Bidirectional replication of adenovirus type 2 DNA.

Copyright© 1976 AmericanSociety forMicrobiology Printed in U-SA.

Bidirectional Replication of Adenovirus Type 2 DNA

MARSHALL S. HORWITZ

Department of Microbiology-Immunology, CellBiology, and Pediatrics, Albert Einstein College of Medicine, Bronx,New York 10461

Received for publication 11 November 1975

After short periods of labeling

with

[3H]thymidine,

recently completed

adeno-virus DNA

molecules

were

isolated

and cleaved with

restriction

endonucleases.

The strands (heavy and light) of

most

of the

restriction

endonuclease fragments

were

separated. The

pattern

of

labeling clearly shows

an asymmetry

of

radioac-tivity

on

the isolated strands of each

restriction

endonuclease piece. The data is

consistent with

replication

proceeding

in

the

5' to 3'

direction

on

each strand.

Thus, there

is an initiation point

placed

at or near

each

end of the molecule.

Adenovirus

type 2 DNA, a linear moleculeof 23 x 106

daltons, replicates

in the nucleusand

produces approximately

100,000

copies/cell

dur-ing

lytic infection

(9, 10).

The

duplex

DNA

has

nonpermuted sequences (6)

and inverted

termi-nal

redundancy

at

ends

(8, 34),

which

are

iden-tical for

100 to 140

nucleotide

pairs (1).

The

inverted terminal redundancy allows the

for-mation

of single-strand

(ss)

circles by base

pair-ing

between both ends ofthe denatured

ss

mole-cules. Since the ends of adenovirus duplex DNA

are

identical,

these

molecules

cannot

be

con-verted

to

covalently linked double-strand

(ds)

circles like those formed

by

bacteriophage

lambda DNA

(10, 34). During

replication,

ss

molecules larger than the

genome

have

not

been demonstrated

(12, 29, 30);

therefore,

no

covalent addition of

progeny DNA to

parental

molecules

occurs.

There

are

small pieces of

ade-novirus

DNA

similar

to

"Okazaki fragments"

(20),

which

can

be dissociated from the

replica-tion

complex by alkaline denaturation

(2, 12,

31, 33).

During

normal viral

replication

it is

not

known if all regions of the

genome are

first

polymerized

into

Okazaki

fragments,

which

are

subsequently

joined. Replicating

molecules

have

a

significantly

higher

buoyant

density

than

parental

viral DNA

(27, 29). Further

evi-dence has shown that the

density

shift

is

caused

by extensive

ssDNA

regions and

not

by

RNA-primer

fragments

as

reported

for

polyoma

DNA

replication (16).

Recently,

a

number

of

models

of

replication, which include bidirectional

growth,

have been

proposed for adenovirus

DNA.

Sussenbach

and

co-workers have

pre-sented

data

that

replication

starts atthe

right

end

(AT

rich) by displacing

the

parental heavy

strand

with continuous

polymerization

inthe 5' to3'

direction

on

the

light-strand template

(7).

After

a

delay, replication

starts on

the

dis-placed

strand at various

internal

points

ormay

startat

the

3'

end of

the template molecule in a

pattern

of

continuous growth in the opposite

direction

to that on the first strand.

Experi-ments

from

our laboratory, in which

adenovi-rusDNA

half-molecules

were produced by

me-chanical shearing, have shown

that there is

bidirectional growth

with two termination

sites,

oneon

the left and another

on the right

half

of the

molecule (13).

The

presentstudy extends these findings by

using

two restriction

endonucleases,

which

have allowed

us to examine nine regionsof the

DNA. The method

to

determine the

origin

and

terminus

of DNA

replication

is

similar

to

that

employed by Dintzis

(5) to

analyze the direction

of

replication

of

polypeptides and

more

recently

by

Danna and

Nathans

(4) to

isolate the

origin

and

direction of replication

of simian virus 40

DNA. When

a

radioactive

precursor is added to

a system

synthesizing DNA, the

label enters

replicating molecules

at a growing point that is at a

different site

in

each molecule. This

as-sumes

that

the labeling procedure does

not

change

the

rate

of

DNA

synthesis by

synchro-nizing

molecules

at any

phase

of the replication

cycle.

The

molecules,

which

are

completed

dur-ing

labeling periods shorter than the total

syn-thesis

time

of the

macromolecule,

will be

pref-erentially

labeled

at

the terminus.

Therefore,

when

completed molecules

are

effectively

sepa-rated from

replicating molecules, the

amount

of

radioactivity

in

various

regions

of the

com-pleted molecules

will reflect theorigin and

di-rection

of

synthesis.

In this report the pattern

of

labeling of

ds

DNAindicates

highest

specific activity

atboth

ends of the

DNA,

which is consistent with a

termination site at

each end of

the

molecule.

While

this

manuscript

wasin

preparation,

sim-ilar results

were

reported

(23, 28).

However, examination

of

the

single-strands

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308 HORWITZ

of each

of these

DNA

fragments

has shown that the heavy and light strands label

asynchro-nously. This difference in labeling ismost

con-sistent with replication in which each strand is

synthesized in the 5'to3' direction starting at

opposite ends of the molecule.

(A preliminary report of these results was

presented at the Cold Spring Harbor Tumor

Virus Meeting, Cold Spring Harbor, N. Y.,

Au-gust, 1975.)

MATERIALS AND METHODS

Cells and viruses. Thesourceof HeLacells, ade-novirus type 2,andthe conditions of infection have been previously described (17). All experiments weredone in suspensionculturesataninput multi-plicity of 4,000virions/cell (100 to200PFU/cell).

Radioactive labeling of cells. At 18h postinfec-tion,theinfected HeLa cellswerecentrifuged for2 min at 1,500 rpm in anInternationalPRJcentrifuge

andresuspendedat aconcentration of 107cells/mlin

Eagle spinner medium with 5% fetal calf serum. After 5 min of temperature equilibrationin a water bath at 37 C, the cells were radioactively labeled

with [3H]thymidine at 0.25 mCi/ml (40 to 60 Ci/

mmol). The incorporation of radioactivity was stoppedby dilutingthe cellsin 7volumes ofice-cold Earle salts, rapidly centrifuging the cells at 1,500 rpm, andadding 0.2%sodium dodecyl sulfatein0.01 MTris-EDTA (pH 7.4). Usingtheseconditions, the incorporation ofradioactivity was linear for 60 min without anyappreciablelagatthebeginningof this interval (14).

Purificationof viral DNA. Intact [3H]thymidine-labeled viral DNA was purified from cells after pre-cipitating large-molecular-weight cell DNA by a modification of the Hirt procedure (11, 29, 30). Cells (3 x 107)weresuspended in 2 ml of 0.01 M Tris, 0.01 M EDTA (pH 7.4) at 0 C. Sodium dodecyl sulfate (0.2%) and 500 ,ugof Pronase(preincubated at 37 C

for2htodigestanyresidual nucleases) wereadded,

and the mixturewasincubated for 15 min at 30 C. The volume was increased to 9 ml by the addition of the Tris-EDTA buffer, which contained 1% sodium dodecyl sulfate. After a 5-min incubation at 30 C, NaCl wasadded to a final concentration of 1 M. The solutionwasleftat 4C for16h,andthe precipitate wasremoved bycentrifugationat12,000 rpmfor 20 mininaSpincoangle 30 rotor. By processing 1.5 x 107to3 x 107cellsina finalvolumeof10ml,80%of newlyreplicated adenovirus DNA was recovered in the Hirt supernatant. Viral DNAwasprecipitated from the supernatant with2volumes of ethanol. The DNA, redissolvedin 1mlof 0.01x SSC (SSC = 0.15 MNaCl+0.015 Msodium citrate), was centrifuged inanSW27 rotorof the Spinco ultracentrifuge (16 h at 22,000 rpm) on 16-ml, 5 to 20% neutralsucrose gradients containing 1 M NaCl, 0.01 M phosphate buffer, and 0.01 M EDTA. The 31S fractions were

pooled, dialyzed against 0.3M NaCl, 0.01 M Tris,

0.01 M EDTA (pH 8.1), and loaded onto 2-ml col-umnsofbenzoyl-naphthoyl-DEAE-cellulose

(BND-cellulose). The ds DNA waseluted with 1 MNaCl, and the DNAcontaining any ssregion waseluted with 1 MNaCl and 2% caffeine inthe same Tris-EDTA buffer (29).The appropriate column fractions

were precipitated with 2 volumes of ethanol and

redissolved in 0.01 x SSC.

['4C]thymidine-labeled viral DNA, which was used as auniformly labeled marker, was purified by disrupting virionwhichhad beenbandedtwice on CsCldensitygradients(12).

Restriction endonucleases. The enzymes from both Escherichia coli(EcoRI)andHaemophilus par-ainfluenzae (HpaI) were purified from bacterial strainsobtainedfrom the ColdSpringHarbor Labo-ratory. The bacteria were grown and the enzymes

were purified as previously described (19, 25). All

endonucleasedigestswereincubatedfor4hat37C in 10 mM Tris-hydrochloride (pH 7.4) with 10 mM

MgCl2, 6 mM KCl, and 1 mM dithiothreitol. The

reaction wasstopped in 0.04 MEDTA; the solution wasadjusted to a final concentration of10%sucrose and 0.1% bromophenol blue. The DNA fragments wereseparated by electrophoresis on cylindrical gels (1.6by 35 cm)of1.4%agarose inTris-EDTA-acetate (TEA = 40mMTris-hydrochloride, 1 mM EDTA, 5 mM sodium acetate) buffer for 16 h at 100 V. The gelswerestained in thesameTEAbuffer containing

0.5 ,ugofethidiumbromide perml, and thebands

werevisualized with aUV-lightsource(25). For separation of the strands ofeach restriction endonuclease fragment ofDNA, the ds fragments were cut outof the agarosegelafterstaining with ethidiumbromide and visualizationbyminimal ex-posure to UV irradiation. The short cyclindrical pieceof gel wasplaced into aglassscintillation vial with10mlof 0.2MNaOH for 2.5 h at room tempera-ture. The NaOH was decanted, and the gel was soaked for a further 2 h in TEA electrophoresis buffer at 0 C. Thegelslicewasplacedback intothe

electrophoresis tube, anda new column ofgelwas

polymerizedovertheoriginalslice. The agarose was

pouredafterequilibrationat60C.After the

polym-erization, thegeltubewasinverted,and the

electro-phoresis wasperformed under identical conditions

as used for the original separation of the ds frag-ments.The gel werestainedwith ethidiumbromide

andthe single strands were visualized. The

sepa-ratedstrands were cut from the gel, and the radioac-tivity wascounted.

Radioactivityinthe gel slices was quantitated by

remelting the agarose in an autoclave and then adding 10mlofscintillation fluid [1 part Triton, 2 partstoluene, 5 g of 2,5-diphenyloxazole (PPO) per liter, and 50 mg of1,4-bis-(5-phenyloxazolyl)benzene (POPOP) per liter], which had been heated to 50 C. The samples, which were shaken immediately after the addition of scintillation fluid, were cooled to room temperatureandcounted in the ambient tem-peraturescintillation counter(Beckman LS 230).

Reagents.

[3Hlthymidine

(40 to 60 Ci/mmol) and

['4C]thymidine

(57mCi/mmol) were purchased from Schwarz BioResearch, Inc. Agarose was obtained from Sargent Welch Co. BND-cellulose was pur-chased from Serva.

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ADENOVIRUS DNA

309

RESULTS

Isolation of recently completed viral DNA

molecules. Eighteen hours postinfection, cells

wereradioactively labeled with [3H]thymidine

and viral DNA wasseparated from host DNA

by using the Hirt procedure.The Hirt

superna-tant, containing replicating and completed

viral DNA molecules, wascentrifugedon

neu-tral sucrosegradients as shown in Fig. 1. The

completed molecules (31S) sediment to the

re-gion in the gradient that peaks atfraction 14.

The heterogeneous population of replicating

molecules sediment faster andappearbetween

fractions1and14(29).The completedmolecules

were further purified free of replicating

mole-')

t0

0. C-)

I

u0

10

C-)

I0 15 20 top

WCTION

NUMBER

FIG. 1. Sedimentation velocitygradients to

sepa-rate replicating from completed viral DNA. Pulse-labeled DNAisolated from the Hirtsupernatantwas

centrifugedonneutralsucrosegradientsasdescribed

inMaterialsandMethods. The gradientswere

frac-tionated into 0.75-mlaliquots, and the radioactivity

wasdeterminedby removing20plfromeachfraction

forquantitatingthetrichloroacetic acid-precipitable DNA.Fractionsintheregionof31S-completedviral DNA (fraction14)werepooledasdesignatedby the

bar,andthe DNAwasdialyzedagainst 0.3M NaCl,

0.01 M EDTA, and 0.01 M Tris (pH 8.1) before chromatographyonBND-cellulose(Fig. 2).Symbols:

0,5-minpulse; 0,15-minpulse.

cules

on

BND-cellulose, which eliminates any

DNA

with

ss

regions. The results of

BND-cellu-lose chromatography of the 5-, 10-,

15-,

and

240-min

samples

areshown in Fig. 2. The first peak

toelute

(fraction 2)

contains

the

completely

ds

molecules, and

the

second peak

(fraction

8)

rep-resents any

molecules with

ssregions.

Greater

than

99.3%

of

DNA, purified from virion

and

similarly treated, elutes

in

the ds region. When

DNA

from the virion

is

denatured by boiling

before

chromatography,

100%

of the DNA

sub-sequently elutes in fraction

8,

the

ss

region

(data

not

shown). With the

shortest pulse

times

examined (5 min),

most

of the DNA from

the

31S region

of

the gradient has

some ss regions,

which

are on

molecules that have

just

initiated

replication.

By 15

min,

the

31S region of the

gradient has

more

labeled

ds than

ss

molecules,

and

by

4

h

76%

of the

labeled

DNA is in

ds

molecules.

Determination

ofspecific activity of ds

re-gions of

pulse-labeled

DNA. The

ds[3H]-

thym-idine-labeled DNA from fraction

2(Fig. 2) was

mixed with

uniformly labeled

[14C]thymidine-containing DNA isolated from the adenovirion.

The DNAs

were

digested either with the

re-striction

endonuclease EcoRI

or a

mixture of

this

enzyme

and that derived from

HpaI. The

patterns

of

digestion for EcoRI and

HpaI

have

been elucidated

(18). The

pertinent restriction

endonuclease

pieces

are

drawn

to

scale

on

the

abscissa of

Fig.

4.

The EcoRI

pieces

are

de-signated

in

capital letters (B

to

F) and the

HpaI

fragments

are

designated

in

lowercase

letters

(e, c,

f,

a).

"C-" and "E-" refer

to

EcoRI

fragments further

digested by

HpaI with

the

loss

of

approximately

1.5%

of the genome.

The results of

a

typical EcoRI

+

HpaI

restric-tion

endonuclease

digest

of the mixture of

[3H]DNA

pulse-labeled for

5 min

and the

[U-'4C]viral DNA

are

shown in

Fig.

3.

Peaks with

the

higher

ratios of 3H- to

'4C-labeled DNA

include

"c,"

"C-,"

and

"e,"

which

are near

both

ends of the molecules

(see

Fig. 4).

A

summary

of results obtained from

5-,

10-,

and 15-min

pulse-labeled DNAs

are

shown

in

Fig.

4.

The

lowest

specific

activity

(3H/14C)

is in

the

region

of fractions

"a"

and

"B" near

the

center

of the

molecule,

and the

specific activity

increases

to-ward both

ends. As

expected,

differences in the

specific activity

of the individual

pieces

are

greatest

for the

5-min

digest,

less

at 10

min,

and

minororabsent

by

15min.

This

pattern

of

specific

activity

clearly

shows

bidirectional

growth

of the

molecule,

but

a

number of

quite

different models could

explain

this

data.

To

differentiate between

numerous

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310 HORWITZ

.10

Ca.2

I

~~~~~~~~~~~~~~~~~~~~~5

5 10 5 10

F R A C T I O N N U M B E R

FIG. 2.BND-cellulosechromatography toseparate replicatingfromcompletedviral DNA. Two-milliliter columnsofBND-cellulosewerepreparedin2.5-mldisposableplastic syringes by wettingthe cellulose in 0.3 M

NaCl, 0.01 MEDTA, 0.01 M Tris buffer (pH 8.1) containing 20% ethanol. The ethanolwas removed by

centrifugation ofthe resin immediatelyafter suspension ofthe cellulose. The BND was poured into the columns andextensivelywashed with thesamebufferwithout ethanol until theoptical densityof theeffluent at260nmwasless than 0.05. TheDNA, which had beendialyzed againstthe columnbuffer,wasloadedonto the BNDand washed with 12 mlofthesamebuffer.Elutionofds DNAwasachieved with 12 mlofthe column

buffertowhich NaCI had been addedtoafinalconcentrationof1M.Aliquots (2 ml)werecollectedduringthe

elation,and

50-pJ

sampleswereremovedforquantitationofthe DNAineachfraction.The columnwasthen

washed with 12 mlof the latter buffer,towhich2%caffeine(designated bythe arrow) had been added. Thess DNA elated after the addition of caffeine andwas quantitated by taking

50-pl

aliquotsfor radioactivity counting. The ds DNA, elatedinfraction 2,wasprecipitatedwith2volumesofethanol. (A) *,5min; 0,10 min.(B) A,15 min; A, 240 min.

ties,

it was necessary to

know if

replication

was

similar

on

both strands. Sharp

et

al.

(24)

have

shown that the

denatured heavy (H) and light

(L)

strands ofmost

of the restriction

endonucle-ase

fragments

can

be successfully separated

on

agarose

gels.

Using

the alkaline denaturation

technique for

DNA

embedded

in

agarose

(see

above),

we

have been able

to separate H and L

strands from the EcoRI

"B."

"C," "D," "E,"

and

"F" piecesand the HpaI "e" and "c" pieces.

Determination of specific activity of ss

re-gions

of pulse-labeled DNA. The ratio of 3H

(pulse)-

to 14C

(uniformly)-labeled

DNA was

de-termined for the separated

strands ofeach

re-striction

endonuclease

piece isolated from

re-cently completed duplex

DNA as

described

in

Materials and

Methods. The

extent of

strand

separation

for several ofthe fragments is shown

inFig. 5.

The strand separation of DNA

dena-tured within the

agarose

gel

is

superior

to

dena-turation in

solution

not

only

because of the

relative

speed

of the former

technique

but

also

because

of

the

decreased

amount

of renatured

DNA

found

after

electrophoresis.

In

most

ex-periments,

asin

the

one

shown

in

Fig.

5,

there

is no

renatured

DNA

detectable by

ethidium

bromide staining. The

assignment

of

strand

specificity (H

or

L) for the isolated strands

fol-lows the designation of Sharp

et

al.

(24). The

faster

moving

band for the EcoRI "B." "C,"

"D,"

"E," and "F" and the slower

band for HpaI "e"

and "c"

(P.

A.

Sharp, personal

communication)

belong

to

the

same

strand, which

is

the heavy

strand

in

alkaline

CsCl gradients. The

HpaI "a"

fragment does

not separate into H and L

strands

under

any

conditions

tried thus far.

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oc B C- D

I, I E-FeI fI

go

6

a-10 20 30 40 50

F R AC T I 0 N N U M B E R

FIG. 3. Agarose gel electrophoresis of a restriction endonuclease digest of the5-minpulse-labeled DNA. The ds viral DNA from fraction 2 (Fig. 2), which was extracted from cellspulse-labeled for5 min with

[3H]thymidine,wasmixed with

["4C]thymidine-containing

DNApurifiedfrom adenovirions. The mixturewas

digested with EcoRI andHpaIfor4h asdescribedinMaterialsand Methods. Afterelectrophoresisofthe DNA on1.4%agarosecolumns for 16 h at 100 V, the gel was stained withethidium bromide and the DNA bands werelocated. The gel was sliced into aliquots starting with acutbetween the 'a" and Sc" piecestoinsurethat these closely migrating fractions were in separate gel slices. The gels were melted, and theradioactivitywas quantitated by scintillation counting. The designation of the restriction endonuclease fragments isas de-scribed in thetext. Thelocationofthefragments and theirrelative sizes areshown on the abscissa ofFig. 4.

Fragments"E-" and "F' donotseparatein thissystem.

In Fig.

6 and 7, a summary of the specific

activities of the isolated strands is shown for

pulse-labeling

periods of 5 and 10

min,

respec-tively. It

is

clear that the specific activity

is

quite

different for the corresponding regions on

both

strands, and this suggests that the

L

strand

(0)

is

replicating from left to right and

the

H

strand

(0)

from right to left.

Single-strand

data for the "e" fragment at the extreme

left end of the molecule is not shown in the

figures

but

is

presented

in

Table 1. The high

specific activity on the

L

strand

of the "e"

frag-.

ment and

the

low

specific activity

on

the

H

strand do not continue the trend of labeling

derived from quantitating replication

on the

other

96%

of

the molecule.

DISCUSSION

The data

is most consistent

with

bidirec-tional

growth,

which is

asymmetrical

oneach

of the strands of

adenovirus type 2 DNA. The

light strand appears

to

initiate

at

or

near

the

left hand end and replicate continuously to the

right. This corresponds to growth

in the 5' to 3'

direction

according to

the

data of

Sharp

et

al.,

who

assigned

the 5' end of

the

heavy

strand to

the right (24).

In

contrast, the

heavy

strand

initiates

on

the

right and

replicates toward the

lefthand end. This model of

replication does

not

require

Okazaki

fragments

to

successfully

propagate

either

chain,

although

several

inves-tigators have

reported the

presence of these

intermediates.

There

appears

to be a

discrepancy

of

labeling

at

the lefthand

end of the

molecule, which

was

detected by

examining

the

HpaI

"e"

piece

repre-senting

4%

of the

DNA.

The

light

"e"

strand

had more

radioactivity

than

expected for

an

origin of

replication.

This could

occur

because

the

origin

of replication

contains

nucleotides

311

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312

HORWITZ

such

as

RNA serving a primer function. Since

the RNA would

have

to

be

excised and replaced

with

deoxynucleotides,

this

region

may be

la-beled by

thymidine with kinetics similar

to a

terminus. This would

occur if

the RNA

were

removed toward the end of the

replication

on

that

particular strand.

Another possible

explanation

is that the

rep-1.2*

0

o

-

0.-e c f a B F D E C

RESTRICTION ENDONUCLEASE FRAGMENT

FIG. 4. Orderoflabeling ofselectedregionsofds adenovirus DNA. From data suchas thatinFig.3, ratios ofpulse-labeled[3H]DNAtouniformly labeled

['4C]DNA

were calculated. Included are the data from the 5-, 10-, and15-min pulse-labeling periods

obtainedeither from digests usingEcoRI alone

(frag-mentsB, F,D,E, C) ortogether with theHpaI

en-zyme (fragments e, c, f, a). This results from the

three time points are normalized to give identical ratios for the "e" fragment. Symbols: 0, 5 min; 0, 10min; A, 15 min.

licating

molecule

is a

circle,

and

that

replica-tion

continues

beyond

the

molecular end to

an

extent

of

4%

of the genome.

Although

linear

adenovirus DNA molecules have terminal

re-dundancy,

it is

of

the

inverted

type,

which

does

3

Or-25k

20

0 15

1 0

05

e c f a 8 F D E C

RESTRICTION ENDONUCLEASE FRAGMENT

FIG. 6. Order

of labeling

(5min) ofselected

re-gions of

ssadenovirusDNA. DNA waslabeled with

[3H]thymidine for

5mininadenovirus-infectedcells

at18hpostinfection. The recently completed

duplex

viral DNA was purified and digested either with EcoRI

for fragments B, F,

D, E,andCorwith EcoRI

+

HpaI for fragments

e, c, f, and a; the DNA was

electrophoresed

onagarose

gels

as describedin

Fig.

3.Selected

fragments

wereprocessedtoseparatethe H and Lstrandsaccordingtothe descriptionin

Fig.

5 andinMaterials andMethods.The specificactivity

of

the

pulse-labeled

DNA

(PH)

in relation to

uni-formly

labeled DNA (14C) wasdeterminedand

plot-tedinrelationtoits mappositionontheadenovirus chromosome.

Symbols: *,

Lstrand; 0,H strand.

t

c,

r..>

I

R

Hpo.

e

[image:6.509.267.451.137.317.2]

Hpii

or

FIG. 5. Strand separation ofrestrictionendonuclease fragments. Pulse-labeled DNA

([3H]thymidine)

was mixedwithuniformly-labeled DNA

(['4C]thymidine)

anddigested eitherwithEcoRI

orHpaI.

Thepieceswere separated onagarose gels, stained, andcutfrom thegel.After soaking thegelslices in 0.2 MNaOHto denature the DNA, the endonuclease fragments werererun onneutralagarosegelsasdescribedinMaterials andMethods. The results ofstrand separationareshown forseveralof thefragments.AlthoughtheHpaI"a" piecedoes notseparate intoHandLstrands, theHpaI "c"and the EcoRI"B"fragmentseparate intotwo

bands. (For otherfragments successfully separated by this technique, see text).

III

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

J. VIROL.

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[image:6.509.62.248.159.360.2] [image:6.509.66.457.472.584.2]

qS 20h

u

I

0

or ₁₀

05K

e c f B F D E C RESTRICTION ENDONUCLEASEFRAGMENT

[image:7.509.62.247.56.265.2]

FIG. 7. Order of labeling (10 min) of selected re-gionsof ss adenovirus DNA. Infected cells were la-beled with [3H]thymidine for 10 min in the same experiment as described in Fig. 6. The DNA was processed and quantitated exactly as described for the 5-min sample.

TABLE 1. Specific activitiesof the heavy and light strandsseparated from the

HpaI

"e"fragment after

5-or10-minlabeling

5min 10min

Strand

"e "c" "e" "C"

Heavy 2.3 3.1 0.8 2.2

Light 7.8 0.5 6.5 0.5

aThe "e" fragment wasobtained from the same

DNAs as shown in Fig. 6 and 7. The "e" heavy strandcorrespondstothe DNAplottedas

(0)

inFig. 6 and 7, and the specific activities (pulse-labeled

PH]DNA/uniformly labeled [14C]DNA) shown were

calculated asforthefigures. Data for the "c" frag-mentfrom Fig. 6 and 7 are shown for comparison.

not

allow the usual

types

of DNA-DNA

interac-tions

at

the ends

to

facilitate circularization of

duplex DNA. Recently,

a

protein that holds the

ends of

adenovirus DNA

together

has

been

pur-ified with the

DNA

from virions (22).

However,

no

circular

forms have

yet

been

recognized

as

intermediates in adenovirusDNA

synthesis.

It

is

also

possible

that

there

is a

nonspecific

5'-exonuclease

digesting

small

portions of

the

DNA. If

these

regions were

repaired,

thymi-dine label would

appearat

the

ends of

the

com-pleted

DNA

molecules. Of the three

proposed

explanations,

only

the model

of a

replicating

circle would also

explain

the

specific activity

of

the "e"

heavy strand, which is lower than

ex-pected for its position next tothe "c"

fragment.

Similar data is not yet available from the

righthand end of the molecule. The HpaI "g"

fragment,which is 1.4% of the genome, is at the

right end. Although the "g" band was

visual-ized with ethidium bromide staining

and

ap-peared in

fraction

44(Fig. 3),the "g"

radioactiv-ity wasalways superimposed on a background

of counts in

fractions

45

through

47, which

makes

exactcalculations difficult. We have not yet been able to separate the strands of the "g" fragment and are approaching the quantitation

of counts on

each of the strands by

hybridiza-tion of duplex "g" fragment with isolated

strands ofthe EcoRI "C" fragment.

It is alsopossible that labeling at the

molecu-lar ends may be complicated by an inability to

separate newly initiated molecules with very

short

replicating

regions from the pool of ds,

31S completed molecules. It is difficult to

de-sign

a

control

experiment

for

this

possibility.

The models of adenovirus replication as a

linear

molecule fail to provide a mechanism for

the synthesis of the

5'

ends of the

DNA. If RNA is a primer in this system, there would not be a

way to fill

the

gap at

the ends

upon

the

removal

of

RNA, because all

the

known

DNA polymer-ases

require

a

primer nucleotide

sequence

be-fore

elongation

can occur.

This

problem

has

been solved by the formation of

concatameres

for thereplication of a molecule such as

bacteri-ophage T7, which

replicates

as a linear DNA

(32).

Concatameres, which

are

joined

molecules

longer than

unit

length, allow the end of

one

molecule

to serve as a

primer

for another.

No such

covalently

linked concatameres have been

found

during adenovirus

DNA

replication,

al-though small quantities of viral DNA

may

sedi-ment

faster than

genome

length

on

alkaline

gradients

(3).

These larger molecules

have

never

been shown

to

be labeled with the

kinet-ics

expected of replicating intermediates,

are

reported

to

be linked

to

host cell

DNA, and

may

be important

in

the

integration of viral

DNA in

host chromosomes even during the lytic

cycle.

The data

presented in this report are consist-ent

with

one

of the models

for bidirectional

replication,

originally reported by Sussenbach et al.

(27).

However,

our

observations

are not

consistent withSussenbach's proposal that

rep-lication

onthe displaced strand

often

begins at

one or several

internal

initiation sites.

Al-though

our

data

do not specify on which

molec-ular end the first

round of displacement

synthe-sis occurs,

Sussenbach

proposed that

replica-tion

always

began at the right end by displac-ing the heavy parental strand. His observation

If1 I- - T

18,

2.5r

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[image:7.509.61.255.367.432.2]

314

HORWITZ

of the displacement of

only the heavy strand

has been

questioned by several

other

investiga-tors (15, 28).

This

asymmetrical

model

of bidirectional

replication

for

adenovirus DNA

is

similar

to

models

reported for the replication of

mitochon-drial DNA (21).

Another similarity between

these

two

systems is

the

relative resistance of

the

replication of both DNAs to

inhibitors of

protein

synthesis (14, 26).

The

uncoupling

of

DNA

synthesis from its

usual strict

dependence

on

new

protein

synthesis may

depend

in

part

on

the

displacement model of replication

shared

by

both mitochondrial and adenovirus DNAs.

ACKNOWLEDGMENTS

This investigation wassupported by Public Health Ser-vice grant CA-11502from the National Cancer Institute. Marshall S. Horwitz is the recipient ofa Public Health Service CareerDevelopmentAward from theNational Can-cerInstitute(1K04CA-35554).

Iwish to thank Arthur Davino for experttechnical as-sistance,JerardHurwitzforhelpful discussions,and Ste-phen Baum,Susan Horwitz, and Matthew Scharff for criti-calreadingof the manuscript. Ialsowish tothank Julian Panforproviding EcoRI enzyme for some of the pilotstudies and MartinFarber for furnishing the bacterial strainsused toproducethe restrictionendonucleases.

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