Identification of EBNA1 amino acid sequences required for the interaction of the functional elements of the Epstein-Barr virus latent origin of DNA replication.

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JOURNALOFVIROLOGY, June 1993,p. 3418-3426

0022-538X/93/063418-09$02.00/0

Identification

of EBNA1 Amino Acid

Sequences Required for

the

Interaction of the Functional

Elements of the

Epstein-Barr

Virus Latent Origin of DNA Replication

KIM GOLDSMITH, LESLIE BENDELL, ANDLORI FRAPPIER* MolecularVirology andImmunology Program, McMaster University,

1200 Main Street West, Hamilton, Ontario, CanadaL8N3Z5 Received 22January 1993/Accepted16 March 1993

Epstein-Barr nuclear antigen 1 (EBNA1) activates DNA replication from the Epstein-Barr virus latent origin, oriP. This activation involvesthedirect interactionof EBNA1 dimers withmultiplesites within thetwo noncontiguous functional elements of the origin, thefamilyof repeats (FR) element and thedyad symmetry (DS) element. The efficient interaction of EBNA1 dimersbound to these two elements in oriP results in the

formation of DNA loops inwhich the FR and DSelements arebound togetherthroughEBNA1.In orderto

elucidate themechanismbywhich EBNA1 induces oriP DNAlooping,wehaveinvestigatedthe DNAsequences and EBNA1 amino acidsrequiredforEBNAl-mediatedDNAlooping. Usingaseries oftruncation mutantsof EBNA1producedinbaculovirusandpurifiedtoapparenthomogeneity,wehave demonstrated that the EBNA1

DNAbinding and dimerizationdomain isnot sufficient to mediate oriP DNAloopingandthatanadditional

region(s) located between amino acids 346 and 450 is required. SingleEBNAl-binding sites, separated by930

bp ofplasmidDNA,werealso shown to support EBNAl-mediatedlooping, indicatingthatthe formation of largeEBNA1complexes, suchasthose observedonoriPFRandDSelements,isnotarequirementforlooping.

EBNA1 is a multifunctional protein essential for the

replication and maintenanceofEpstein-Barr virusgenomes

during latent infection (30). EBNA1 activates the latent

origin ofEpstein-Barr virus DNA replication, oriP, to

ini-tiateasingleround ofreplicationpercellcycle (1,29).This activation involves a direct interaction of EBNA1 with

multiple 18-bp palindromic recognition sites present within the two noncontiguous functional elements of oriP, the

family ofrepeats(FR) and the dyadsymmetry(DS)elements

(19, 22). These elements are separated by approximately 1

kb inoriP, but origin functionisindependentofthisspacing (22). The DS element contains fourEBNA1-binding sites,a

65-bp region of dyad symmetry, and the initiation site for

latent-phase DNAreplication (9, 27). The FR element

con-tains20EBNA1-bindingsites and,when boundbyEBNA1,

activates DNAreplication from the DS element, enhances transcription from avariety ofpromoters, andgoverns the

stable segregation of oriP-containing plasmids during cell

division (12, 21, 25). The mechanism by which EBNA1

functions inanyoftheseprocessesis notyetunderstood.

In order to gain a better understanding of the EBNA1

proteinand themechanism(s) by which it functions,wehave

overproduced EBNA1 by using thebaculovirus expression system(bEBNA1) and purified it to apparent homogeneity (6). PurebEBNA1wasshowntobeadimer in solution and

tobindthemultipleEBNA1-binding sites inoriPas adimer.

bEBNA1binding tothe DS element caused localized struc-tural distortions ofthis element consistent with bEBNA1-induced bending of the DS element (8). Recently, we used electron microscopy to examine the interaction of pure bEBNA1 withorP(7). The resultsindicatedanordertothe

assemblyofbEBNA1 onto orP,with abEBNAl complex first being formed on the FR element of orP. At higher bEBNA1 concentrations, interaction of bEBNA1 with the

* Correspondingauthor.

DS element was observed in the form of looped DNA

molecules, inwhich the FR and DS elements of orPwere

joinedthroughabEBNA1complex, causingtheloopingout

of the 1 kb ofinterveningDNA. EBNA1-mediatedlooping of the FR elementtothe DS elementwasalso reported by Suet

al.(24) andwasfoundtostabilize theinteraction ofEBNA1 with the DS element. These results suggest that

EBNA1-mediated looping oforiP maybe an important step in the

activation of the DS elementbythe FRelement.

DNAloopinghas been described foravarietyof transcrip-tionsystemsas amechanismby whichenhancerorrepressor

elements act at a distance to activate or repress promoter

elements (reviewed inreference 13). DNAloopinghasalso been shownto regulate DNA replication in theEscherichia

coliplasmidsR6K andP1.ForplasmidR6K,thereplication

initiatorproteinmediates DNAlooping betweenthe-yand cX originsto activate replication from the aorigin orbetween

the y and 1Boriginsto activatereplicationfrom the 13origin (15-17). The initiatorprotein ofthe P1 plasmid negatively regulates replicationby mediatingDNAlooping between the

controllocus and thereplication origin(4). Althoughit isnot yetclearwhether similar looping mechanisms playarole in

the initiation of DNA replication ineukaryotes, the multi-component nature of eukaryotic origins suggests that the interaction of origin elements by DNA looping maybe an

important step in the regulation of replication from these origins (reviewed in references 5 and 10). In keeping with

this hypothesis, the UL9 origin-binding protein of herpes simplexvirus type 1 has been shown to mediate looping

betweenbindingsites I and IIwithin

oris

(11).

Inorderto further understand EBNA1-mediatedlooping

of oriP and itsfunctionalsignificance,wehave beenmapping

theEBNA1 amino acidsresponsible for theassociationata

distance of DNA-bound EBNA1 dimers. To this end, we

haveoverproducedaseriesoftruncationmutantsofEBNA1

in baculovirus, all of which retain the DNA binding and

dimerization domain (2, 23), and have purified them to 3418

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apparent homogeneity. Here we examine their ability to mediate interactions between theoniPFR and DS elements, aswell as between individual EBNA1-binding sites.

MATERIALS ANDMETHODS

DNA. TheoriP-containing DNA used for electron micros-copy wasgenerated by digesting pGEMoriP, which contains the complete oniP sequence in pGEM2 (Promega), with XmnI andHindIl (6). This yields a 3.16-kb DNA fragment containing oriP and a 1.84-kb vector fragment which are easily distinguished by size in the electron microscope. For enhanced ligation efficiency assays, pGEMoriP was linear-ized with MluI, which cuts between the FR and DS elements of oriP. The linearized plasmid was then extracted with phenol-chloroform (1:1) and ethanol precipitated prior to being used in ligation efficiency assays. To construct a DNA fragment containing twoEBNA1-binding sites, pGEMdyad, which contains only the DS element of onP (7), was digested with AvaI to remove a 123-bp fragment containing three EBNA1-binding sites of the DS element. The large (3-kb) AvaI fragment of pGEMdyad was religated to form

pGEMsl, which contains one EBNA1-binding site

corre-sponding to the DS element site 1 (20). pGEMs2 was

generated from pGEMsl by excising the 1.16-kb

PvuII-to-SspIfragment of pGEMsl, containing the EBNA1-binding

site, and ligating it into the unique EcoRI site of pGEMsl (after the recessed 3' ends of pGEMsl had been extended with DNA polymerase I Klenow fragment). The resulting

pGEMs2 plasmid contains two copies of EBNA1-binding

site 1 of the DS element, separated by 930 bp of plasmid DNA. A2.33-kb DNA fragment containing the two EBNA1-bindingsites was excised from pGEMs2 with PvuII and SspI

and purified from low-melting-point agarose prior to being

used in electron microscopy.

Construction ofEBNA1 truncation mutants. EBNA1 DNA fragments coding for bEBNA39,

-346,

and

-395

were excised fromplasmid p205 (kindly provided by Bill Sugden [30]) with NcoI, TaqI, and BsaI enzymes, respectively, in addition to

MscI.Therestriction fragment used to construct bEBNA330

was generated by partial digestion of p205 with TaqI and

complete digestion withMscI.The ends ofEBNA1

restric-tion fragments were made blunt by extending 3' recessed ends with the DNA polymerase I Klenow fragment (for

bEBNA39,

-330,

and-346) orby removing 5' overhangs with

mung bean nuclease (for

bEBNA395).

EBNA1 fragments

wereligated tothebaculovirus transfervectorpVL941-SW

after linearization of thevectorwith Ncol and extension of the 3' recessed ends with the DNA polymerase I Klenow fragment as previously described (6). As a result, the EBNAl-coding sequenceswereplaced next to the ATG start site of the vector, resulting in proteins composed only of

EBNA1 sequences. For

bEBNA451,

the SmaI-to-MscI

re-strictionfragmentofp205containing EBNA1 sequences was

ligatedto pVL941-SWafterlinearization of the vector with

XbaI and extension of the 3' recessed ends with the DNA

polymerase I Klenow fragment. The resulting

bEBNA451

proteincontainstwoamino acids(Ala Leu)atitsNterminus

inadditiontoEBNA1 sequences.

bEBNA330619

was

gener-atedbyreplacingtheSacII-to-Spel fragmentofpVL941-SW/

EBNA330, containing the C-terminal 36 amino acids of EBNA1, withaDNA linkercontainingastopcodon. For all

EBNA1 fragments cloned into pVL941-SW, the DNA

se-quences spanning the N-terminal and C-terminal EBNA1 truncationswereverifiedbydideoxysequencingwith

Seque-nase(United StatesBiochemicals).

Generation of recombinant baculoviruses. Recombinant EBNA1 baculoviruses were generated by recombination of pVL941-SW transfer vectors containing EBNA1 sequences with wild-type baculovirus (Autographa californica nuclear polyhedrosis virus) in Spodoptera

ftugiperda

(SF-9) cells as previously described (6). SF-9 cells were propagated as monolayer cultures in Grace's medium (GIBCO Laborato-ries) supplemented with 0.33% yeastolate, 0.33% lactalbu-min hydrolysate, and 10% fetal bovine serum.

Purification of bEBNAl mutants from insect cells. SF-9

monolayer cultures were infected with recombinant baculo-virusescontaining EBNA1 fragments and harvested 46 to 48 h postinfection as previously described (6). For

bEBNA395

and -451. infected SF-9 cells were washed in phosphate-buffered saline and then extracted for 30minon ice in 20 mM Tris-HCl (pH 7.5)-135 mM NaCl-1% Nonidet P-40-1 mM

MgCl2-10%glycerol-2 mMphenylmethylsulfonyl fluoride at

107 cells per ml.Extracts were then clarified by centrifuga-tion at12,000 x g for 30minat4°C. bEBNA39,

-330,

-346, and -330-

604were

extracted from infected SF-9 nuclei as previ-ouslydescribed forbEBNA1 (6). All bEBNA proteins were purified by chromatography on a heparin-agarose column (Bio-Rad) and then on a DNA affinity column containing EBNA1-binding sites as previously described for bEBNA1 (6). Pure bEBNA1 proteins were then concentrated by loading onto a 1-mlheparin-agarose column in column buffer (20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid] [pH 7.5], 0.5 mM EDTA, 20% glycerol) containing 350 mM NaCl and eluting in column buffer containing 1 M NaCl. The concentrations of pure bEBNA1 proteins were determined by the method of Bradford (3).

Electron microscopy. DNA containing oriP (10 fmol as DNA molecules) or two EBNA1-binding sites (20 fmol as DNAmolecules) was incubated with 480 fmol or 1.4 pmol of homogeneousbEBNA1 truncation mutants, respectively, in 20 ,ul of 25 mMTris-HCl (pH 7.5)-300 mM NaCl for 10min at25°C. The higherconcentration ofbEBNA1 proteins used with DNAcontaining two EBNA1-binding sites was neces-sary in order to achieve binding of these proteins to the singleEBNA1 sites, for which the bEBNA1 proteins have an affinity lower than the affinity they have for the FR and DS elements. Reaction mixtures were then diluted sixfold with 20 mM Tris-HCl (pH7.5)-280 mM NaCl-4 mM spermidine and stored on ice (15 to 30 min) prior to being spread on glow-charged carbon-coated copper grids (400 mesh) with-out fixation. Grids were stained with 5% aqueous uranyl

acetate,rotary shadowed with tungsten, and observed with a

JEM-1200EXII electron microscope. Length measurements were performed on projected negatives with a Numonics Graphic Master digitizing tablet.

Enhanced ligation efficiency assays. Homogeneous

bEB-NAl truncation mutants (1 to 4 pmol as dimer) were incu-bated with 45 fmol of a DNAfragment containing the FR and DS elements of oriP in 100 ,ul of 10 mM Tris-HCl (pH 7.5)-300 mMNaCl-20mMMgCl2-2 mMspermidine-lOmM dithiothreitol-2 mM ATP for 10 min at 25°C. Reaction mixtures were diluted twofold prior to the addition of 1 Weiss unit of T4 DNA ligase (GIBCO BRL) and a 5-min incubation at 15°C. Reactions were stopped by the addition ofEDTA to 10 mM and were ethanolprecipitated with 5

jig

of glycogen (Boehringer Mannheim) as a carrier. Samples wereresuspended in 9 ,ul of 1 mMTris-HCl(pH8.0)-0.1 mM EDTA-1% sodium dodecyl sulfate (SDS) and subjected to electrophoresis on a 0.8% agarose gel containing 0.5 ,ug of ethidium bromide per ml.

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3420 GOLDSMITH ET AL.

EBNA1

bEBNA1

nuclear DNAbinding/

(GIy-Arg) Gly-Gly-Ala badclocaization dimeazation acidc

5 1 1

90328 458 604 641

66

A

bEBNA

39

bEBNA

330

bEBNA346 bEBNA395

bEBNA

451

bEBNA330-619

330 641

346 641

395 641

451 641

330 619

FIG. 1. bEBNA1 truncation mutants. EBNA1 amino acids presentin eachmutantareshown. Thepositionsof the DNA binding

anddimerization domain, thenuclearlocalizationsignal, and other

salient features ofthe EBNA1 polypeptideare indicated.

RESULTS

Mappingof EBNA1 sequencesessentialfor oriPDNA loop-ing.Inordertomaptheregion(s)of EBNA1which mediates

the association of DNA-bound EBNA1 dimers, we have

constructed a series oftruncations ofEBNA1, all of which retain the DNAbinding anddimerization domain (Fig. 1) (2, 23). Like bEBNA1, all of the truncationmutants lackmost

of the Gly-Gly-Ala repeat region of EBNA1 previously shown to be nonessential for EBNA1 function (28, 30). bEBNA1 mutant proteins were expressed in insect cells, using the baculovirus expression system, and purified to apparent homogeneity. Thepure bEBNA1 mutant proteins

used in subsequent experiments are shown in Fig. 2.

bEBNA395 and bEBNA451 are observedto migrate as dou-bletsinsomeSDS-polyacrylamide gels,indicating

heteroge-neous forms of these proteins. All of the pure bEBNA1

mutantswere confirmed to bind

oniP

FR and DS elements and single EBNA1-binding sites by gel retardation assays

(datanot shown).

The interaction of the bEBNA1 mutants with

oniP

was

examined by electron microscopywith theoriP-containing DNAfragment(10 fmol) shown in Fig. 3 (topdiagram) and equimolar quantities (480 fmol) of the bEBNA1 truncated proteins. This ratio represents a twofold molar excess of

bEBNA1 proteins to EBNA1-binding sites. Like bEBNA1,

bEBNA39

-3309

-346, and -330-619 mediatedtheinteraction of

(kda)

z I?

CDCD LO_ o

LLJa) C) sJt a) e

Do C CO CO CO IT CO

97 66

45-31

[image:3.612.69.286.82.219.2]

-22

FIG. 2. Purified bEBNA1 mutant proteins. Purified bEBNA1

proteins (1.2

,ug)

weresubjectedtoelectrophoresison an SDS-12%

polyacrylamide gel and visualized by Coomassie blue staining.

Molecularsizes (in thousands)are onthe left. Lanes: 39, bEBNA39;

330,bEBNA330; 346, bEBNA346; 395,bEBNA395; 451,bEBNA451;

330-619,bEBNA330

619-FR and DS elements within oniP molecules to

generate

looped structures (Fig. 3), as well as interactionsbetween

onPmolecules resulting in the formation of

multiple

DNA

complexes (Fig. 4). Although the

frequency

with which looped DNAmolecules were induced

by

bEBNA39,

-330,and

-346wasreduced

compared

with that

by

bEBNA1, the total

fraction of DNA molecules bound

by

these mutants was

similarly reduced (Table 1).When the

frequency

of

looped

molecules was calculated as a percentage of total

protein-bound DNA molecules, the values obtained for

bEBNA1

and

bEBNA39,

-330,

-346,and

-346-619

werevery similar

(Table

1, values inparentheses).Therefore,

although

amino acids 7 to 346 appear to affect DNA

binding,

they

arenot

required

for DNA looping, and neither are amino acids 620to 641.

bEBNA395

mediated looping of

oniP

molecules at a lower frequency than theother bEBNA1 mutants

(Table

1;

Fig.

3)

and did not induce intermolecular interactions

resulting

in the formation of multiple complexes

(Table

1).

This result indicates that

bEBNA395

is impairedin its

ability

tomediate interactions between FR and DS elements. For

bEBNA451,

looped andmultiple DNA structureswere notobserved ata

frequency above that seen in theabsence of

protein,

and for those that were observed, the DNA interactions did not appear to be mediated by protein (Table

1).

These results were not affected by a twofold increase in theconcentration

of bEBNA451 in the binding reaction mixture or

by

a

decrease in the NaCl concentration in the reaction mixture to 150 mM (data notshown). We interpret the resultsofthe electron microscopy study to indicate that a

region

of EBNA1 essential for oniPlooping lies between amino acids 346 and 450 and that this region spans amino acid 395.

For allbEBNA1 mutants,onPmolecules withabEBNA1 complex on the FR element were the most

commonly

observed protein-DNA complexes

(Fig.

5;

Table

1).

As

reportedpreviouslyforbEBNA1, all of the bEBNA1 mutant proteins formed a single discretecomplexonthe FR element as opposed to a linear array ofbEBNA1 dimers

spanning

the

20 EBNA1-binding sites of the FR element

(7).

This

obser-vationsuggests that the bEBNAldimersbound to

adjacent

sites within the FR element are

associating

to form a

higher-order bEBNA1 complex. For the bEBNA1 mutants

that were not impaired in the ability to mediate FR-DS

interactions, binding to the DS element within

onP

was

observed only in looped molecules (Table

1).

Linear

oniP

molecules with bEBNA1 bound to both the FR and DS elements were observed with bEBNA1 mutants that are

defective

(bEBNA451)

or impaired

(bEBNA395)

for FR-DS interactions.

Assessment of

bEBNAl-mediated

interactions oforiP

ele-ments by enhanced ligation efficiency. A second

approach

taken to assess the ability ofbEBNAl mutants to mediate binding of oriP FR and DS elementswasbased on a

ligation

enhancement assay developed byMukherjee et al.

(16,

17).

For this assay, a DNA fragment in which the FR and DS elements of oniP were located closetoeach endof the DNA molecule was generated by linearizing pGEMoriP between the FR and DS elements of oriP with MluI

(Fig. 6,

top diagram). The interaction of DS and FR elements

through

EBNA1 thus brings the cohesive ends of the DNA molecules together, resulting in increased ligation

efficiency.

When these DNA fragments were incubated with the pure

bEBNAl proteins and incubation was followed by a short

(5-min) ligation reaction, bEBNAl andbEBNA39,

-330,

-346,

and

-330-619

were found to induce the formation of DNA

concatemers compared with control ligation reactions

with-out bEBNAl (Fig. 6). In the reactions shown in

Fig. 6,

1

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bEBNA1

bEBNA330

* t;-.'.

X

~ /

.r

-!

bEBNA39

bEBNA346

X...,~~~~~~~~~~~~-6-D

bEBNA395

bEBNA330-619

FIG. 3. LoopedDNA structuresinducedbybEBNAlproteins boundtoboth the FR and DS elements oforiP.bEBNAlproteins were

incubated withaDNAfragment (topdiagram) containingoriP.DNAlengthsareshown in base pairs.Bar= 0.1

itm.

pmol of bEBNA1 represents stoichiometric levels of

bEBNA1 dimerstoEBNA1-bindingsites inoriP. The induc-tionof DNA concatemers inthisassayindicates that multi-ple DNAmolecules arebeingboundtogetherthrough these

bEBNAl mutants. The induction of DNAconcatemers by

bEBNA346

required more protein (4 pmol) than the other bEBNAl mutants. This increased

protein

requirement is

likely due to the unstable DNA binding properties of

bEBNA346, since gel retardation and nitrocellulose filter

binding assays have indicated that the interaction of

bEBNA346 withtheonP elements isconsiderablylessstable

than that ofbEBNAl orthe other bEBNAlmutants

(data

not shown).

bEBNA395

and

bEBNA451

did not confer

en-hanced ligation efficiency to the DNA molecules at any

concentrationtested. NeitherDNA concatemers,indicative

ofintermolecularinteractions,norcovalently closedcircular

products, indicative of intramolecular interactions, were

induced by these mutants, indicating that bEBNA395 and

bEBNA451

donotmediateFR-FR, FR-DS, orDS-DS

inter-actions inthis assay. Identical resultswere obtainedwhen

bEBNA451

was bound to the DNA fragment in 150 as

opposed

to300mM

NaCl

(data

not

shown).

Single

EBNAl-binding

sites support bEBNAl-mediated

DNAlooping. ThebEBNA1-mediated

looping

of DS and FR elementsinvolves bEBNA1

complexes

composed

of

multi-ple bEBNA1 dimers bound to

multiple

EBNA1-binding

sites. In order to determine whether individual EBNA1 dimersbound to

single

EBNA1-binding

sitescould interact

tomediate DNA

looping,

we constructed aDNA

fragnent

containingtwo

EBNA1-binding

sites

separated by

930

bp

of

plasmid DNA

(Fig.

7, top

diagram).

Whenthis DNA

frag-ment wasincubatedwith

bEBNAl

andobserved

by

electron

microscopy,loopedstructureswereobservedat a

frequency

[image:4.612.165.460.74.356.2]

..

FIG. 4. Multiple

complexes

inducedby bEBNA1

proteins

bound

tooriP DNA.

bEBNA1 (a),

bEBNA39(b), bEBNA330 (c),

bEBNA346

(d), and

bEBNA330--19

(e)wereincubated with the DNA

fragmnent

containing

oriP

shown inFig. 3. Bar= 0.1

iLm.

FR -I

i

H 70+1101 980 i 600 1300

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TABLE 1. Complexes formed between bEBNAlmutantproteins and oriP DNAa

bEBNA1 %observedstructure(s) No.of

mutant FR-DS FR-DS Multiple Unbound structures

(looped) (linear) FR DS complex DNA scored

None 0.5 0 0 0 0.5 99.0 200

bEBNA1 14.1 (20.7)b 0 36.5 0 17.4 32.0 241

bEBNA39 7.9(19.9) 0 18.8 0 12.9 60.4 240

bEBNA330 4.6(17.0) 0 13.8 0 8.7 72.9 253

bEBNA346 4.7(16.2) 0 18.2 0 6.1 71.0 214

bEBNA395 1.9(7.6) 1.4 21.0 0 0.5 75.1 209

bEBNA451 0.9(3.0) 3.3 22.3 0.9 0.5 72.1 211

bEBNA330619 10.1(21.3) 0 21.4 0 16.0 52.5 238

abEBNA1proteins(480 fmolasdimer)wereaddedto10 fmolof theoriP-containingDNAfragmentshown inFig.3.

bValues in parentheses indicate the percentage ofprotein-bound DNA that containedDNAloopsbetweentheFRandDSelements.

similar to that with oriP DNA (Fig. 8a to c; Table 2). Measurements of the lengths of DNAarms and loops indi-cated thatloopingoccurredthroughthetwoEBNA1-binding sites (Fig. 7). These results indicate thata bEBNA1 dimer bound to a single EBNA1-binding site can stably interact with asecond DNA-bound bEBNA1 dimerata distanceto

formaDNAloop.ThefrequencywithwhichmultipleDNA

molecules were bound togetherthroughbEBNA1,however, was substantially reduced (approximately eightfold)

com-pared with results with oriP DNA (Table 2). In DNA

molecules in which a single EBNA1 site was bound, bEBNA1dimerswereobservedtobindtoeither of thetwo

EBNA1-binding siteswith approximatelyequal frequencies

(Fig. 8d and e; Table2).

When the bEBNA1 mutants were examined for their

abilityto mediate looping of the DNA fragment containing twoEBNA1-binding sites, thefrequency of loops observed for each mutant wasvery similar to the frequency of loops inducedonoriP, with the exception of that forbEBNA395, whichwasnotobserved to induce interactions between the

two single EBNA1 sites(Fig. 8ftom; Table 2). Inkeeping

with the results with oriPDNA,

bEBNA451

was unable to mediate looping on DNA containing two EBNA1-binding sites. Since the small size of thetruncatedbEBNA1 dimers

on thesingle binding sites made their detection by electron microscopy difficult or impossible

(bEBNA451

dimers are below the theoretical size limitof detection of50 kDa for this method [26]), we did not quantify linear DNA molecules bound by thebEBNA1 mutantproteins.

bEBNA1

.-.... .7 . ..

bEBNA346

bEBNA39

bEBNA395

.'"'',

OrS

'

bEBNA330

..,'.X,...\*.. .

)EBNA451

bEBNA330-619

1" I,r

4, .. ;. . .. .-.

FIG. 5. bEBNA1proteins bound to the FR element ofonPDNA.bEBNA1proteins were incubated with the DNA fragment containing oriPshown inFig.3. Bar = 0.1 j±m.

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4F

H-710-4 -- 3025 -- F

280-z

bEBNAl 39 330 346 U 395 451 330-619

IrI- A DA

L21

I41

24

n i 9 d i 94 1 2 4 1 2 4 L0 2 1 2 4 1 2 4 1 2 4

] *

_ L

_ ccc

FIG. 6. Detection ofmultiple complexes by enhanced ligation efficiency. ADNA fragmentcontaining the FR and DS elements (top diagram)wasincubated with bEBNA1 proteins (1, 2,or4pmolas

dimer)orwithout protein (0). DNA lengthsareshown in base pairs. Afterthe addition of ligase, sampleswere incubated for 5 minat

15°Cpriortoethanolprecipitation andagarosegelelectrophoresis in

0.5,ug of ethidium bromideperml. Thepositions of the linear DNA fragment(L)aswellascovalently closed circular (ccc) and

concate-meric (*)ligation productsareindicated. Lane L contains the linear oniP substrate DNA thatwasused inligation reaction mixtures.

DISCUSSION

Wehave used aseries of truncationmutantsofbEBNA1

and two independent methods (electron microscopy and enhancedligationefficiency)tomap aregion of EBNA1 that

1-2241H 930 --- F1 132

12-5

10-E

° 6

E4

Z

2- 12-.2

10-9

-

-E-'b

6-

i4-I

a-E

3E

LONG ARM

12

12~0140'60ec sLooI100 200 14 It600SW 20002M0

Distance from end(bp)

SHORT ARM

D0 st

fr0o 7e00n

SO 900 0 1100

Distance fromend(bp)

200 400

L0o

op

1000g12D(14bp

60 20002200

Looplength (bp)

FIG. 7. DNAlength measurements oflooped structuresformed between bEBNA1 and DNAcontaining twoEBNA1-binding sites. bEBNA1wasincubated withaDNAfragment (topdiagram) contain-ingtwononcontiguous18-bp EBNA1-bindingsites(open boxes).

*

7-WW

X

A;

:'

A: ,': :':

FIG. 8. Complexesformed between bEBNA1 proteins and DNA containingtwoEBNA1-binding sites. The DNA fragment containing thetwoEBNA1-binding sites shown in Fig. 7wasincubated with bEBNA1 (a to e), bEBNA39 (f and g), bEBNA330 (h and i), bEBNA346a and k),orbEBNA330619(1 and m) and then prepared forelectronmicroscopy.Loopedstructures(atocand ftom) and linear structureswith bEBNA1boundtotheend(d)orthemiddle (e) EBNA1-bindingsiteareshown. Bar= 0.1 pm.

is required to mediate the interaction of the DS and FR

elements oforiP. The N-terminal 345 amino acids and the

C-terminal 22 amino acids of EBNA1were notfoundtobe requiredfor intra-orintermolecular interactions between the

FRandDS elementsineitherassay, nor weretheyrequired

to mediate interactions between single EBNA1 sites at a

distance. AbEBNA1mutantthatlackedthefirst450 amino

acids ofEBNA1 butretained the DNAbinding and

dimer-ization domain was found to be defective in mediating

interactions between the FR and DS elements in both

assays, as well as between single EBNA1 sites. These

results indicate that the DNA binding and dimerization domain of EBNA1 is not sufficient to mediate oriP DNA

loopingundertheconditions tested. Anothermutantlacking

thefirst394 amino acids of EBNA1(bEBNA395)wasfound

tohave a limited abilityto mediateDNA looping between FR andDS elements inoniP byelectronmicroscopy,but it did not mediate detectable intramolecular interactions

be-tween the FR and DS elements in the enhanced ligation

efficiency assay. This discrepancy is likely due to the

in-creased separation distance of the FR and DS elements in

the DNAfragmentused in theligationassay.bEBNA395did notmediateintermolecular interactions betweentheFR and

DS elements in eitherassay, and itwasalsonotobservedto

mediate DNA looping between single EBNA1 sites sepa-rated by 930 bp. These results suggest that the affinity of

DNA-bound bEBNA395 dimers for each other is reduced comparedwith thatofbEBNA1. Our resultsareconsistent

with thepossibilitiesthat(i) bEBNA395containsonlypartof

the domain that mediates the interaction of DNA-bound

protein:

(pmol)

L _

ccc _m

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[image:6.612.64.302.75.275.2] [image:6.612.333.550.76.330.2] [image:6.612.128.247.448.691.2]

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[image:7.612.59.556.92.209.2]

TABLE 2. Complexesformed between bEBNA1 mutantproteins and DNA containing two EBNA1-binding sitesa

% observedstructure(s) No. of

bEBNAlbEBNAl

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~structures

mutant Two sites Two sites Middle End Multiple Unbound scored

(looped) (linear) site site complex DNA

None 0.5 0.5 1.9 0.5 0.5 97.1 209

bEBNA1 12.1 3.5 18.2 15.2 2.2 48.8 231

bEBNA39 9.8 224

bEBNA330 3.3 211

bEBNA346 3.0 202

bEBNA395 0 209

bEBNA451 0.5 203

bEBNA330619 8.5 200

a bEBNAl proteins (1.4 pmol asdimer)wereaddedto20 fmol of theDNAfragmentcontainingtwosingleEBNA1sites shown inFig.7.Samplescontaining the bEBNAl truncationmutants wereonly scored for looped DNA molecules(see text).

EBNA1 dimers, (ii) interactions between DNA-bound EBNA1 dimers involvetwo domains, onelocated between

amino acids 346 and 394 and a second located between

amino acids395 and 450, and (iii) theregion thatmediates

interactions betweenDNA-bound EBNA1dimersis located between aminoacids 346and394,but in its absenceasecond

region between amino acids 395 and 450 mediates limited

homologous interactions. Allof the bEBNA1proteinsused

inthisstudyarecomposed solelyof EBNA1sequences(with

theexception ofbEBNA451, which containstwoadditional

amino acidsattheNterminus)andwerepurifiedtoapparent homogeneity to eliminate the possibility that protein

se-quencesfused tobEBNA1 mutants orproteins copurifying

with bEBNA1 proteinsmay beresponsible forthe

interac-tions thatwehave observed.

Milman and Hwang (14a)hadpreviouslyreportedthatan

E. coli-produced fusion protein containing EBNA1 amino acids 450 to 641 and 36 amino acids from the X N protein

caused intermolecular interactions between synthetic EBNA1-binding sites at a low frequency. The equivalent EBNAl fragment (bEBNA451) lacking the X N sequences

and produced in insect cells was not found to mediate

intermolecularinteractions betweenEBNA1-bindingsites in

anyofourassays. Thisdiscrepancycould be duetoanyor

all of thefollowing possibilities: (i)theANproteinsequence

in the EBNA1 fusion protein mediates the interaction of DNAfragments observed by Milman and Hwang, (ii)

differ-encesinposttranslational modificationsof the EBNA1 frag-mentproducedinE. coli and insect cells affect itsabilityto

mediate DNA interactions, and (iii) the protein-mediated

DNAinteractions detectedbytheagarosegel

electrophore-sisassayofMilman andHwangareofanaturedifferentfrom thosedetectedbytheassayspresentedhere. Toaddress the

lastpossibility,wehave testedpartially purified preparations

of theE. coli-produced X N-EBNA1 fusion protein (kindly

provided by David Mackey and Bill Sugden) inourenhanced

ligation efficiencyassayandfound that the fusion protein did notmediate inter-orintramolecular interactions betweenFR

and DS elementsin thisassay(datanotshown). Therefore,

the DNAinteractionsobserved by Milmanand Hwangtobe mediated by the X N-EBNA1 fusion protein are clearly differentfromthebEBNA1-mediatedDNAinteractions pre-sentedhere.

As reported previously for bEBNA1, none of the bEBNA1 mutants that mediated oriP DNA looping were observedby electron microscopytobindtotheDS element within onP inthe absence of DNA looping, althoughallwere abletostablybindto DNAcontaining only theDS element

(7).LinearDNAmolecules with bEBNA1 boundtoboththe

FR and DS elements within oriP were only observed in

mutantsthatwereimpairedfor DNAlooping.These results

are consistent with the hypothesis that the interaction of

bEBNA1 dimersbound to the FRand DS elementswithin

oriP is extremely efficient, occurring concurrent with or

immediately following binding of bEBNAl to the DS

ele-ment. In contrast, the interaction of bEBNA1 dimers in solutionunder identicalconditionswas notdetected by gel filtrationorglycerolgradient analysis (6),suggestingthat the

domain(s) of EBNA1 responsible for the association of

DNA-bound EBNA1 dimers becomes exposed or altered

uponbindingof EBNA1 toDNA.

The enhanced ligation efficiency assay that we used to assessthe ability of bEBNA1 mutants to mediate

interac-tionsbetween FRand DSelementswasbasedontheligation

enhancement assaydeveloped byMukherjee et al.

(16, 17)

that was used to demonstrate the ability of the rr initiator

protein to mediate intramolecular interactionsbetween the

on 1 and on y sequencesof

plasmid

R6K. Theinteractions

thatwedetectedwith bEBNA1 inthis assaywere

intermo-lecular, resultingin the formation of DNAconcatemers.The

reason thatintramolecular associations of FR and DS ele-ments(resultingincovalentlyclosedcircularproducts)were notdetectedin this assayislikely duetothehighefficiency with which DNA molecules bound by these proteins are

sequestered into multiple complexes. Electron microscopy

data of onP DNA boundbythese bEBNA1proteinsindicate

that looped and multiple complexes were observed with

similar frequencies. However, since we have found that

multiple complexes often contain as many as 20 DNA

molecules, they account for many more DNA molecules

than do single looped molecules. Therefore, although bEBNA1 does mediate looping between the FR and DS elements in the DNA moleculeusedforligation assays(8a),

looping within a single molecule resulting in covalently

closed circular products was likely not detected because

mostof theloopedDNA molecules were bound into multiple complexes.

Our electronmicroscopy data indicate thatbEBNA1 can mediate loop formation between individual EBNA1-binding sites separated by 930 bp of DNA (spacing similar to that found between the DS and FR elements in onP). Since

EBNA1binds toitsrecognition site as a dimer (2, 6), these

results indicate that single DNA-bound EBNA1 dimers can interact tomediate DNA looping and that the formation of

largerbEBNA1 complexessuch as those formed on the FR

and DS elements isnot aprerequisiteforbEBNA1-mediated loop formation. Unlike the interaction of

bEBNA1

with onP,in whichoneelement(FR)is boundprior to interaction J. VIROL.

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with the second element

(DS),

bEBNA1 was observed to

bindtoeither ofthetwo

single binding

siteswithinthe DNA

fragment

used for these studies withequal frequency,

indi-cating

thattherewas no

preferential

order offillingof these

twosites. This resultwas

expected,

since thetwo

EBNA1-binding

sitesareidentical.Like theinteractionsobservedon

oniP DNA,

the

binding

ofbEBNA1toboth of thesinglesites withina

single

molecule in the absence ofloopingwasrare,

indicating

the efficient intramolecular interaction of the DNA-bound bEBNA1 dimers. Intermolecular associations

between

single

DNA-bound bEBNA1 dimers were

signifi-cantly

reduced

compared

with intermolecular interactions between bEBNA1

complexes

formed on DS and FR ele-ments. This result suggests that the

affinity

of

single

DNA-bound

bEBNA1

dimers for each other is less than that of

larger

bEBNA1

complexes

and/or

that each DNA-bound

bEBNA1 dimer can be involved in

only

one

homologous

interactionat adistance. Stable intermolecular interactions

between

single

DNA-bound EBNA1 dimers and EBNA1

complexes

formedonthe FR element have been

previously

reported (14).

Our results suggest that at least part of the

region

of

EBNA1

responsible

for the

homologous

association of

DNA-boundEBNA1 dimersmapsbetween amino acids 346 and 394 and that additional amino acids between

positions

395 and 450 are also involved. This

region

of EBNA1 includes a basic

domain,

a nuclear localization

signal,

a

serine-rich sequence

containing

one or more

phosphoryla-tion sites

(18),

anda

proline-arginine-rich

sequence

spanning

aminoacid395. Weare

currently investigating which,

if any, of these sequences are

important

for the interaction of

DNA-bound EBNA1 dimers at a distance. At present, we

cannotexclude the

possibility

that the

region

of EBNA1 that

we have found to mediate DNA

looping

in our bEBNA1 truncationmutantsmaynotbe

required

to mediate

looping

in the contextof the

full-length

EBNA1

protein.

This

possi-bility

will be addressed with

internally

deleted and

point-mutated EBNA1

proteins.

The interaction of distant DNA elements

through

an

initiator

protein by

a DNA

looping

mechanism has been

previously

demonstratedtobeessential for

replication

from

al

and

03 origins

in

plasmid

R6K andcanbe reducedfour-to

eightfold by

a

single

amino acid

change (proline

to

leucine)

in

the initiator

protein (15).

Similarly,

we

predict

that the

deletion of amino acids essential forEBNA1-mediatedonP

looping

willeliminatethe

ability

ofEBNA1toactivateDNA

replication

from

onP.

In this respect, it is

interesting

to consider the mutational

analysis study

ofEBNA1conducted

by

Yates and Camiolo

(28),

in

which,

of the amino acids in the

region

ofEBNA1that wehave foundtobe involved in oriP

looping, only

those between amino acids 346 and

384,

392 and

396,

and 403 and 432 have not been shown to be nonessentialfor transient

replication

fromonP.

Therefore,

if

EBNA1-mediated

looping

ofonPis essential for

replication,

amino acids essential for

looping

would be

expected

tomap

within these areas.

ACKNOWLEDGMENTS

Weare

grateful

toKlaus Schultes for

help

with

sample

prepara-tion for electron

microscopy.

We alsothank Jim

Smiley

and Mike O'Donnell for critical

reading

of the

manuscript.

This workwas

supported by

agrant from the National Cancer Institute of Canada and

by

aMedical Research CouncilofCanada

studentship (to K.G.).

L.F.isaResearch Scientist of the National Cancer Institute of Canada.

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