Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis.

(1)

Replication

Process

of the Parvovirus

H-1.

VII. Electron Microscopy of Replicative-Form DNA Synthesis

IRWIN I. SINGER AND SOLON L. RHODE III*

PutnamMemorial Hospital Institute for Medical Research, Bennington, Vermont 05201

Receivedfor publication 11 June 1976

The geometry of replicative form (RF) DNA synthesis of theH-1 parvovirus

wasstudied withthe electron microscope usingformamideor aqueous variations

of theKleinschmidt spreading procedure. H-1DNAwasisolated from human or

hamster cells infectedwith a temperature-sensitive mutant, tsl, whichis

defi-cient inprogenysingle-stranded DNAsynthesis at therestrictive temperature

(S.L.Rhode, 1976),thusminimizingpossible confusionbetweenRFandprogeny

DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as

determinedby gel electrophoresis, ethidium bromide staining, autoradiography,

and digestionwithendoR EcoRI, was high. H-1 RF DNAs werelinear

double-stranded molecules, 1.53 ,m in length. H-1 RIs of RF DNA replication were

double-stranded, Y-shaped molecules, with thesame length as RF DNAs. The

replicationorigin was localized no more than 0.15 genome lengths from one end

oftheRFDNA, withreplication proceedingtoward the other end at a uniform rate. SimilarRFand RI molecules of dimer size were also observed. The length

of H-1 single-stranded DNA extracted from purified virions was measured

relative to thatof

OX174

and it had a verysimilar contour length, so that the

molecularweightof H-1single-stranded DNA wouldbe at least 1.48 x 106 to 1.59

x 106 (Berkowitz and Day, 1974).

The parvovirus H-1 contains a single- ceedsfrom the end containing the origin to the

stranded(ss) DNA(22),and iscapable ofauton- opposite terminus at a uniform rate. The

omousreplication(12, 20, 21).During infection, branched RI DNA appeared to be entirely ds.

a double-stranded (ds) replicative form (RF) Dimer-length RF DNA molecules andRIswere

DNA is synthesizedand replicated semiconser- alsoobserved. The lengths ofH-1viral andRF vatively at a nearly exponential rate (13). Prog- DNArelative to those of

pX174

weremeasured,

eny ssDNA is produced simultaneously, pre- and the molecular weight of H-1 ssDNA was

sumably by displacement fromRF DNA engag- determined tobe at least 1.48 x 106 to 1.59 x

ing in asymmetric DNA synthesis (14), and 106,similar to the value obtained by gel

electro-encapsidatedinto virions. Inthisstudywehave phoresis (16).Additional data on the location of

examined H-1 replicative form DNA and its the origin ofreplication has been obtained by

replicative intermediates (RI DNA) with the partialdenaturation mapping, and will be

pre-electron microscope to define the geometry of sentedinthe followingpaper of this series(19).

RF DNA replication. To minimize any

confu-sionwith progenyviralDNAsynthesis, we an- MATERIALS AND METHODS

alyzed the replicating DNA of a temperature- Virus and cells. Parasynchronouscultures of

sec-sensitivemutantof H-1,tsl, which isdeficient ondary hamster embryo fibroblasts or human NB

in progeny ssDNA synthesis, but not in RF cells were prepared and infected with tsl or

wild-DNAreplication (15). type (wt)

H-1

as previously described (16).

Esche-Previous studies using ethidium bromide- richia coli H-502 and H-4714, and

OX174

(wt) and

CsCl density gradient centrifugation, velocity am3 werekindly provided by R. L. Sinsheimer.

sedimentation, and gel electrophoresis pro- Viral DNApreparation. H-1 virion DNAwas

ex-duced noevidenceforcovalentlyclosed circular tracted from purified virus (12) labeled with H-1 RF DNA(13). Electron microscope visualiz- [3H]thymidine ([3H]TdR) by lysis in 0.2 N NaOH and ationof H-1 RF DNA reveals it to be alinear

centrifugation

inanalkalinesucrose

gradient

(16).

ationlofuH-i

R. DAm

reveal.

itatosbe

a

lin

OX174

viral DNA was prepared as in (11), and

molecule,

1.53

um

in

length.

Analysis of RIs XX174 RF DNA was produced as outlined by

John-shows thattheyareY-shaped, with the origin son and Sinsheimer (8).

H-1

RF DNA was labeled

ofreplicationlocated no more than 0.15 genome andprepared as previously using the Hirt extraction

lengthsfrom oneend, and that replication pro- withPronase digestion (16). The Hirtsupernatants

713

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714 SINGER AND RHODE J. VIROL.

containing viral DNA were extracted with phenol stubs and their attached grids. The samples were

after RNase treatment and before fractionation in shadowed by melting a 2-cm segment of platinum-the preparative neutral sucrosegradients. Benzoyl- palladium (80:20) wireina vacuumof2x 10-5mm of ated DEAE-cellulose (BDC) chromatography was Hg; a visible droplet of molten metal was

main-carried out asdescribed (16). tained on the tungsten electrode for the 1-min Gel electrophoresis. Agarose gel electrophoresis shadowing period while the table rotatedat120rpm. incylindrical gels has been described (16). Vertical The grids were shadowedata50angle 5cmfrom the gel electrophoresis was performedon anEC470 elec- metal source, and were then easily removed from trophoresis cell (E-C Apparatus Corp., Philadel- their stubs with fine forceps. Micrographs were phia, Pa.) with a gel (0.3 by 12 by 16 cm) of 1% made with a JEM-7 electron microscope (J.E.O.L. agarose.The gel and electrode buffers were buffer E Co., Inc., Medford, Mass.) equipped with liquid-(40 mM Tris[pH 7.2]-20 mM Na acetate-1 mM nitrogen-filled cold traps at the diffusion pump and EDTA) asdetailed (5). Electrophoresis was carried on topofthe objective lens pole piece (immediately out withaconstant voltage of 100Vat16°C untilthe beneath the specimen), and a30-,tmgold foil objec-bromophenol blue marker was near the bottom of tive aperture. The lens currents and high voltage the gel. The gels were stained in E buffer with 5 ,ug (80kV) were turned on at least 1 h before studying of ethidium bromide per ml for1handphotographed grids at a magnification of 14,200, calibrated fre-under illumination with long-UV light. Autoradi- quently with a grating replica (E. Fullam, Inc., ographs were made of the gelafter vacuum drying Schenectady, N.Y.). The intermediate lens current with Kodak no-screen X-ray film NS2T, exposed (which controls magnification on this instrument) for 24 hat23°C. was neverchanged once a micrograph of the calibra-Electron microscopy. Viral DNA, which had tion replica was made; the output magnification been banded to equilibriuminCs2SO4, was dialyzed remained constant throughout the course of this against 0.1MTris(pH 8.5)-O.01MEDTA, broughtto work. Care was also taken to use a low

electron-0.2 M inNa acetate, precipitated with ethanol, and beam current (not exceeding 10

AA)

and a reduced dissolved in 50 to 100

gl

ofthe latter buffer for amount ofcondensor illumination to minimize dam-electron microscopic study. Most of the DNA was age to the specimens. The electron micrographs prepared with the formamide technique. The spread- were enlarged 10 times by projection so that the

ingsolutior.contained10llofwater,10

gl

of an0.5- DNA molecules could be accurately traced; their

mg/ml solution of cytochrome c (Sigma, type III, in contour lengths were measured with a Dietzgen 0.5 M Tris-hydrochloride[pH 8.5]-0.05 M EDTA), planimeter, and expressed as the mean plus or

mi-5

gl

of DNA sample in 0.1 M Tris-hydrochlo- nus the99% confidence interval (CI). ride(pH 8.5)-0.01 M EDTA, and 25 ,ul of formamide

(Matheson Scientific, Inc.). Immediately after thor- RESULTS ough mixing, the entire50

gl

of DNA solutionwas

spread onto a freshly prepared hypophase of 20% Purification ofH-1 RF and RI DNA from formamide in 10 mM Tris-hydrochloride(pH 8.5)-i infected cells.

H-1

DNA was extracted from mMEDTA, in a Teflon-coated trough as described

tsl-infected

cultures of parasynchronous hu-by Davis et al. (4). DNA was also spread hu-by the man NB cells or

secondary

hamster embryo aqueous method asoutlined by the latterauthors. fibroblastsbythe method ofHirtaspreviously Parlodion-coated 300-mesh copper grids were diled bythe

met

supertas wre sub-touchedto thehypophase surface 1 min after DNA detailed (16). The Hirt supernatants were sub-spreading; no talc was used. The grids were attached jected to

velocity

sedimentation in a prepara-to stubs to avoid damaging their parlodion films tivesucrose

gradient,

andfractionswerepooled when handling them with forceps, sampling the asillustratedinFig. 1. Pool A contains primar-DNA, or during staining, dehydration, and shadow- ily monomer RF DNA, and pool B contains ing. To dothis, microscope slides were dipped into a monomer RF DNA, dimer RF DNA, and RI

1%solution of parlodion in amyl acetate (wt/vol) and

molecules,

which sediment more rapidly than allowedtodry vertically. The parlodion films were monomerRF DNA (16). Mock-infected cultures stripped ontoadistilledwatersurface, and portions

simer

extrA cted

cultures

having asilver-gold interference color were picked

were

similarly

extracted, and

theyieldof radio-up with awire loop, air dried, and placed onto the labeled DNA in these regions of the sucrose surface of agrid resting on a stub; theoverhanging gradient was less than 2% of that from H-1-edgesof the parlodionfilm firmly held the grid on infected cultures. The

homogeneity

of the ra-the stub. This method minimizes damage to the diolabeled DNA to competitive hybridization parlodionfilm, which increases length variation of (13), or to cleavage by bacterial restriction en-the adherent DNAmolecules; contamination of the donucleases (16) indicated that the

H-1

DNAs lowersurface of thegrid by the various solutions is were at least 90% radiochemically pure. How-alsoprevented. Thegridswere stainedimmediately ever it

after DNAsampling with freshly prepared 50 HM

e,

S

possible

that

mock-infected

cultures uranyl acetate-50 ,MHCl in90% ethanol (pH3.9)

are

inadequate controls for

purity. For exam-for 30s, followed by a 10-s rinse in 90% ethanoland ple, the cytopathic effects ofH-iinfection might dehydration with 2-methylbutane for 10 s. Shadow- induce

degradation

ofcellular

DNA,

although ing wasaccomplished with a Denton vacuum evapo- this was not observed when cells with prela-rator andarotary table, which accommodated the beled DNA were infected (13), orcontamination

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SYNTHESIS

l w 0 ' _ with

long-UV

light (Fig. 2a). As shown before

15 _ A (16), the predominant bands are the monomer

RF DNA in the mixtureofA and B and in A

alone, dimer RF in B alone, and thepartially

cleaved dimer RF, EcoRI-A, dimer B, and B

fragments in the digest of A-plus-B mixture.

Thesestained DNAbandswereshownto

corre-10 spond to the radiolabeled DNA by an

autora-diograph of the gel (Fig. 2b). Thus, after the

sucrosegradient step, thepreparations consist

largely of monomer and dimer H-1 RF DNAs,

x asjudged by electrophoreticmobility and

speci-CL lficityofcleavage with EcoRI. It should be noted

I / thatthemonomerand dimerRFbands,aswell

asthose of the EcoRI-A and -Bfragments,

ap-5/

pear as doublets in the ethidium

bromide-stained gel, butnot inthe autoradiogram. This

difference in resolution isprobably due to our

deliberate overexposure ofthe autoradiogram

to

visualize the

light

bands

in

the

EcoRI

digest.

B I A The occurrence of two distinct

EcoRI-B

frag-0 , ments has

already

been documented

(16);

the

5 10 15 20 25 possible existence of doublet monomer and

di-FIG. 1. Preparative sucrosegradient

of

PH]BUdR

mer RFs and EcoRI-A fragments iscurrently

containing H-1 RF DNA. Parasynchronous cultures under

investigation.

Also,thematerial remain-of hamster embryo cells infected with tsl H-1 at a ing at the origin of thelanes containing DNA multiplicity of infection of 5 to 10 PFU/cell were from fraction Bof the sucrose gradientis pre-incubated at 39.5°C. The cultures were treated with sumed to be entangled molecules not greater FUdR (10.5

m.g/ml)

14 to 14.5 h p.i., and labeled than dimer size, since electron microscopy

with[3H]BUdR as inResults. Viral DNA extracted never revealed longerDNAs in fraction B, and

by the Hirt method was redissolved in 50 mM thisentrapmentisnotconsistentlyobserved. It Tris(pH 7.5)-i mMEDTA and treated with pan- ispossible that this material is associated with creatic RNase (50

mg/ml)

for 30 min at 37°C. RNase

was removed by phenolextraction and the DNA was

protein,

but we

believe

that this is unlikely,

precipitated with 0.15 MNaCl and 2.5volumes of sice theDNA was digested withPronase, and

ethanol at-20°C for 16 h. The DNA was redissolved extracted with phenol.

inthegradient buffer and sedimented in a 5 to 20% The DNA prepared for electron microscope sucrosegradient for 18h at24,000 rpm,4°C,in an analysis was density labeled with [3H]bromo-SW27 rotor as done previously (16). Fractionsofi ml deoxyuridine([3H]BUdR). Specifically, H-1 tsl-werecollectedthrough the bottom ofthe tube, and 20- infected NB cultures at39.5°C were incubated

pi

aliquots used to determine the positions of radio- with medium containing 5'-fluorodeoxyuridine activity.Regions were pooled as illustrated, precipi- (FUdR) and BUdR

(10-5

M) 14 to 16 h p.i. tated with ethanol, and redissolved in 20 mM and then FUdR with [3H]BUdR (2

gCi/ml,

Tris(pH

8.0)-i

mM EDTA-0.15% Sarkosyl before a X 106 M) 16

to

18 h

p.i. Similarly,

ts-l

isopycniccentrifugation in

CsSoe4.

The directionof

infected

hamster

embryo

cells at

39.5°C

were

sedimentation isfromrighttoleft. incubated with FUdR and BUdR 14 to 14.5 hp.i., and then FUdR and[3H]BUdRat 14.5 to

with

degraded unlabeled

cellular DNAmaybe 16 hp.i. After Hirtextraction and sucrose

gra-inexcessof the viral DNA. Such contamination dient centrifugation, the A and B pools (from

wasruledoutby comparing

radiolabeled

DNA Fig. 1) werebanded to equilibrium in Cs2SO4

tototal DNA byagarosegel electrophoresis. H- gradients (Fig. 3A and B). All DNA greater

1tsl viralDNAlabeled for 12 to16hpostinfec- than hybriddensity (arrow indicates adensity

tion (p.i.) with 32p was prepared through the of 1.440g/cm3)waspooled,anddialyzedagainst

sucrose gradientstep. Equal portions ofpool A 10 mMTris(pH

8.5)-i

mM EDTA forstudy with

and

pool

B (Fig. 1) were analyzed separately theelectronmicroscope.Inthis way,the

prepa-andcombined bothwith and withoutdigestion ration was enriched formolecules that had

rep-with endoR EcoRIinaslabgel(3 mmby12cm licated one or more times in the presence of

by 16 cm) of1% agarose. The gel was stained BUdR, and controlexperiments indicatedthat

with ethidium bromide, and the fluorescent 98% ofcontaminating light DNA wasremoved.

DNA was photographed under illumination Thecompositionofthe final dense H-1 DNA

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,..-~U-_~~~~~~~~

F

L

FIG. 2. Verticalslab-gelelectrophoresis ofH-iDNAaftercentrifugationinneutralsucrose. Parasynchron-ousNBcultureswereinfectedwithtslH-iat39.50Candlabeled with32P04from12to16hp.i.(16). The viral DNA was extracted, treated with RNase,and subjectedtopreparative centrifugation in a neutralsucrose

gradient.Fractions werepooledasinFig.1,andthe DNAwasdissolvedin 100p.lof10mMTris(pH7.5)-b mMNaCl-0.1 mMEDTA. Aliquotsofl10

MI

ofpoolsAand Bwerecombined andadjustedto50 mMNaCl,10 mM

MgCl,,

and1 mMdithiothreitol, anddigested with100 UofEcoRIfor1 hat370C. The reaction was

stopped byadditionof20

tii

of2.5%sodiumdodecylsulfate-50%glycerol-10 mMEDTA.Slab-gel electropho-resiswascarriedoutasdescribedinMaterials and Methods. Thegel contained, from lefttoright:poolB(20

pi),poolA (20pI), EcoRI-digestedA +B(10/.dof each) mixture,and A +B(20

PIl

each) mixture.Twenty

microlitersofeachsamplecontained theyield ofDNAfromabout2 X107NB cells. Thegelwasstained with ethidium bromide, and theDNAwasvisualized withlong-UVlight(a),andbyautoradiography(b). It has been shown(16) that thespecificH-iDNAspeciesobservedare(indescendingorder): dimer RF (DIRF),RF withattached EcoRI-Bfragment(RF +RIB,partialdigestofDIRF), RF,EcoRI-Afragment(RIA),dimer EcoRI-Bfragment(DI RIB),and EcoRI-Bfragment (RI B).

preparation

was examined

by

agarose

gel

elec- Relative contour

lengths

ofH-i and

OX174

trophoresis (Fig.

4A) and found to be almost viral ssDNAs.

Preliminary

electron

micro-entirely

monomerwithsmall amountsofdimer

scopic

examination ofH-i ssviral DNAshowed

RF DNA. The 13H]TdR-labeled virion DNA that it is

linear,

and hasacontour

length

simi-used in this

study

was also

analyzed by gel

lartothat of circular pX174 viral DNA. Since

electrophoresis

as shown in

Fig.

4B. The elec- we wanted to determine the molecular

weight

tropherogram

of thelatter

preparation

isdomi- ofH-i ssDNA relative to

4)X174

DNA

by

mix-nated

by

a

homogeneous

peak,

with a smaller

ing

these

preparations

and

measuring

the ratio

portion

of

faster-migrating species

assumed to of their

lengths

onthesame

grid,

itwas

neces-be

fragmented

molecules. It should be noted sary to ensure that

significant

breakage

of

thatthe virion ssDNA

migrates

faster than the

OX174

circleswasnot

occurring

sothatbroken ds RFunder these conditions and that there is (linear)

4X174

ssDNA would not be confused

no evidence of virion DNA in the RF DNA with

H-i

ssDNA. We therefore examined

preparation

(Fig.

4A). A very small

peak

of

4X174 ssDNA,

and found that the extent of

uncertain

significance

atthe

electrophoretic

po- circle

breakage

was9%.These viral DNAswere

sition of RF DNA is noted in the virion DNA then mixed such that the concentration ofH-i

electropherogram.

This could arise from an- DNA was twice that of the

4X174;

the

maxi-nealing

of V strands to traces (0.25%) of C mum amount of linear

4X174

DNA

contami-strands,

butthis hasnot yetbeen proven.

nating

the H-i viral DNA

pool

was therefore

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RF DNA

717

l l mum molecular weight of H-1 viral ssDNAis

10 A. D1.48 x 106 to 1.59 x 106, based on the 4X174

l1.50 ssDNA

molecular-weight

determination of1.59

/1

45 x 106(1). In addition, we did not observe

circu-1.45 larization (evidence of terminal

self-comple-1.40

mentarity)

after incubation of H-1 viral DNA

5

I5

- under

annealing

conditions

(50%

formamide-50

{\1.3 mM Tris-5 mM EDTA for 1.5 h at 23°C),

fol-lowed byimmediatespreading from50%

form-- amideonto20%formamide; theelectrophoretic

x JC\4 profile also was

unchanged

after this

treat-E O - ' ' s

~

t8 ment.

X0- B. ,

Geometry

ofH-1 RF and RI dsDNA's.

Re-1.50 gions of the

Cs2SO4

gradients containing

puta-tiveRI and RF(Fig. 3A andB), which exhibited

1\.45

[3H]BUdR-substituted

H-1 tsl DNA ofgreater

1.40 than hybrid density, were chosen for electron

5 \ - l.40 microscope study, since it would be unlikely for

1-.35 the host cell to produce any DNA of this density

duetothe semiconservative nature of cellular

DNA synthesis, and the short

labeling

times

employed. The

fully

substituted DNA from

pool

_ s s , < s B (Fig. 3B) contained ds linear RF DNA of

1 5 10

15

20 monomerand dimer

lengths (Fig.

5B),

and

Y-Tube Number

FIG. 3. Isopycnic centrifugation of H-1 tsl DNA

A.

moromerPF

pools A and B from Fig. 1. The viral DNA pools 6 prepared by velocity sedimentation in the neutral

sucrosegradient (Fig.1)weresedimentedto

equilib-riumingradientsofCs2SO4aspreviously described 4_ (16). (A) Pool A; (B) pool B. Centrifugation condi-tionswere48hat35,000 rpmin atype 40fixed-angle rotor at 10°C. Fractions of 0.2 ml were collected 2

through the bottomofthetube, and

10-p.

aliquots 2

weredriedon25-mmfilterpaper disksforassayof dimer

radioactivity. DNA greater thanhybrid density (ar- RF rows) waspooled, dialyzed against 0.1 M Tris(pH 0

8.5)-10mMEDTA, adjustedto0.3MNaCl,precipi- virionDNA tatedwithethanol,and redissolved in 50to100

pl

of E

0.1 M Tris(pH8.5)-10 mM EDTA forelectron mi-croscopy. The direction ofsedimentation is from I

right to left. 10_

3% (DNA spreading solution contained 50%

formamide; hypophase contained20%

formam-ide). Figure5A is arepresentative

micrograph

5

of this mixture, and histograms of the

mea-sured H-1 and 4X174 viral DNAs

(Fig. 6)

ex-hibit thesamemaximumpeaks(at 1.0,Lm)and

verysimilarcontour

length

variation.Both H-1

and4X174 viral ssDNA's hada mean

length

of 0

1.0 amunder these formamide-spreading con- 1 20 40 60

ditions. We also

spread

a mixture ofH-1 and - Fraction +

4X174

ssDNA's from a solutioncontaining 30% FIG. 4. Analytical gel electrophoresis of

[3H]-formamide

ontoa

hypophase

with 10% formam-

BUdR-containing

H-1 RF DNA and

[3H]TdR-ide. Under these coditions, themean

length

of labeled H-1ssviral DNA.H-1RF

DNA,

prepared

as

Hde.

Under

these96

codition,0

umen

e 143 g-+- 99 in Fig. 3 (mixture ofpools A and B), and

[3H]TdR-H-I

ssDNAwas0.96 ± 0.03z (n = 143 + 99 labeledvirion DNA were analyzed in a cylindrical

CI) and thatofX174DNA was1.03 ± 0.02

ymI1

gel (0.6 by 15 cm) of 1.4% agarose (16). Electropho-(n = 123). Since gel electrophoresis revealed retic conditions were 30 Vfor 17.5 h at23C. (A)H-1

some

fragmented

molecules

(Fig. 4B),

themini- RFDNA; (B) H-1 virion DNA.

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4

14~~~~~~~~~~~4

FI. Elcto

mirgah f-

N

pedfo

0 ommd

no2%fraie

a m

V I R'Sieff

( *

of

l * t .24 : v D

J~~-~~ <<'s S

,z,-1*

E~

; S--*".'U

Ir 3

(Fig.5a7Ta

m,

m.;;*.; r

length

wa~1.53 -+ 0.0 um -n= 1 1) that of th1 2.9 0r2um( 2 o h

ie.Iiily

>-t

ie

;* 2 2 s t &*

N-.,

FIG 5 ElectronmicrographsofH-1DNAspreadfrom 50%formamide onto 20%formamide.Bar =nIzm.

(A)Mixctureof linear ss wtH-i andcircular ss4X174 viral DNAs; (B)tsl H-i ds RFDNA from pool B (Fig.

3B) (synthesized at the restrictive temperature,39.5°C), exhibiting monomer- and dimer-length molecules; (C)tsiH-i (39.5°C)ds RFmonomer(i)and ds RIsreplicated14%(2) and52%o(3)obtained from pool B (Fig. 3B) .

shaped RIs of both monomer and dimer size branch to that of the unreplicated base) were (Fig.

5C

and 7A-D). The mean RF monomer 1.55 ± 0.10

gum

(n = 51) for the monomer, and length was 1.53 ± 0.04,um (n = 111); that ofthe 2.94 ± 0.22

,rm

(n = 12) for the dimer. Initially, dimer was 3.10 ± 0.28 ,um (n = 16). Gorre- tsl H-i RIs were purified from synchronized

sponding

mean RI

lengths

(each length

was hamster

embryo

fibroblastsorhuman NBcells.

calculated by

adding

the

length

ofthe

longest

The RIs of thesetwo groups wereidentical in

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---vE ,-I .--- 20%I I of the length of the longer of the two

20 - arms; RF DNA lengths varied within 20% of the

mean RFlength). Histogramsofthese RF and RI molecules exhibit analogous contour length 1S

*X-174

distributions ofmonomerand dimer DNA

sub-populations (Fig. 8),

but the

percentage

of RI

dimers (19%) was 1.4 times higher than thatof

RFdimers (13%). The molecular weight ofH-1

10_ monomer ds RF is 2.95 x 106, based on our

measurements of purified circular ds RF

OX174

spread separately under replicate conditions: X

1.65 ± 0.05 ,m, n = 15. Analysis of the

mon-@5. omer RF DNA peak (pool A) of the sucrose

gradient after Cs2SO4 purification (Fig. 3A)

X yieldedverysimilar

results, except

that theRI

M o ~ E § § @ l @ | § contentwassixfold lower than in the RI

region,

%

30

_

pool B

(Fig.

3B).

E _- Since it has been

reported

that ssDNA

ap-z pearsthinner and more twisted than dsDNAin

H-1 formamide preparations (4, 23), we expected to

observe obvious differencesinstrand

morphol-20 - ogy between RF, RI, and viral DNAs.However,

the appearance of H-1 ds RFs and RIs was

indistinguishable from that of ssH-1 or

OX174

viralDNAs (Fig.5A-C); therefore,the

possibil-ity that H-1 RI DNAs contain ss regionscould

not be excluded on the basis of their appearance

usingformamide-spreading solutions. We

con-sequently

studied

H-1 RI and RF DNA

mole-cules

(isolated by BDC

chromatography,

16)

using the aqueous technique, which causes ss

o

L/ r-. .0 . regionsof the DNAtocondense into bushes (4).

0.6 l.0o.5 All RI (Fig. 7C) and RFtsl H-1 DNA was fully

[image:7.501.48.242.63.399.2]

Length(pm) extended and lacked bushes with this

method,

FIG. 6. Histograms of the contour lengths of cir- so that

H-1

RI and RF DNA must be largely

ds;

cularsskX174 and linear ss H-1 wt viral DNAs. the mean RI length was 1.73

0.15 gm

(n

7),

These DNAs were combined in a spreading solution andthat of theRFwas1.46

±0.13

um(n 28)

with 50% formamide, and cospread onto a hypo-

ungthis thnique.

Sgb e ro

phase containing 20%formamide; all micrographs using this technique. Single bushes were

ob-were taken from a single grid. The mean lengths of served on molecules of RF length when aqueous both H-i and X1 74 viral DNAs were 1.0

gm

(n = preparations of wild-type H-1 RI DNA were 50, 99% CI = +0.04,um for X1 74; n =97, 99% CI examined;

these

moleculesare

presumably

in-=

±+0.05

,m for H-1).Notethat theH-1histogramis termediates in progeny ssDNA replication.

skewed toward the lower end ofthe range, corre- A frequency distribution of

H-1

RIs spread sponding to the leading shoulder ofthe main analyti- from 50% formamide onto 20%

formamide,

and

cal-gel electrophoretic peak (Fig. 4B). ordered according to the proportion replicated,

isshownin Fig. 9; only unambiguously forked

size and distribution of replication fork posi- molecules whose

lengths ranged

from 1.0 to2.0

tions, andweretherefore

pooled

for final

analy-

,um

(the

monomer

length

range)wereincluded.

sis. Replicating molecules

constituted

33% of The amount of

replication

varied from 0.15 to

this RF

population.

Seventeen percent of the 0.88 genome

lengths;

the random distribution of

measuredRIpool exhibitednodifference inthe replicationforks indicated that the rate of DNA

lengths oftheir daughter branches, whereas

replication

was uniform

throughout

this

por-the remainder of por-the RIs had slightly different tionof the H-1 genome. The originof

replica-daughter-branch lengths, whosevariation was tion is thus within 0.15 genome units of an

approximately

that expected due to methodo-

end(s)

of the DNA. No

"eye"

structures were

logical errors,

determined

by measuring the seen, and

replication

was observed to

proceed

distribution of RFmonomerlengths (75% of the

unidirectionally throughout

73%of the RF

mol-RIshad

daughter-ann

lengths

that differed

by

ecule. Of additional

significance

isthe

finding

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4...,,

0

7 Eet mcorphs ofre' sl - U-DNA(l f-0 lt

Aure

of

i -

r

. w r 50 fmi

FIG.

7Fg.3).Eletro

mtrorahmoofesRrpcaintsolatH-ib BDNA

(isolatedfomgculture

atd

theparestritiv

tempera-microscopy bytheaqueousmethod, which is 60%replicated.Note that allportionsofthe moleculearefully

extended, and thatno bushesarevisible. (D)tsl ds RImonomerreplicated18%,from poolB (Fig.3B).

thatnoneofthe dimer

length

RImoleculeswas

former

method,

H-i

DNAwas

purified

by

sub-more than 50%

replicated.

Very

similardistri- stitution of

[3H]BUdR

for TdR

during

more

butions of the

replication

fork

position

were

than

one round of

replication,

and

isolating

obtained with the aqueous

spreading

tech-

molecules

greater

than

hybrid

density

from

nique.

Cs*SO,

gradients.

Unlike cellular

DNA,

viral

DISCUSSION

RF

DNA

undergoes multiple

rounds of

semi-DISCUSSION

~~~conservative

replication

during

the short

period

In this

study,

we examined

H-i

viral,

RF,

of

density

labeling,

so that

selecting

DNA and RI DNA

by

electron

microscopy.

Viral greater than

hybrid

density

excluded 98% of DNA was

prepared

from

purified

virions

by

any

contaminating light,

cellular DNA.

Simi-alkaline sucrose

gradient

centrifugation.

Gel

larly,

RIDNAmolecules become

radiolabeled,

electrophoresis

of virion DNA revealedit to be

and

the

purity

ofthese

preparations

appears

largely homogeneous

in

migration

withasmall

high

on the basis of control

experiments

with

proportion

(approximately

16%) of faster

(pre-

mock-infected

cultures,

H-i-infected

cells

using

sumably

shorter)

molecular

species,

possibly

displacement hybridization

(13),

and

homoge-generated

by radiolysis.

RFand RI DNA was

neity

in

gel

electrophoresis

before and after

extracted from infected cultures

by

the Hirt restriction endonuclease

digestion

(in

this

pa-method,

sedimented in neutral sucrose gra- per). The second method of

purification

utilized

dients,

and

purified

further

by isopycnic

cen-

the

tendency

of RI molecules to bind to BDC

trifugation

in

Cs2SO4,

or

by

BDC

chromatogra-

tightly by

virtue of their ssDNA

components,

phy

and

isopycnic

banding

in

CsCl.

With the

and

their

elutability

therefrom with caffeine.

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DNAs from

Cs2SO4

density gradients.

Further-30 _ : more,thelengths ofH-1RF and RI DNAs have

consistently been the same (1.5

gm)

through-25 _ RF out our electron microscope analysis, and this

DNA also exhibits a

specific

pattern

of

loops

after partial denaturation; the lengths of its

20 . :

EcoRI

fragments are similarly uniform after

Kleinschmidt preparation (19). These results

would not be possible if our RF and RI DNA

were heavily contaminated with random-sized

fragmentsof cellular DNA.

10- _Our results show that the RF dsDNA of tsl

H-1

replicates

viaalinear

Y-shaped

(branched)

RI DNAthatappearstobe

largely ds;

thesum

ofthe lengths of the unreplicatedportionof the

RI plus one replicated branch is equal to 1.5

E,m,

the RF

length.

The initiation site for this

z n RF replication islocatedwithin 15% of one or

JLl1RI both ends of the RFDNA;the replication fork

5_ , 8

appeared

toproceed alongmostof the RI

mole-cule at a uniform rate. Wedid not observe any

ss

regions

in numeroustsl H-1 RIDNA

mole-07 Lo 20 30

'5 'o

cules

using

the aqueous

technique,

which

Length Am) makes such regions appear as bushes (4). How-FIG. 8. Histograms of the lengths of linear tslH-1 ever, small ss sections may be present in

H-1

ds RF DNA molecules, and Y-shaped ds RI DNAs RIs because they were preferentially retained (length = sum of longest branch plus unreplicated

against

salt elution from a BDC column, base), isolated fromhamster embryofibroblasts or which separates dsDNA molecules from those human NBcells(Fig. 3B) at the restrictive tempera- c

sspres

dsDNA Branchedfrom Rls ture, and prepared for electron microscopy by

contaiing

ssregions (16). BranchedlinearRIs the 50%/20%formamide technique.Both RF and RI have been similarly visualized using electron populations exhibit subgroups of monomer and di- microscopy for parvovirus LU III (18), ade-merlengths having very similar variation. novirus 2 (2), adenovirus type 5 (6), herpes

simplexvirus type 1 (HSV-1) (17),and the

bac-This method has proved successful for isolating teriophage T7 (24). ssDNA segments were

con-RImolecules in a variety of DNA viruses (17), spicuous in the RIs of adenovirus 5 and phage

andwaspreviously usedtopurify H-1 RI DNA

T7,

butwere notobserved inadenovirus2and

(16). HSV-1although theRIs of both ofthese viruses

Itispossiblethat mock-infected culturesare werealsopurified withasimilarprocedure

us-unsuitable controls for nonviral DNA contami- ing

benzoylated naphthoylated

DEAE-cellulose

nation, sincethe cytopathic effects of H-1 infec-

chromatography.

Replicative loops ("eye"

tionmay causefragmentation of cellular DNA.

forms)

werefoundinHSV-1and T7 in addition

We

previously found

no

evidence for

such an to

Y-shaped

RIs. We didnotobserve

replicative

effect (13), using host cells with

prelabeled

loops

inH-1 RIDNAmolecules. This is

proba-DNA. Asa further testfor cellular DNA con-

bly

due tothe location of the initiation

site-tamination,the RF and RI DNA regions (pools very close to the end of the DNA

(within

0.23

Aand B)of thesucrosegradientweresubjected ,m), sothatany putative

loop

would be

tran-to agarose

gel electrophoresis,

and the total sient and inconspicuous; the

replication

of at

DNAwasidentified by staining with ethidium least73%of the H-1genome wasobservedtobe

bromide. The

predominant

areasstainedcorre- unidirectional.

However,

we can not exclude

spondedtothelabeled H-1monomerand dimer the possibilitythat anend of the RFmolecule

RF DNA, as determined by

autoradiography.

might

be

replicated by

asecond fork

moving

in

Thus, there is noevidence for significant con- a direction opposite tothat observed. The

ac-taminationof theseH-1 RFand RIDNAprepa- companyingpapers in this series (16, 19) also rations with unlabeled cellularcontaminants, present evidence

indicating

thatthe initiation

unless thisputativeDNAisthe size of H-1 RF, sitefor RFreplicationislocalizednear aunique

and has anEcoRI cleavage site at the same endof theRFDNA molecule.

locationas inH-1 RF.Thisunlikelypossibility RFand RI molecules of dimerlength (3.0 ,um)

was dealt withby selecting BUdR-substituted werealso foundinourstudy. However,not one

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(10)

raonTT ofGenome

40.(

35I

30r

25

O.

FIG. 9. Ordered distribution of replication fork positions in tsl H-i ds-RI monomer DNA molecules purified fromhumanNBcellsorhamsterembryo fibroblastsat39.50C(Fig.3B). RIs between1.0and2.1 gm long(sumofthelongestarmplustheunreplicatedbase) werenormalized andrankedaccordingtotheamount

ofreplication that had occurred. Thepositionofthereplicationfork isdistributedrandomlyalongthe H-i

genomeregionbeginning0.15fractional unitsfromoneendoftheDNA andterminating0.12unitsfromthe other end.

ofthe measured RI dimers had

replicated

more branched molecules due to chance end-to-side thanhalf of its

length.

Sincethe

majority

of RF

apposition

oftwo RF molecules would be pro-dimers are

composed

ofmonomers linked

by

portionaltothe numberof molecules that

inter-hydrogen

bonding

rather than

by

covalent sected each other. Based on the resolution of bonds

(16),

it is

likely

that dimer RI DNAs the

method,

the

width,

andthemean

lengths

of dissociate when the

replication

fork reaches a the

molecules,

weestimate that 5%ofthe total gap at thecenter of the molecule.

intersecting

molecules would appear to be One-fourth of the observedH-i RI molecules branched Rls. The number of

X-shaped

inter-had sister branches whose

lengths

differed

by

secting

molecules in the hamster

embryo

RF 20 to 45%, which was greater than the 20% DNA

preparation

was26, so that the

expected

variation observed in RF

lengths.

This differ- number of branched

Y-shaped

molecules dueto

ence could be caused

by

either

radiolysis

or chancewas1.3.Since 34

Y-shaped

RImolecules

photolysis

due to

incorporation

of

large

were observed in this

preparation,

it is very

amounts of [3

H]BUdR,

by

the

asymmetrical

unlikely

that this artifact accounts for more

distribution of small ssDNA

regions

in

daugh-

than a small

percentage

of the H-i RI DNAs. ter arms, or

by

the

larger

relativeerrorsintrin-

Also,

the distribution of RI DNA

lengths

was

sic to

measuring

shorter

lengths.

Some of the very similarto that of the RF

molecules;

this

Y-shaped

DNAsweobserved couldbe artifacts would be

unlikely

if a

significant

number of

resulting

from the chance association of the end branched moleculeswereduetochance

end-to-ofoneRFmolecule with the side of another RF side

overlapping.

DNA. The number of molecules

appearing

as The mean

length

ofss viral DNA extracted

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[image:10.501.148.379.66.379.2]

(11)

from purified wt H-1 virions and prepared in NewYork.

the presence of 30 to 50% formamide was at

5-

Edgel, M. H.,C. A. Hutchison III, andM.Sclair.1972. least

0.96

to 1.0 am. The molecular weight of SpecificendonucleaseRfragmentsofbacteriophage least 0.96 to 1.0 ,um. The molecular weight of XX174deoxyribonucleic acid. J. Virol.9:574-582.

this ssDNA is 1.48 x 106 to 1.56 x 106, based on 6. Ellens, D. J., J. S. Sussenbach, and H. S. Jansz. 1974.

the measured length andthe previously deter- Studiesonthe replication of adenovirus DNA. III.

mined molecularweight of 4X174ssviral DNA Electron_61:427-442.microscopy of replicating DNA. Virology

(1)present as aninternal standard. The latter 7. Garon, C.F., K. W. Berry, and J. A. Rose. 1972. A

values agree closely with the figure of2.95 x unique form of terminal redundancy inadenovirus

106 obtained for themolecular weight of

H-1

ds DNA molecules. Proc. Natl. Acad. Sci. U.S.A.

RF

DNA,

which wascalculated independently 69:2391-2395.

using

'OX174

ds RF DNA as a reference, and 8. Johnson, P. H., and R. L. Sinsheimer. 1974. Structure

using ~X174 ds RF DNA as a reference, and of anintermediatein the replication of bacteriophage

that of 3.26 x 106, measured with respect to 4X174 deoxyribonucleic acid: the initiation site for

specific lambda phage fragments using gel elec- DNAreplication.J.Mol. Biol.83:47-61.

trophoresis

(16). It is

of

interest to notethat the 9. Koczot, F., B. J. Carter, C. F. Garon, and J. A. Rose.

meanlengthofH.1 ss viral DNA is signifi- 1973. Self-complementarity of terminal sequences

mean lengthz of H.-i ss viral DNA iS signii- within plus or minus strands of

adenovirus-associ-cantly shorter than that of the corresponding ds ated virus DNA. Proc. Natl. Acad. Sci. U.S.A.

RF DNA when spread under the same condi- 70:215-219.

tions using 50%formamide

(ss/ds

ratio =0.67). 10. Mayer, F., A. J. Mazaitus, and A.

Puthler.

1975.

Elec-tron microscopy of simian virus 40DNA

configura-The

ssDNA/dsDNA

length ratios ofa number tion under denaturationconditions. J. Virol.

15:585-of DNA viruses are significantly less than unity 598.

(3, 7, 10). Finally, we did not observe the circu- 11. Pagano, J. S., and C. A. Hutchison. 1971. Small

circu-larization ofssH-1 viral DNA after incubation lar viral DNA: preparation andanalysis,p. 79-123.In

K.Maramorosch and H.Koprowski(ed.),Methods in

under annealing conditions, which occurs in virology, vol. V. Academic Press Inc., NewYork.

the caseof another parvovirus, adenovirus-as- 12. Rhode, S. L. 1973. Replication process of theparvovirus

sociated virus, and is thought to result from H-1.I. Kinetics inaparasynchronouscell system.J.

terminal

self-complementarity

(9).

Inthe pre- Virol.11:856-861.

terminalself-complementarity (9). Inthepre- 13. Rhode, S. L. 1974. Replication process of the parvovirus

vious paper, evidence was presented that one H-1.II.Isolation and characterization ofH-1

replica-end of the RF DNA molecule, which is a repli- tive form DNA. J. Virol. 13:400-410.

cation terminus, is self-cohesive, andmaygive 14. Rhode, S. L. 1974.Replicationprocessof theparvovirus

rise todimer molecules in "tail-to-tail" linkage

H-1. III.

Factors affecting

H-1

RF DNA synthesis.J.

Virol. 14:791-801.

(16). On thebasis of this study, itappearsthat 15. Rhode, S. L. 1976. Replication process of theparvovirus

the end near the origin of replication is not H-1. V. Isolation and characterization of

tempera-complementary

to the other endasinthecase ture-sensitiveH-1mutantsdefectiveinprogenyDNA

of adenovirus-associated

virus. Further evi- synthesis. J. Virol.17:659-667.

16. Rhode,S. L.1977.Replicationprocess of theparvovirus

dence documenting the dissimilarity of the ter- H-1.VI.Characterizationofareplication terminusof

miniof H-1RFDNAispresentedinthe follow- H-1replicative-form DNA. J. Virol.21:694-712.

ing paper on the partial denaturation mapping 17. Shlomai, J., A. Friedman, and Y. Becker. 1976.

Repli-ofH-1 RF molecules. cative intermediates_{Virology 69:647-659.} of herpes simplexvirusDNA.

ACKNOWLEDGMENTS 18. Siegl, G., and M. Gautschi. 1976. Multiplication of par-vovirusLuIIIin a synchronized culture system. III. Thiswork was supported by Public Health Service grant

Repicatin

ofsviraloDNA.dJcuViro

1781-5.e

CA-07826-11 from the National Cancer Institute, and a

Replication

of

viral

DNA. J.

Virol.

17:841-853.

CA-erous2gift

from the

aiona

anc

.

ins

a 19. Singer,I.I., and S.L.Rhode.1977.Replicationprocess

geeosgif from

th Gie

Fonation.

of the

parvovirus H-i.

VIII.

Partial

denaturation

Wegratefullyappreciate theexcellenttechnical assist-

mappingpandolocalizatonVofItheareplicationurign

anceof RobertCostantinoandJessica Bratton, and thank H-i

lcative-for

DN th

elictron

m

icros

Kay A. 0.Ellem andHelene Toolan for critically reading

JH

rol.

21:724-731.

this

_{secetaiaduie.'}

manuscript, andVirginiaHaas and JaneenPratt for 2

VTrol.

21:794-731.

20. Toolan, H. W. 1968. The picodnaviruses: H,RV and secretarial duties. Lami, and M. S. Hopkins. 1969. Single-stranded LITERATURE CITED DNAfrom theparvovirus

H-1.

Virology39:617-621.

ogy,vol. 6. Academic Press Inc., NewYork. 1. Berkowitz, S. A., and L. A. Day. 1974. Molecular 21. Toolan, H. W. 1972. The parvoviruses, p.410-425.InF.

weight of single-stranded fd bacteriophage DNA. Homburger (ed.), Progress in experimental tumor High speed equilibrium sedimentation and light scat- research, vol. 16. Kager, Basel.

tering measurements.Biochemistry 13:4824-4831. 22. Usategui-Gomez, M.,H. W.Toolan,N.Ledinko,F. Al-2. Bourgaux-Ramoisy, D., J. Robin, and P. Bourgaux. Lami,and M. S.Hopkins.1969.Single-strandedDNA

1974.ReplicatingDNAof adenovirus type2.Can. J. from theparvovirusH-1.Virology39:617-621. Biochem. 52:181-189. 23. Westmoreland,B.C.,W.Szybalski,and H. Ris. 1969. 3. Bujard, H. 1970. Electron microscopy ofsingle-stranded Mapping of deletions and substitutionsin heterodu-DNA. J.Mol.Biol. 49:125-137. plex DNA molecules ofbacteriophagelambdaby elec-4. Davis, R. W., M.Simon,and N. Davidson. 1971. Elec- tronmicroscopy.Science 163:1343-1348.

tron microscopeheteroduplex methods formapping 24. Wolfson,J.,and D. Dressler. 1972.Regionsof single-regions of base sequencehomologyinnucleicacids, p. stranded DNAinthe growing pointsofreplicating 413-428.InL.Grossman and K. Moldave(ed.),Meth- bacteriophage T7 chromosomes. Proc. Natl. Acad. odsinenzymology, vol. XXI. Academic PressInc., Sci. U.S.A.69:2682-2686.