can be negatively affected by stem-loop formation within it even when the deletion mutant strand of the heteroduplex can

accommodate T-antigen binding when present in the

homoduplex configuration (8). Therefore, data consistent with at least one of these possibilities exist. Clearly, simple

formation of a symmetric heteroduplex hairpin structure, one strand of which is wild type, is not enough to

accom-modate specific stable T-antigen binding. Whatever the

detailedbasis for the failure of T-antigen bindinginthis case,

these data again suggestthatthe conformation ofsequences at andnearthe SV40 replication origincan have significant

effects upon the ability of Tantigen to bind there. Hence, if such structures form in vivo, they may have important regulatory effects on the initiation of autonomous viral genome replication or the regulation of the early and late viraltranscription units orboth.

Wesincerely appreciate the helpof Margaret Bradley in purifica-tionofTantigen and inotherdiscussions, and the assistance ofAnn Desaiinthepreparation ofthemanusript.

D.G.T. is the JamesM. FaulknerFellow of the Medical Founda-tion, Boston, Mass. This work was supported by

Public

Health ServicegrantCA15751 fromtheNational Institutesof Health.

LITERATURE CITED

1. Bergsma, D.,D.Olive, S. Hartzell, and U. Subramanian. 1982. Territorial limits and functionalanatomyof thesimianvirus40 replication origin. Proc.Natl. Acad. Sci. USA 79:381-385. 2. DeLucia, A. L., B. A. Lewton, R.Tjian,and P.Tegtmeyer. 1983.

Topography of simian virus 40 A protein-DNA complexes: arrangementsofpentanucleotide interaction sitesattheoriginof replication. J. Virol.46:143-150.

3. DiMaio, D., and D. Nathans. 1980. Cold-sensitive regulatory mutantsofSV40. J. Mol. Biol. 140:129-142.

4. Galas,D.J., andA.Schmitz. 1978. DNAfootprinting: asimple methodfor detection ofprotein-DNA binding specificity. Nu-cleic AcidsRes. 5:3157-3170.

5. Lewton, B.A., A. L. DeLucia, and P.Tegtmeyer. 1984. Binding of simian virus40 AproteintoDNAwithdeletionsattheorigin ofreplication. J.Virol. 49:9-13.

6. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labeled DNA withbase-specific chemical cleavages. Methods Enzymol. 65:499-580.

7. Myers, R., S. Rio,A.Robbins, and R. Tjian. 1981. SV40gene expression is modulated by the cooperative binding ofTantigen toDNA. Cell 25:373-384.

8. Tenen,D.G.,D. M.Livingston, S.-S. Wang,andR.G. Martin. 1983.Effect ofastem-loopstructurewithin the SV40 replication origin upon SV40Tantigen bindingtoorigin regionsequences. Cell 34:629-639.

9. Tenen,D.G.,T.Taylor,L.Haines,M. K.Bradley, R. Martin, and D. M. Livingston. 1983. Binding of SV40 large Tantigen fromvirus-infectedmonkeycellstowild-typeandmutant viral replicationorigins. J. Mol. Biol. 168:791-808.

10. Tooze, J.1981. DNA tumorviruses:molecularbiology of tumor viruses, 2nd ed., part 2/revised. Cold Spring Harbor Labora-tory, ColdSpring Harbor,N.Y.

VOL. 56, 1985