Epstein-Barr virus nuclear antigen forms a complex that binds with high concentration dependence to a single DNA-binding site.

JOURNALOFVIROLOGY, Feb. 1987, p. 465-471

0022-538X/87/020465-07$02.00/0

Copyright X) 1987, American SocietyforMicrobiology

Epstein-Barr Virus Nuclear Antigen

Forms

a

Complex That Binds

with High Concentration

Dependence

to

a

Single DNA-Binding

Site

GREGORY MILMAN* AND EUNSEONG HWANG

Department of Biochemistry, The Johns Hopkins UniversitySchoolofHygieneandPublic Health,

Baltimore, Maryland 21205

Received 9 July 1986/Accepted 17October 1986

A bacterially synthesized 28-kilodalton carboxyl-terminal fragment (28K-EBNA) of Epstein-Barr virus nuclear antigen shows highly concentration dependent binding to monomer, dimer, and trimer copies of

synthetic DNA-bindingsite

5' GATCTAGGATAGCATATGCTACCCCGGGG 3'

3' ATCCTATCGTATACGATGGGGCCCCCTAG 5'

inbacterial plasmids. Therateof the binding reaction is independent of the number of sites, but dependent uponthelengthof theDNA containingthesites. These dataareconsistentwith 28K-EBNA locating its binding

sites bya processoffacilitated transferorsliding along the DNA. The highly concentration dependent binding

suggeststhat multiple 28K-EBNA monomerpolypeptides formacomplex beforeor duringbinding. Binding

occursequallywell at 24and37°C,butnot at0°C. A28K-EBNAcomplex boundtoasinglesite has unoccupied

bindingsitescapable of interacting withadditionalDNA molecules. Such interactionisconfirmed byagarose gelelectrophoresis of protein-DNAcomplexes whichindicate thata28K-EBNA complex forms bridges between

twoDNA molecules. A bridge between the two binding regions in theEpstein-Barrvirus origin of plasmid

replication(oriP) wouldformaloopstructurewhich could beanimportantfeature for the regulatory function

ofauthentic Epstein-Barr virusnuclearantigen.

Epstein-Barr virus (EBV) transforms human B lympho-cytesinto permanently proliferating cell lines (19, 25). EBV

DNAreplicatesinthese cellsasamulticopy circularplasmid orepisome. Thecis-acting signals which enable EBV DNA plasmid replication are located on an 1,800-base-pair (bp) region(oriP)in the EBVBamHI Crestriction fragment (20, 23).oriPcontainstwolociessentialtoitsfunction (16). The leftend of oriP contains 20copies of 30-bp tandemrepeats which have a 12-bp palindromic consensus sequence TAGCATATGCTA. The right end of oriP contains four copiesofrepeatsnearlyidenticaltotheconsensus sequence, and the DNA sequence in this region can be drawn as a

116-bp stem-loop dyad symmetry structure reminiscent of other originsofreplication.

EBVnuclear antigen(EBNA-1) coded inthe EBV BamHI Krestrictionfragmentis theonlyviralproductrequiredfor

oriP-dependent plasmid replication (24). EBNA-1, like

polyomavirus and simian virus 40 T antigens, appears to performpleiotropic functions. Inadditionto arole in repli-cation, the protein may act as a transactivating

transcrip-tionalenhancer(16).Theamino half of EBNA-1polypeptide

contains a repeated Gly-Gly-Ala sequence whose length varies in different EBV isolates. Although these repeats

compriseoverathird of the B95-8 strainEBNA-1sequence, they do not seem necessary for EBNA-1 to function in plasmid replication (24). Phosphorylation and dephos-phorylation of EBNA-1 may regulate the physiological ac-tivityof theprotein (9).

Thepurificationandcharacterizationof EBNA-1 has been difficult because of the scarcity of the protein in cells. For

*Correspondingauthor.

example, Sculleyandco-workers(18) obtained only 5 ,ug of paritally purified EBNA-1 after2,500-fold enrichment from 20 g ofRaji cells. Moreover, their purification procedures required solubilizationindenaturingreagents(4 Mureaor6

Mguanidine hydrochloride) whichmay permanently inacti-vatethephysiological activities of EBNA-1. Higher levels of

EBNAmay be obtained from COS-1 cells transfected with DNAcodingfor EBNA-1 under thecontrolofasimian virus 40 virus promoter(17) orfromherpes simplexvirus

recom-binantscontainingachimeric EBNA-1 construct (10).

Our approach to characterizing EBNA-1 has been to synthesizeinbacteriaa28-kilodaltoncarboxyl-terminal frag-ment(28K-EBNA) whichpossesses propertiesof the intact molecule. The 28K-EBNA polypeptide is recognized by

anti-EBNA antibodies in humanserum(14) and is usefulin diagnosis of EBV related diseases (8). Filter-bindingassays

and DNase I footprinting demonstrated that 28K-EBNA

specificallybinds to therepeat sequencesinoriP(15).

To understand better the interaction of 28K-EBNA with its DNA-binding sequence, we constructed a synthetic EBNA-1-binding site containing the concensus palindrome sequencefrom oriP. Thesyntheticsequencewasinsertedin monomer, dimer,and trimerformsinto the Escherchia coli plasmidpUC8 (13). Inthispaperwedescribetheanalysisof 28K-EBNA binding to these synthetic repeats by filter binding assays and the examination of the association of

28K-EBNA with DNAbyagarose gelelectrophoresis.

MATERIALS AND METHODS

Bacteriallysynthesized

28K-EB?iA.

Thebacterialsynthesis

andpurificationof 28K-EBNAhavebeendescribed(14). To

summarize, the 2,236-bp SmaI subfragment of the EBV

(P3HR-1) BamHI fragment K was inserted into the

high-465

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PLASMIID DUC8

PvuI1 Pvull Ndel

1 430 2722

308

PLASMIID DUC8 (308 bD Pvul! 4ra nent)

EcoRI Hindll!

Pvull BamHl Pvul

Iwu I

1 187 308

177 207 PLASMID DRI (337 bD Pvull fraQment)

186 216 Ndel Hindll

Pvull EcoR! BamHI Pvul

I ~~~~~~~~~II I

I 1177 216 337

201 236

FIG. 1. RestrictionmapofpUC8 and pRl.

expression bacterial plasmid pHE6. The resulting plasmid, pNAK28, causes E. coli to produce large quantities of

28K-EBNA, apolypeptide containing the carboxylterminal

191 amino acids ofEBNA-1. The 28K-EBNA polypeptide waspurified to greaterthan 95%homogeneity by

phospho-cellulose andhydroxyapatite column chromatography.

Construction of synthetic EBNA DNA-binding sequences. DNA oligonucleotides 5' GATCTAGGATAGCATATGCT ACCCCGGGG 3' and 5' GATCCCCCGGGGTAGCATAT

GCTATCCTA 3' were synthesized on an oligonucleotide synthesize (Applied Biosystems), purified by acrylamide gel electrophoresis, and isolated from the gel. The oligomers wereannealed by heating to90°C and slow coolingtoroom temperature. The annealed oligomers were ligated into re-peatswith T4ligaseandthencleavedwithBamHIandBglII toproducetandem multiple repeat sequences.

32p incorporation into plasmid DNA. Plasmid DNA was

cleaved withPvuII, and theresultingblunt-endedDNAwas

end labeled with Klenow DNA polymerase by using only [32P]dGTP in the reactions (12a). The 32P-labeled small 300-bp fragments were purified by electrophoresis in 1.5%

low-melting-pointagaroseandseparated from theagaroseby phenol-chloroform extraction and ethanol precipitation.

End-labeled DNAwasusedfor the binding studies analyzed

by agarosegel electrophoresis.

DNAwasalsolabeled withT4 DNA polymerase (12a). A

2-pg sample of plasmidwascleaved with 30 U of Hindlll in

20 pulof T4polymerase buffer (33 mMTris acetate, 66mM

potassium acetate, 10 mM magnesium acetate, 0.5 mM

dithiothreitol, 0.1 mg of bovine serum albuminper ml, pH 7.9). The reaction mixturecontaining the cleaved DNAwas

incubated with 3.7 U of T4 polymerase (New England Biolabs) for exactly 9 minat 37°C to digest approximately 300 to 400 bases. The digestion was halted, and the DNA

was resynthesized by the addition of [32P]dATP and the other three unlabeled deoxynucleotide triphosphates. The labeledDNAwasseparated fromunincorporated[32P]dATP by column chromatography on Bio-Gel A-0.Sm (Bio-Rad

Laboratories). DNA labeled byT4polymerase wasused for

nitrocellulosefilterbinding assays.

Mobility retardation in agarose electrophoresis.

PvuII-cleaved pUC8 DNA was mixed with PvuII-cleaved pRl, pR2, orpR3 DNA before bindingtoprovide anhomologous

comparison DNA lacking an EBNA-1-binding sequence.

The DNAs and 28K-EBNA were mixed in 4 ,u ofbuffer

containing40 mMTrisacetate(pH

8.3)-260

mMNaCI-1 mM

MgCl2-1

mM mercaptoethanol-1 mM EDTA-12.5 ,g of

bovine serumalbumin per ml. The reactionswere incubated for 1 hat 24°C and then mixed with 1

RI

ofelectrophoresis

buffer(40 mM Tris acetate, pH 8.3) containing 50%glycerol

andbromphenolblue indicatordyeandplacedin the wells of

a 1.5% low-melting-pointagarose (Bethesda Research

Lab-oratories) gelinamini-gelapparatus(Hoeffer).Thesamples

were electrophoresed at 5 W (approximately 100 V) for

approximately 2 h until the tracking dye reached

approxi-mately 1 in. (ca. 2.5 cm) from the end of the gel.

PvuII-cleaved lambda DNAwasusedto provideDNA size

mark-ers.Thegelsweresoakedin0.5pgof ethidium bromide per mlfor 10 min and thenphotographed. Forautoradiography,

thegels were dried ontoWhatman no. 1 filter paper witha

vacuumgeldrier(Hoeffer) without heatfor1hand thenwith heatfor 15 min.

Nitrocellulosefilterbinding of 28K-EBNADNAcomplexes. Samples of28K-EBNA and 3 fmol of each 32P-labeled DNA (1,000 to 10,000 cpm) were incubated in 5 pI of buffer containing50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid) (pH 7.5),200 mMNaCl,5 mMMgCl2,

1 mMdithiothreitol, 250

pg

of BSA perml,and 125

pLg

of E. coli tRNA per ml. Nitrocellulose

(Schleicher

and Schuell Co.; BA85)was cut into0.5-by0.5-cm squares and soaked for 30 min in 0.5 M KOH, rinsed 10 times in water, and

equilibrated in 100 mM Tris hydrochloride (pH 7.5). The filters were placed on a fritted glass holder attached to a

vacuum. The28K-EBNA DNAmixtureswereappliedtothe

nitrocellulose, andthe filterswere washed with 1to 5 mlof

buffercontaining25 mMHEPES(pH7.5), 150 mM

NaCl,

5

mMMg2Cl,and10% glycerol.Thefiltersweredried,and the

amount of bound 32P-DNA was determined by scintillation counting.

RESULTS

Plasmidscontaining EBNA-binding sequences. Thetandem EBNA-1-binding sites inoriPcontain different left(AGGA)

andright (CCCR)consensus sequences surroundinga12-bp palindromic repeat. The two synthetic DNA strands were

annealed toproduce astructure with similar asymmetry:

AGGA PALINDROME CCC

5' GATCTAGGATAGCATATGCTACCCCGGGG 3'

3' ATCCTATCGTATACGATGGGGCCCCCTAG 5'

Theannealed DNA monomers were ligated with T4 ligase to

form high-molecular-weight, randomly associated DNA

re-peats. Toobtaintandem repeats forinsertion intoplasmids,

theannealed monomer repeats wereconstructed with half of

a

BglII

site ononeend and halfofaBamHIsite on the other.

A nontandemligationeventforms acomplete site for one of these enzymes. The high-molecular-weight ligated DNA was cleaved with both restriction enzymes to produce only tandem repeats.

The tandem repeats wereinserted into the BamHl site of

plasmid pUC8 (13), and plasmids pRl, pR2, and pR3 were constructedcontainingone, two,and three synthetic EBNA

binding sites, respectively. The distance between synthetic EBNA-binding sites is the same as that in the "tandem repeatregion" (15) of authentic EBV DNA. The number of

insertedrepeats wasdetermined by cleavage withPvuIIand

electrophoresisof the cleaved DNA on 1.5% agarose. Maps ofimportant restriction sites in pUC8 and pRl are shown in

Fig. 1.

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HIGHLY CONCENTRATION DEPENDENT EBNA BINDING TO DNA Rate of 28K-EBNA binding is independent of number of

sites on the DNA. HindIII-cleaved, 32P-labeled linear plas-mids pRl, pR2, pR3, and pUC8 were incubated at 24°C with 2.6 ,ugof 28K-EBNA per ml for different times. The percent DNA bound as a function of time is shown in Fig. 2A. The

time course ofbindingfor all three plasmids was the same. The binding reaction appears to follow first-order kinetics, and the curve is a regression logarithmic fit to the data. A

linear curve resulted when the average percent DNA

un-boundfor all three plasmids was plotted on a semilog scale (Fig. 2B). Under these experimental conditions, the half-time for binding of 28K-EBNA to the plasmids was

approx-imately 5 min.

Onceformed, the binding complex was very stable. The amount of DNA bound was not greatly affected by dilution

of 28K-EBNADNAcomplexes in solutions containing up to

A1oo

0

z

0 Q)

80

60

40

20

0

0 5

Tix.

B

100

0

0

z 10

a

[image:3.612.312.554.86.222.2]

I.)

TABLE 1. StabilityofEBNA-DNAcomplexa

Treatment % of DNA bound

100 mM NaCl... 100

200 mMNaCl... 100

400 mMNaCl... 74

733mMNaCl... 0

1mMP04- ... 10O 7mMP043 ... ... 86

67 mMP043 ... ... 67

333mMP04-3... ... 47

pH7.5 ... .. 100

pH 5.0... .. 79

pH 9.5 ... .. 72

aSamplescontaining3fmolof32P-labeledpRl and464 pmol of28K-EBNA (2.6 ,ug/ml)in 5ILIwereincubated for 1h at24°C. The incubation mixtures werethendilutedwith 10p.lof solution containing sodium chloride, sodium phosphate (pH 7.5), 100 mM sodium acetate (pH 5.0), or 100mMsodium carbonate (pH 9.5) to attain the final concentrations listed above. The mixtures were incubated for10 min, and the percentages ofDNA bound compared with undiluted samples were determined by the nitrocellulose filter-binding assay.

X411'

400 mM NaCl, in buffers at pH 5 and 9.5, or in high

A concentrations of

phosphate (Table

1).

Factorsdetermining the rate of 28K-EBNA binding to DNA.

The binding time was dependent on the length of DNA

containing the binding sequence. The 2,751-bp 32P-labeled pRlplasmid described above was cleaved withPvuIItogive

a 236-bp fragment containing the binding site. The 28K-EBNAbindingto this smallerfragment also displayed first-order kinetics withahalf-time forbinding of8 min(Fig. 2B). The rateofthebinding depended upon both the

concen-tration of 28K-EBNA and the temperature (Fig. 3). At a

j I 28K-EBNA concentration (13.2

p.gIml)

that was fivefold

10 15 20 higher than that used in the experiment shown in Fig. 2, the

binding of 28K-EBNAtopRl occurredapproximately five-e (Miri) fold faster. The half-time for binding at this 28K-EBNA

,~.l concentration was a little under 1 min. In contrast to the

100

0

z

m

a

4.1

U

0 5 10 15 20

Timrle (Mimi)

FIG. 2. Rateof28K-EBNA bindingtopRl, pR2,andpR3.(A)

Linear32P-labeled plasmids pRl (-), pR2(*), andpR3 (A)were incubated for the indicated timeswith 2.6 ,ugof28K-EBNA perml,

and thepercent DNAboundwasdeterminedbythenitrocellulose filterbindingassay.(B) The2,751-bplinearpRl (D)plasmidand the 236-bp PvuII-HindIII-cleaved pRl (A) fragment were incubated with 2.6 ,ug of 28K-EBNA per mlforthe indicated times,and the percent DNA unboundwasdeterminedbythenitrocellulose filter-bindingassay.

80

60

40

20

0

0.v'I I0 A

0 10 20 30 40 50 60

Timrl e (Miri)

FIG. 3. Factorsaffectingtherateof 28K-EBNAbinding. Linear 32P-labeledplasmid pRl was incubated at24°C (*) and 37°C (-) with 2.1 ,ugof 28K-EBNA per ml and0°C (A)and24°C(V)with 13.2 ,ug of 28K-EBNA per ml, and the percent DNA bound at the indicated times wasdetermined bythe nitrocellulose filter-binding assay.

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468 MILMAN AND HWANG

100 high concentrations of DNA, suggesting that this bandarises

from interaction between two DNAmolecules.

28K-EBNA cross-links DNA molecules

containing binding

I 0

80

/

sites. 32P-labeled PvuII fragments from pUC8 and

pRl

were

/ _ mixed with excess unlabeledPvuII

fragments

from

pUC8,

0 pRl, pR2, orpR3. The DNA mixtureswereincubated with

60

28K-EBNA,

and the

resulting

DNA

species

were

analyzed

by

agarose

gel

electrophoresis (Fig. 6). InFig. 6A,the small unlabeled PvuII fragments for pUC8, pRl, pR2, and pR3

4 40 appear as heavily staining bands in lanes H, G, F, and E. The

32P-labeled

pRl

and

pUC8

PvuII

fragments

are visible as lightly staining bands in the same lanes. Incubation with u

$4 20 _ _ 28K-EBNA causedadecrease in the intensities of the bands

X) containing binding sites. The 28K-EBNAcaused nochange

in the heavily staining pUC8 PvuII band in lane D or the

o _ I I lightly staining pUC8 bands in lanes A through C. Binding of

28K-EBNA caused the clearappearance of two new bands

0 2 4 6 8 10 12 14 only in lane C. The autoradiograph of the gel in Fig. 6B

EBNA Coriceritratiora (Gg/rml) demonstrates that incubation with 28K-EBNA caused no

[image:4.612.66.305.71.263.2]

changeinthemobilityof thepUC8 small PvuII fragment, but FIG. 4. 28K-EBNA binds withhigh concentrationdependence. greatly decreased the intensity of the

pRl

small PvuII

Linear32P-labeled plasmids pRl (K), pR2 (*), andpR3 (A) were fragment in lanes A through D from that in the absence of incubated for 30 min at 24°C with the inciated concentrations of 28K-EBNA in lanes E through G. When the 28K-EBNA

28K-EBNA, and the percent DNAbound was determinedby the bound tothe

pRl

PvuII

fragment,

a

fast-migrating complex

nitrocellulose filterbindingassay. (at an apparentmobility of 468bp)was produced in all four

DNA mixtures (lanes A through D), but a slow-migrating

results at 24°C, binding at0°C was barely detectable even

after 60 min. Ata slightly lower 28K-EBNAconcentration

(2.1

pug/ml),

the rate ofbindingdecreased

dramatically.

The

binding did not differgreatly between 37 and 24°C, and at both temperatures the apparent half-time of binding was

approximately 70 min. The percent DNA bound at 24°C

continued to increase for 120min, and at0°C there wasno

detectablebinding (data not shown).

Binding of 28K-EBNA ishighlyconcentrationdependentat a single binding site. Figure 4 illustrates the sharp depen-dence ofbinding on the concentration of28K-EBNA

pro-tein.Different concentrations of28K-EBNAwereincubated

for 30 min at24°Cwith linearplasmids pRl, pR2, pR3, and

pUC8.NobindingtopUC8DNAwasobserved. Thebinding

of 28K-EBNA to the plasmids with one, two, or three repeats was virtually identical.

Essentially

no

binding

was

observed below2.1 ,ugof28K-EBNA permlevenafterlong time periods, and complete binding occurred at

concentra-tions of2.6 ,ug/mland higher. Binding at2.1 ,ug/ml hadnot

reached equilibriumduring the 30-min periodofincubation,

and the twofold difference inbinding between Fig. 3 and 4 resultedfrom the large rate differences for small variations in 28K-EBNAconcentrationin thishighlysensitive range. The results are similar to those previously observed for

28K-EBNAbindingtoauthenticoriPsitesinpHEBO (15;

unpub-lished observations).

Interactionof28K-EBNA withsynthetic repeat sequences.

Figure 5 illustrates the binding of 28K-EBNA to a single

syntheticrepeat. Amixture ofPvuII-cleavedpUC8 and pRl

was incubated for 60 min with 28K-EBNA, and the DNAs

were separated by electrophoresis in 1.5% agarose and stained with ethidium bromide. The 308-bp fragment is

derivedfrom pUC8, and the 337-bp fragment is the homol-ogous fragment from pRl containing the 29-bp synthetic

insert. The 28K-EBNA bound only to the pRl 337-bp

fragment, shiftingits mobility to the two new positions with apparent mobilities of 370 and 820 bp (Fig. 5, lane B). The relativeintensitiesof the two new bands did not change with

28K-EBNAconcentration. The upper band appeared only at

FIG. 5. Binding of 28K-EBNA toasingle repeatinpRl. Lane A containedPvuII-cleaved lambda DNAformolecular weight markers (sizes: 343, 468, 532, 636, 1,708, and 2,296 bp). Lanes B and C contained 110 fmoleach ofPvuII-cleaved pUC8 and pRl DNAs. Lane Balso contained 13.3 pmolof28K-EBNA. The bottom right arrowindicates thepUC8 308-bp fragment, and the top right arrow indicates the pRlhomolgous 337-bp fragment containing the 29-bp EBNA-binding site. The two left arrows indicate the two new positions (apparent mobilities of370 and820bp) of the pRl fragment boundto28K-EBNA.

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HIGHLY CONCENTRATION DEPENDENT EBNA BINDING TO DNA

A B

CD E F

G H

M

____--

ABC DE FG H

A

B

C

FIG. 6. 28K-EBNA cross-links DNA molecules containing binding sites. (A) Ethidium bromide-stained 1.5% agarose gel. Lane M contained PvuII-cleaved lambdaDNAfor molecular weight markers. Lanes A through D contained 13.3 pmol of 28K-EBNA. Lanes A through G contained approximately6.5fmol each of32P-labeled(300 cpm) 308 and 337-bpfragments fromPvuII-cleavedpUC8 andpRl.In addition, 55 fmolof unlabeled PvuII-cleaved pUC8 DNA was included in lanes D and H, PvuII-cleaved pRl DNA in lanes C and G,

PvuII-cleavedpR2DNAinlanes B and F; andPvuII-cleavedpR3 DNA in lanes A and E. (B) Autoradiograph of gel in (A) exposed for 18 h.(C) Autoradiograph ofgel in (A)exposed for 120 h.

complex (at 870 bp apparent mobility) was formed only in

the presence ofexcess unlabeled pRl. An autoradiograph exposedforalonger time (Fig. 6C) reveals that the labeled

pRl PvuII fragment formed slower-migrating complexes

withunlabeled pR2 and pR3 PvuII fragments (lanes A and

B), but formed no complex with the pUC8 PvuII fragment

(lane D).

DISCUSSION

Thefacilitatedtransferorslidingofabinding protein along

aDNA molecule is themechanism proposedfor thelocation

of

binding

sitesbymanyregulatory proteins (e.g.,E. coli lac

repressor) (2). The rate-limiting step is the

low-affinity,

nonspecificassociation ofthebinding proteintoDNA.After

nonspecific binding, the protein rapidly migrates along the DNAto locate a high-affinity specific binding site. A facili-tated transfer mechanism is

required

to accountfor observed

rate constants

(1010

M

s-')

which are 100- to

1,000-fold

greater than diffusion-controlled reactions. A calculated

one-dimensional diffusion coefficient of10-9 cm2s-1 forthe lac repressor enables the

protein

to scan 1,000

bp

pers.

Two kinetic experiments suggest that the 28K-EBNA

polypeptidelocatesits

binding

site

by

facilitated transfer. In

the firstexperiment (Fig. 2A), therateof 28K-EBNA binding isindependent ofthe numberofhigh-affinitybinding sites on

aDNAmolecule. Asinglesite in pRl boundby28K-EBNA issufficienttoretain DNAonnitrocellulose.The presence of additionalbinding sitesinpR2orpR3 has little influenceon

the time required for binding. All three plasmids bind to

28K-EBNA with apparent first-order kinetics and with the

same rateconstant. Theseobservations are consistentwith the rate-limitingstep beingthe low-affinity bindingtoDNA

predicted by the facilitated transfermechanism.Then,even

though the time required for28K-EBNA to scanthe DNA

forhigh-affinitysites maydecrease with increaseddensityof

sites,nosignificant

change

would be

expected

inthe overall

rate of DNAbinding by 28K-EBNA.

In the second experiment (Fig. 2B), the rate ofbinding dependsuponthelengthofthe DNAcontainingthebinding

site. The timerequiredfor 28K-EBNAtobind halfthe DNA

is 5 min for a 2,751-bp DNA

(linear

HindIII-cleaved

pRl

DNA) and 8 min for a 236-bp DNA (the same DNA also

cleaved withPvuII).These observationsarealso consistent

with a facilitated transfer mechanism. The 10-fold lower

concentration for the shorter DNA causes alower rate for

the low-affinity, nonspecific binding. The rate may not be

ABC D

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[image:5.612.78.527.75.424.2]

10-fold lower because the nonspecific 2,515-bp DNA frag-ment in the reaction could facilitate the transfer of the binding protein from one DNA moleculeto another.

The 28K-EBNA concentration is 2.6 ,ug/ml in the rate

studies shown in Fig. 3. This concentration is slightly above the threshold required for binding. A fivefold increase in 28K-EBNAconcentration from 2.6 to 13.2

jig/ml

produceda

corresponding fivefold increase in the rate of binding to

DNA (Fig. 3). However, a small decrease in 28K-EBNA concentration from 2.6 to 2.1

jig/ml

causes a 14-fold de-crease inbinding rate. Therateofbindingisapproximately

the same at 24 and 37°C degrees, but is greatlyinhibited or

totallyblocked at0°C, suggestingthataggregation does not

take place at0°C.

The sharp transition fromboundtounbound DNAwithin a narrowrangeof28K-EBNAconcentrationdoesnotresult fromdepletionofproteinin thereactionbybindingtoDNA. At a 28K-EBNA concentration of 2.1 ,ug/ml in the binding

reactions, the molar amount of protein (374 fmol) is more

than 100-fold in excess of that of the DNA (3 fmol). The

excess 28K-EBNA protein is active and capable ofbinding

atleast 0.1fmol of DNA per fmolof28K-EBNAat2.6

jLg/ml

(datanot shown). Once thebinding complexis formed,it is very stable (Table 1).

Previous studies (15; unpublished observations) of 28K-EBNAbindingtoauthenticoriPsitesin theplasmid pHEBO

(20) indicated a step function dependence on 28K-EBNA

concentration. The apparent cooperativity of 28K-EBNA

binding topHEBO couldresultfrominteraction among the

multiplebinding sites. Anexaminationof therateofbinding

toasinglesite(Fig. 3) indicatesalargeincrease in therateof

the binding reaction for a small increase in 28K-EBNA

concentration.The same sharpbinding dependenceon 28K-EBNA concentration is foundforplasmids containing one, two, or three binding sites (Fig. 4). The concentration of

28K-EBNA required for binding in these experiments is almost identical to thatobserved forbinding to pHEBO in

earlier studies. Therefore, if the step function

dependence

results from cooperativity, the interactions must require only asingle binding site. Cooperativity at a singlebinding

site suggests that the 28K-EBNA forms a complex either beforebinding oratthebinding site.

Thespecificity ofEBNA-1as aregulatoryprotein depends

upon the fidelity of the EBNA-DNA interaction. High fi-delity requires EBNA-1 recognition of EBV DNA-binding sites in the presence of human genomic sequences. If it occurredrandomly, a12-bpbindingsitewould be presentat

approximately 60 copies in a

109-bp

genome. An even

greater number of EBNA-binding sites probably exist

be-causethereisambiguity in the consensusbindingsequence. EBNA-1 may achievebinding specificity in the presence of

these pseudo-binding sites by forming complex structures which link clusters of binding sites. Because these clusters

areextremelyunlikely to occur by chance, the nucleoprotein

structuresformed can provide high-fidelity recognition (5). There are many examples of procaryotic complex nucleoprotein structures. The lambda phage Int protein interacts with approximately seven sites distributed over 230 bp and condenses the linear sequence of 800 A (ca. 80 nm) into a compact three-dimensional structure of 140 A in diameter (3, 4). Similarly, lambda phage 0 protein binds to

eight sites within 105 bp at the lambda origin of replication

(6),andE.coli DnaA protein binds to four sites in 240 bp in the oriC origin of replication (7). Clusters of binding sites which probably form nucleoprotein complexes also are

foundin manyeucaryotic viruses (21).

The EBV oriP contains a cluster of 20 tandem repeat EBNA-1 binding sequences.Twoother

regions

in the EBV genome contain clustered arrays of

EBNA-1-binding

sites. Four sites occur in the dyad symmetry

region

oforiP, and

two additional sitesoccur in the BamHI

Q

restriction

frag-ment (15). Binding ofEBNA to these clustered sites could result inorganized

nucleoprotein

structures.A consequence of a 28K-EBNA complex

binding

to a

single

site is that additional

unoccupied

DNA-binding

sites would be present

on the

complex.

These empty sites should then be able to

bind additional

copies

of the

binding

sequence. If these

additional

binding

sequences were on different DNA

mole-cules,

binding

would cross-link the DNA. The

following

experiments

demonstrate that 28K-EBNA can cross-link

twoDNA strands

containing

binding

sites.

The agarose

gel

electrophoresis mobility

retardation

ex-periment

exhibited in

Fig.

5 demonstrates that 28K-EBNA

binds

specifically

tothe

337-bp fragment

of

pRl

and not to

the

homologous

308-bp fragment

of

pUC8.

The

only

differ-encebetween these

fragments

isthe inserted

29-bp

synthetic

binding site. The

binding

of 28K-EBNA to the

337-bp

fragment causes adecrease in the

intensity

ofthis band and the appearance oftwo new bands ofslower

mobility.

The upper band is not observed when low concentrations of

32P-labeled

DNA are used for

binding,

which

implies

that

this band results from interactions between two DNA

strands.

Proof that the

slower-migrating

band in

Fig.

5 represents

cross-linking

ofpRlPvuII

fragments

is

provided

by

the data

in

Fig.

6. 32P-labeled PvuII

fragments

from

pRl

form dif-ferent

mobility complexes

with 28K-EBNA

depending

upon the sizeoftheunlabeled

fragments

in thereaction.The three

different-sized

species

observed are

explained by

cross-linking of the labeledpRl

fragment

toPvuII

fragments

from

pRl, pR2, or pR3 and no

cross-linking

with the PvuII

fragmentfrom

pUC8.

These DNA

cross-linking

data

provide

confirming

evidence for amultimeric 28K-EBNA

complex.

These studies of

28K-EBNA-binding

kinetics and

associ-ation with DNA suggest mechanisms for

regulation by

full-length

native EBNA-1. If an EBNA-1

complex

cross-links DNA in

vivo,

thenonefunction ofEBNA-1 could beto link the two

regions

essential for

replication-the

DNA sequences in the tandem repeats with those in the

dyad

symmetry

region. Cross-linking

would forma

loop

structure which

might

be an

important

feature for the

regulatory

function. For

example, topoisomerase activity

couldunwind the DNA in the

loop

and

provide

active sites for

replication

or

transcription.

This process could be mediated

by

the

EBNA-1 amino-terminal domain

directly

or

by

proteins

which interact with this domain.

The EBNA-1 amino-terminal domain is linked to the

carboxyl-terminal DNA-binding

domain

by

a

poly(Gly-Gly-Ala) region of variable length in different EBV isolates.

X-ray diffraction studies ofpoly(Gly-Gly-Ala) indicate that

the

polymer

aggregates into long fibers where the repeat

sequences are

fully

extended (9.3 A per

tripeptide)

(1, 12, 22). In a multimeric EBNA-1 complex, the 230-amino-acid

poly(Gly-Gly-Ala) region

of eachmonomercould associate

into

a rope structure over 2,000 A in length which could extend over 600 bp in distance. An extended EBNA-1 structure would enable the protein complex to interact in processes at considerable distances from the DNA-binding

site,

a

generally

accepted property of a trans-activating

eucaryotic

regulatory protein. In this regard, EBNA-1 may

besimialrtothe GAL4proteinofSaccharomycescerevisiae

(11).

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HIGHLY CONCENTRATION DEPENDENT EBNA BINDING TO DNA ACKNOWLEDGMENTS

This workwasaidedby grant MV-287fromtheAmerican Cancer Society, and Public Health Service grants ES03131 from the Na-tional Institute of Environmental Health Sciences and GM32950 from the National Instituteof General Medical Sciences.

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