Conserved Sequences at the Origin of Adenovirus DNA Replication

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0022-538X/82/110530-08$02.00/0

Copyright C 1982,American SocietyforMicrobiology

Conserved Sequences

at

the

Origin of Adenovirus

DNA

Replication

BRUCEW.STILLMAN,* WILLIAMC.TOPP,ANDJEFFREY A.ENGLER Cold Spring Harbor Laboratory, ColdSpringHarbor, New York 11724

Received 23 April 1982/Accepted 13 July 1982

The origin ofadenovirus DNA replication lies within an inverted sequence

repetition at either end ofthe linear, double-stranded viral DNA. Initiation of DNA replication is primed by adeoxynucleoside thatis covalentlylinked to a protein, which remains bound tothe newly synthesizedDNA. We demonstrate

thatvirion-derivedDNA-protein complexes fromfive human adenovirus

serologi-cal subgroups (A to E) can act as a template for both the initiation and the

elongation of DNA replication invitro, using nuclear extracts from adenovirus

type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein

complexeswere not asactiveasthehomologousAd2DNA,mostprobablydue to

inefficientinitiation by Ad2replicationfactors. In an attempt toidentify common

features which may permit this replication, wehave alsosequenced the inverted

terminal repeated DNAfrom human adenovirus serotypes Ad4 (group E), Ad9

and AdlO (group D), and Ad31 (group A), and we have compared these to

previously determined sequences fromAd2 and Ad5 (group C), Ad7 (group B),

and Adl2 and Adl8(group A) DNA. In all cases, the sequence around theorigin

ofDNAreplicationcanbe divided intotwostructural domains: aproximalA*

T-richregion which ispartially conservedamongtheseserotypes,andadistalG* C-rich region which isless wellconserved.TheG*C-richregion containssequences

similartosequences presentinpapovavirus replication origins.The twodomains

may reflect a dual mechanism for initiation of DNA replication:

adenovirus-specific proteinpriming of replication,andsubsequentutilizationofthisprimer by

hostreplication factors forcompletion ofDNAsynthesis.

Adenovirus DNA replicates by astrand

dis-placement mechanism from origins of

replica-tion that are located withinaninvertedrepeated

sequence at each end of the linear

double-stranded DNA (see the review by Winnacker

[37]). Replicationisinitiatedateitherorigin,and

elongation ofDNAchains proceedstoward the

opposite end of the molecule, displacing the

nontemplate DNA strand (type I replication [16]).Thedisplaced strand is thenreplicatedas a

result of a separate but analogous initiation

event (type IIreplication).

The viral DNA has a 55,000-dalton protein

(terminal protein, TP) covalentlylinked toeach

5' terminus (21-23) via aphosphodiester bond

between the

13-OH

of a serine residue in the

protein andthe5'-OHof the terminal

deoxycyti-dine residue (2, 9). The terminal protein is

synthesized as an 80,000- to87,000-dalton

pre-cursorprotein (pTP) (2, 31) which iscleavedto

TP late in infection (6). ThepTP is encoded by

early region E2b on the adenovirus genome,

which forms part ofacomplextranscriptionunit

encoding otherreplication proteins (31).

Intra-cellular DNA (6, 8, 14, 29, 30, 36) and nascent

DNA strands replicated in vitro (2, 28) also

contain protein linked to each 5' terminus of

DNA, and this protein has been identified as

pTP (2).

Recentdevelopments have demonstrated that

pTPfunctions in the initiation of DNA

replica-tionby covalently binding the first

deoxynucleo-tideresidue,which then provides a 3'-hydroxyl

groupforsubsequentchainelongation (7, 11, 17,

20, 33). This mechanism is broadly consistent

witha model originally proposed by Rekosh et

al. (21). A DNApolymerase activity is

associat-edwith aprotein complex consisting of pTP and

a 140,000-dalton protein (11). The parental TP

thatis covalently attached to the templateDNA

is not required for the formation of the

pTP-dCMP complex; rather, a specific DNA

se-quence has to be located at the end ofa linear

molecule (33).

The inverted terminal repetitions (ITRs) ofa

number of adenovirus serotype DNAs have

been sequenced and include human

adenovir-uses type 2 (Ad2) (1, 25) and AdS (27), both

group C viruses; Adl2 and Adl8 (26, 32, 35;

C. F.Garon,R. P.Parr,R. K.Padmanabhan, I.

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VOL. 44, 1982

Roninson, J. W. Garrison, and J. A. Rose,

Vi-rology, in press), bothgroupAviruses; and Ad3

and Ad7 (10, 35), both group B viruses. The

sequenceofthe ITRs ofsimianadenovirus SA7

(35), mouse adenovirus FL (34), and the avian

CELO virus (P. Alestr6m, A. Stenlund, P. Li,

and U. Pettersson, Gene, in press) have also

beendetermined.

Challberg and Kelly (5)have described a

cell-free system that is capable of initiation and

completion ofoneround oftypeIreplicationon

anexogenously added templateDNA. Since this

system faithfully mimicsthe mechanism oftype

I replication in vivo, it provides a convenient

means for studying the sequence requirements

forthe initiationof DNA replication. We

com-pared the ability of the DNA-protein complex

fromrepresentative adenovirus serotype groups

A to E to initiate andelongate DNAreplication

in heterologous adenovirus type 2-infected

HeLacell nuclear extracts, andwefound thatall

DNA-protein complexes tested did replicate to

some extent. The replication of Ad7

DNA-pro-tein complex in an Ad2 nuclear extract was

reported previously (28). As a first attempt to

determine the DNA sequencesrequiredfor the

initiation of adenovirus DNA replication, we

extended the analysis ofITR sequences to

in-clude representatives of group D (Ad9 and

AdlO)and group E (Ad4) humanadenoviruses,

as well as another memberof group A(Ad3l).

Thesecombineddatahave enabledustoidentify

the DNA sequences that are common to

differ-ent human adenovirus serotype DNAs which

mayallow DNAreplication in vitro.

MATERIALSANDMETHODS

Cellsandvirus. HeLa S3 cells weregrown in

sus-pension in Joklik modified Eagle Spinner medium

(GIBCO Laboratories) containing 5% calf serum

(FlowLaboratories, Inc.). A549cells,derived from a human lung carcinoma, were obtained from Walter A. Nelson-Rees (Naval Biosciences Laboratory, Naval

Supply Center, Oakland, Calif.)and grown as

mono-layer cultures in Dulbecco modified Eagle medium

(GIBCO) containing10ocalfserum.Ad2 was grown in both HeLa and A549 cells. Adenovirus serotypes

Ad4, Ad7, Ad9,AdlO, andAd3l wereobtained from

the American Type Culture Collection, plaque

puri-fied,andgrown in A549cells.

PurifictionofvirusandDNA. Infectedcells

show-ingextensivecytopathic effect werecollectedby low-speed centrifugation, frozen and thawed, and suspend-ed in cell lysis buffer (0.5% Nonidet P-40-10 mM

MgCl2-10mM Tris [pH7.9]) at about 5x107cells per

ml.After 10min on ice, the cell debris was removed by centrifugation, and virus was purified by two succes-sivebandings in CsCl as described previously (18). Viral DNA was purified by digestion of purified virus

by previously self-digested pronase (1 mg/ml) in the presence of 0.5% sodium dodecyl sulfate (SDS), fol-lowed by phenol and CH3Cl-isoamyl alcohol (24:1)

ADENOVIRUS DNA REPLICATION 531

extractions and ethanol precipitation. DNA-protein complex was prepared as describedpreviously (28).

Labeling and DNA sequencing of adenovirustermini.

The 3' labeling of thetermini of Ad4, Ad9,AdlO,and

Ad3l DNA was performed as described byChallberg

and Englund(3), except that the Klenowfragmentof DNApolymerase I (NewEngland Biolabs)was used instead of T4 DNA polymerase. DNA (30 to 50 1Lg) isolated from phenol-extracted virions was used in eachlabelingexperiment. [a-32P]dGTP(400Ci/mmol, Radiochemical Centre, Amersham,England)wasused forlabeling. Inaddition, each reaction contained0.3 mMdATP, 0.3 mMTTP, and 1 ,uM dGTP. Labeling

was at 12°C for 3 h. Afterlabeling, DNA samples were digested with restriction endonuclease RsaI (New EnglandBiolabs) before being loaded on an 8%

poly-acrylamide gel (40:1, acrylamide-bisacrylamide) in TBE(135 mM Tris OH-45 mM boricacid-2.5 mM

disodium EDTA). Purification oflabeled DNA from unincorporated nucleotidetriphosphates, extractionof DNA from polyacrylamide gels, and nucleotide se-quencing have beendescribedby Maxamand Gilbert

(19).

DNA repUcation in vitro. Nuclear extracts were prepared from Ad2-infected HeLa S3 cells as de-scribedpreviously (5). Reactionconditionshavebeen described previously (28); each 50-,ul reaction con-tained 80 ng ofundigestedorHindlll(NewEngland Biolabs)-digestedDNA-protein complexand was incu-bated for 1 h at30°C.Thereaction wasstopped bythe additionof EDTA to 15 mM andofpronase to 1mg/ml

and further incubated for 1 h at 37°C. The DNA

products were then analyzed by either neutral or

alkalineagarosegelelectrophoresis.

Formation ofpTP-dCMPcomplexes. Apartially puri-fied protein fraction containing active pTP was pre-pared by columnchromatography throughdenatured

DNAcelluloseasdescribedpreviously (33). Reaction

conditions have also beendescribedpreviously;each

reaction,which contained 40 ,ug ofDNA-protein

com-plex,wasincubated for 1 h at30°C.Theproductswere analyzed on 10%o SDS-polyacrylamide gels as de-scribedpreviously(33).

RESULTS

DNAsequencing of terminal repetitions of Ad4,

Ad9, AdlO, and Ad3l. The sequences of the

termini, determinedby thechemical degradation

procedures of Maxam and Gilbert (19), are

shown inFig. 1B. Weinferthat thefirst

nucleo-tide of each ITR isdC, since preliminary

experi-mentsshowed that

[a-32P]dGTP

gavethe highest

incorporation of radioactivity into adenovirus

DNA incubated with the Klenow fragment of

DNA polymerase I (data not shown). Where

determined, every other human adenovirus

DNA had adC residue at the 5' end.

The ITRs ofAd4 (group E), Ad9 (group D),

and Ad31 (group A) are 116, 158, and 148

nucleotideslong,respectively. The ITR of AdlO

is identical to that of Ad9, at least to nucleotide

135.The ITRsof the group C (Ad2 andAdS)and

groupB adenoviruses (Ad3 andAd7) have been

shown to be 103 and 137 nucleotides long,

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532 STILLMAN, TOPP, AND ENGLER J. VIROL.

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respectively. Two other group A adenovirus DNAterminihavebeensequenced; the ITRsof

Adl2 and Adl8 are both 165 nucleotides long

and arequite homologous. The ITR of Ad3l, on

the other hand, isshorter and exhibitsimperfect

homology with Adl2 and Adl8 in only two

sections: nucleotides 1 through 68 and 120

through 148oftheAd31 sequence. For

compari-son,the DNAsequences of the ITRs fromAdM2,

Ad7, and Ad2 are shown in Fig. 1B. It is

noteworthy that whereas Adl2 and Adl8 grow

theleast well of the 20 virus serotypes wehave

propagatedon A549cells,Ad31 growsasrapidly

astheviruses ofsubgroup B,thoughnot aswell

asthe groups C, D,and E(datanotshown).

DNA replication in vitro. Since adenovirus

DNAreplicationstarts atthe endsof the DNA,

only the terminal fragments of restriction

en-zyme-digested adenovirus DNA-protein com-plexreplicatespecificallyin vitro(13).We have

used this assay to determine whether

DNA-protein complex fromAd4, Ad9, and Ad3l will

replicate in vitro, using nuclear extract from Ad2-infected HeLa cells. First, the terminal

restriction fragments were identified by their

inability to enter agarose gels without prior

digestion withpronase. DNA-protein complexes

were digested with HindIII, and samples were

subjected to electrophoresis in an agarose gel

before and after digestion with pronase (Fig.

2A). The terminal fragments bound to TP were

retarded in the gel and only migrated in the

correct position after pronase digestion, thus

identifying the terminal HindlIl fragments as B

and C of Ad4, A and Dof Ad9, and C and G of

Ad3l. The terminal HindIlI fragments of Ad2

are G and K. HindlIl-digested DNA-protein

complex from either Ad2, Ad4, Ad9, or Ad31

was incubated with Ad2 nuclear extract under

standard reaction conditions, digested with

pronase, andsubjected toelectrophoresis on an

agarose gel(Fig. 2B). In each case, the terminal

-G+H

b--1 ? :.

[image:4.491.45.439.310.610.2]

.~~~~~~~~~~~--G

FIG. 2. Replication ofheterologous DNA-protein complexes in vitro. (A) DNA-protein complex derived from eitherAd2, Ad4,Ad9,orAd3l virionswasisolated,digestedwithHindIlI, andsubjected toagarosegel electrophoresis either before (-) or after (+) digestion with pronase (1 mg/ml). Ad9 fragment F and Ad3l

fragmentsJ toS(Y.Sawada,T.Yamashita,and K.Fujinaga, personal communication)werenotvisible. Thegel

wasstained withethidium bromide andphotographed. (B) DNA-protein complexfrom eitherAd2, Ad4, Ad9,or

Ad3lwasincubated withextractfrom Ad2-infectedHeLacells. Theproductswerethendigestedwithpronase

and subjected to electrophoresis through a 0.8%agarose gel. Thegel was dried and autoradiographed. The

positionsof the terminalfragmnentsareshown;theHindlIll digestfor both Ad2 andAd3l waspartial.

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restriction fragments were preferentially la-beled, indicating that each DNA-protein

com-plex had the capacity to act as template for replication in vitro. TheHindIIIdigests of Ad2

andAd3l were both partial, and replication of

theterminal and subterminalfragments is

indi-cated (Fig. 2B). Ad7DNA-proteincomplexwas

previously shown to replicate by using an Ad2

nuclear extract (28).

Todetermine whetherreplication in vitro

re-sulted in genome-length DNA molecules, uncut

DNA protein complex from either Ad2, Ad4, Ad7, Ad9, or Ad31 was used as template in an

Ad2-infected HeLa cell nuclear extract. The

products were digested with pronase and

ana-lyzed by electrophoresis on an alkaline agarose

gel (Fig. 3). In eachcase,full-lengthproduct was

observed, but the amount of product of each

heterologous template was reduced relative to

the homologous Ad2 template. In a number of

experiments with different preparations of

DNA-protein complexfrom each serotype,

dif-ferentamounts ofproduct were observed. This

has also been observed with various

prepara-tions ofAd2complexandreflectsthe amount of

proteolyticdegradation and the concentrationof

thestockof each DNA-proteincomplex

(unpub-FIG. 3. Alkaline agarosegelelectrophoresisofthe

replication reaction products. Undigested DNA-pro-tein complex from either Ad2, Ad4, Ad7, Ad9, or

Ad31 wasincubated inareaction mixturecontaining

nuclearextractfromAd2-infected HeLa cells for1hat

37°C. Thereactionproductswere digestedwith

pro-naseandsubjectedtoelectrophoresis througha0.7% alkaline agarosegel.Thegelwasdried and

autoradio-graphed.

2 4 7 9 31

-fl (20K)

87 K-4 . i-m (85K)

-ma

(66K)

-Z (48K)

FIG. 4. Formation ofapTP-dCMP complex with various DNA-protein complexes as template. Undi-gested DNA-proteincomplex from either Ad2, Ad4,

Ad7, Ad9, orAd31wasincubated in areaction

mix-ture containing [a-32P]dCTP and a partially purified

extractfromAd2-infectedHeLacell nuclei (33)for 1 h

at30°C. Thereactionwasstopped bytheaddition of EDTAto 10 mM and then was subjected to

electro-phoresis through a 10% SDS-polyacrylamide gel as

describedbyLaemmli (15). MarkerproteinswereAd2 virionproteins, and the molecular weightsareshown. The gel was dried and autoradiographed with an

intensifyingscreen.

lished data). However, Ad4, Ad9, and Ad3l

DNA-protein complexes isolated concurrently

with Ad2 complexconsistently incorporated

ap-proximately10-fold fewerpicomolesof

deoxyn-ucleosides than did Ad2DNA-protein complex,

whereasAd7 template showed65% ofthe level

ofincorporation seenin Ad2(28).

InitiationofDNAreplication. Ithas been

pro-posed (21) that the first step in theinitiation of

adenovirus DNAreplication is theformation of complex betweenpTPand dCMP, the5'

termi-nal nucleotide of Ad2 DNA (1, 25). Such a

covalent complex was found when extracts of

adenovirus-infected HeLa cells wereincubated

with

[a-32P]dCTP

andtemplate DNA(7, 17, 33).

We detected the formation of a complex

be-tween Ad2 pTP and dCMP whenheterologous

DNA-protein complexes wereusedastemplate.

DNA-protein complexes derived from Ad2,

Ad4, Ad7, Ad9, andAd3l virionswere

incubat-edwiththepartiallypurifiedAd2 nuclearextract

and

[a-32P]dCTP,

and the reaction products

wereanalyzedby SDS-polyacrylamide gel

elec-trophoresis (Fig. 4). EachDNA-proteincomplex

facilitated the formation of an 87,000-dalton

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[image:5.491.254.449.79.288.2] [image:5.491.118.190.373.595.2]

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pTP-dCMPcomplex;however, the homologous

Ad2complex wasbyfar themostactive.

Com-plex was not formed when either [a-32P]dATP, dGTP, orTTPwasused (datanot shown). The

relativeefficiency of pTP-dCMP complex

forma-tion for eachheterologous template was

reflect-ed in the relative efficiency of replication as

assayed by specific replication of the terminal

restriction fragments (Fig. 2B) and by the

pro-ductionoffull-lengthproduct(Fig.3),suggesting

that pTP-dCMP complex formation may be a

rate-limitingstepin thereplicationof viral DNA.

DISCUSSION

ChallbergandKelly (5)described the isolation

of a soluble nuclear extract from

adenovirus-infectedHeLa cellsthatwascapable of

replica-tion ofexogenousDNA.Replicationwas

depen-dent upon thetemplateDNAhavingthe terminal

protein bound to each 5' end, since template

DNA that hadbeendigested with protease was

inactive (4). Residual peptides present on

pro-nase-digested DNA template probably inhibit

the initiation reaction (33). It was therefore

suggested thatboththe TP and DNAsequences

at the origin of replication facilitated the

initia-tion of DNA synthesis, with the TPinteracting

with one or more of the proteins in the soluble

nuclear extract. The apparently strict

require-mentforthetemplateDNAtocontain

covalent-ly bound protein made it impractical to

deter-mine DNA sequence requirements attheorigin

byin vitromutagenesis. However,the

availabil-ity ofanumber of adenovirus serotypes, each

having differentDNA sequencesattheoriginof DNAreplication, suggestedaway toovercome

this problem of analyzing DNA sequence

re-quirements forthe initiation ofreplication. We

therefore tested the ability of heterologous DNA-protein complexes from anumber of hu-man adenoviruses, representingfive serological

groups, to act astemplate forboth theinitiation

and theelongation ofreplication in nuclear

ex-tractsderived from Ad2-infectedcells. All

heter-ologous DNA-protein complexeswereactive for

both initiation and elongation of replication in

Ad2 nuclear extracts, but they werealwaysless

active than thehomologoustemplate.Template

DNAsfrom Ad4, Ad9,andAd31formeda

pTP-dCMP complex with low efficiency, but the

complex was sufficient to primespecific

replica-tion from both termini of these DNAs, as

as-sayedby the replication oftheterminalHindIll

restriction enzyme fragments. Thus, we

con-clude that DNA sequences within theITR that

are conserved between these viruses and Ad2

may play an important role in the initiation

reaction. It was not practical to reverse the experiment and use Ad2 DNA-protein complex

as a

template

in aheterologous adenovirus

nu-clear extract, since all such viruses we have

used growpoorly inourHeLacells.

Aproblemposedbythese resultsis the

possi-bleinteraction of the DNA-bound

heterologous

TP with the Ad2 pTP that is present in the

nuclear extracts. We do not know to what

degreethiswillaffect the initiationreaction,and

thuswedrawnoconclusionsas totheefficiency

with which specific sequences within the ITRs

of thedifferent adenovirus DNAs support DNA

replicationin the Ad2 system. We concludeonly

that there is sufficient sequence homology to

correctlyinitiatereplicationatsome

frequency.

However, during the course of these experi-mentsitbecame clear that the TP isnotrequired

forinitiation of replication inapartially

fraction-ated replication extract, since aplasmid DNA

containing the ITR of Ad5was active for

pTP-dCMP complex formation (33). The plasmid

DNA was only active when the origin was

located at the end of the linearized DNA. In

addition, the plasmid DNA was active when it

was heat denatured, again provided that the

origin was located at the end of the molecule.

Thus, interactions between pTP and the TPon

theparental DNA are not obligatory, but they

still may affect theefficiency of initiation.

Thehuman adenovirus serotypes 1 to38 have

been subdivided into five groups on the basis of

DNAhomology thatwasderived from solution

hybridization studies with virion DNA (12).

DNA sequences of the ITRs from group A(Adl2

andAdl8), group B (Ad3 and Ad7), and group C

(Ad2 and AdS) have been determined, and

re-gions ofhomology have been noted (26, 35). The

replication studies suggest that sequences in the

ITR which are important for DNA replication

have been conserved; thus, we have extended

the DNA sequence analysis to include the ITRs

from group D (Ad9 and AdlO) and group E(Ad4)

humanadenoviruses and the ITR of an

addition-al group A virus (Ad3l). Recently, the

se-quences of the ITRs for Ad4 (0. Tokunaga, M.

Shinagawa, and R. Padmanabhan, Gene, in

press) and Adl8 (Garon et al., in press) were

determined. Sequences of the ITRs for some of

the known human adenoviruses are shown in

Fig. 1B.

The ITR for each human adenovirusDNA can

be divided into an A * T-rich region of 50 to 52

base pairs, which occurs at the termini of each

DNA molecule, and a G * C-rich region that

varies in lengthbetween 50 and 110 base pairs.

Indeed, A - T- and G * C-rich sequences are

also found in othereucaryotic origins of

replica-tion, most notably those in the papovaviruses

(reviewed by Seif et al. [24]). Within the ITR,

DNA sequences in the first 50- to 52-base pair

A * T-rich region are themost highly conserved

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among all human adenoviruses (Fig. 1B). This

region is alsopartially conserved in the

adeno-viruses of the lowerprimates (35), rodents (34),

andbirds (Alestrometal., in press). A

consen-sus sequenceforthis regioncanbederived that

conforms with the known sequencesfrom allof

the humanadenoviruses(Fig. 1A).

Within this A T-rich region, there is a

per-fectly conserved sequence of 10 base pairs

(ATAATATACC), and all or part of this

se-quence is alsopresentin the ITR of nonhuman

adenoviruses. In addition, where it has been

determined, all human adenoviruses contain a

5'-terminal dC, which is the base towhich the

TPis covalently attached. Thesequenceof 6to7

bases that lies between the terminal dC and the

conserved10-base sequence isoneoftwotypes:

either relatedtotheAd2sequence orrelatedto

theAd7 sequence. Within this region, all

sero-typeDNAscanbederived from either the Ad2

orAd7 sequencesby base substitutionsor

dele-tion(Fig. 1A). Infact, different isolates of

sero-typesAd2, Ad4, and Ad7 showsequence hetero-geneity within this region (Fig. 1A). At the other

end of the 10-base, perfectly conserved

se-quence is a 9- to 11-base region that has

di-verged, which is followed by a region that is

partially conserved among all human serotype

DNAs.

The G * C-rich region, on the other hand, is

not conserved and is also variable in length.

However, twofeatureswithin this regioncanbe

identified that are common toall ofthe human

adenoviruses. The first is theoccurrenceofone

or morecopies ofasequence verysimilartothe

sequence GGGxGGAG, which ispresent twice

at the origin of replication of human BK virus

(24) and which is alsofoundatthe simian virus

40andpolyoma virus (mouse)origins of

replica-tion (24). All humanadenovirusesand the simian

virus SA7 (35) have the sequence GGGCGGat

leastonce in the ITR. Aspreviously suggested

(24), thispapovavirusconsensussequencemight

play an important function as a recognition

signal foraprotein involved in DNAreplication.

A second feature of the G * C-rich region is the occurrence of the sequenceTGACG, which

occurs ator nearthe endof allhuman

adenovi-rus ITRs; related sequences occur in murine

(FL) andavian(CELO)adenoviruses (35;

Ales-trom etal., in press). It is noteworthy thatthis

sequenceisnotalwaysatthe endoftheITR,as

previously observed (26); itcanbefound within

theITR (Ad9, AdlO, andAd3l) orjust outside

the ITR(Ad4).Afunctionhas not beenascribed

tothis conserved sequence.

Itislikelythat theseconserved features

with-intheITRplayseparaterolesin theinitiationof

DNAreplication. Onepossible role for the

high-ly conservedA * T-richregionwouldbetoserve

as an origin for the adenovirus-specific protein

priming of DNA synthesis. The complex

be-tween pTP and the 140-kilodalton protein (11) maybindto DNAsequenceswithin thisregion.

The conserved sequences within the G * C-rich

regionaresimilarto thosefound in other

eucary-otic origins and may serve as cellular DNA

polymerase recognition sites. Thus, the two

structural domains may reflect two functional

sites that initiate DNA replication. The recent

demonstration that a linearized plasmid DNA

containing the ITR atthe end of the molecule

canfunction for initiation of DNA replication in

vitro (33) will enableus todefinemoreprecisely

the adenovirus origin of replication by site-directed mutagenesis.

ACKNOWLEDGMENTS

We thankJohn L. Hierholzer for verifying the serotypes of viruses used in this study and J. A. Rose and R. Padmanabhan forcommunicating sequence data before publication. We also thankMichael Mathews and Fuyuhiko Tamanoi for discus-sions and critical reading of the manuscript and Elizabeth Woodruff,DianeSmith, and Mark Hoppe for technical assist-ance.

Thisresearch was supported by grants from the National CancerInstitute(CA-13106) and from the AmericanCancer Society(RD-126). J.A.E.is a Special FellowoftheLeukemia Society ofAmerica.

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