Polyoma virus minichromosomes: a soluble in vitro replication system.

0022-538X/81/060805-10$02.00/0

Polyoma Virus Minichromosomes:

a

Soluble In Vitro

Replication Systemt

BRIAN B. GOURLIE, MARC R. KRAUSS, ALICIA J. BUCKLER-WHITE, ROBERT M. BENBOW,

ANDVINCENT PIGIET*

McCollum-PrattInstitute and theDepartment of Biology, Johns Hopkins University, Baltimore, Maryland 21218

Received21October 1980/Accepted17February 1981

Polyoma virus minichromosomes were isolated from infected 3T6 cells by

hypotonic extraction of isolated nuclei. The kinetics of in vitroDNA synthesis in

thenuclearextractwassimilartothat observed with intact nuclei.The majority

of theproducts ofin vitro DNAsynthesissedimented with replicative interme-diate (RI) minichromosomes and migratedastwobands (RI-a andRI-b)on 1.4%

agarosegels.Thekineticsofdeoxynucleotide monophosphate incorporationinto

thesespecieswasconsistentwith the existence of several rate-limitingstepsin in

vitro replication by polyoma minichromosomes. Electron microscope analysis

showedthat the RI-a band consisted almost entirely of RI structures ranging

from 46to87% replicated, with one-half ofall structures 67 + 4% replicated.

TheRI-b materialwas morecomplex, consisting ofaandastructureswith tails

ranging from7 to 114%ofpolyomagenomelength and, less frequently, of linked

andmultiplylinked dimeric structures.

As a foundation for the eventual

reconstitu-tion of chromatin replication in vitro, we have

studied thereplicationof isolatedpolyoma

min-ichromosomes. These small, well-defined viral

genomeshave a chromatinstructure similar to

thatof the host (3, 7, 20) and provide anexcellent

model foreucaryotic replication (5, 22, 28, 29).

With theexceptionof the virus-coded Tantigen,

which is responsible for initiating each new

roundofDNAreplication(26), the hostprovides

allof themachinerynecessaryfor viral

replica-tion.

In this paper, wehave characterized

endoge-nous DNA synthesis by isolated minichromo-somesand have shown the accumulationof

rep-licative intermediates (RI-a and RI-b). In the

second paper (13), the electronmicroscope was

usedtoquantitateand characterize DNAspecies associatedwithmature (form I) and

replicating

(RI) minichromosomes. In the third paper (8),

we show that several enzyme activities

impli-cated in DNAreplicationcosediment with

pol-yomaminichromosomes.

MATERIALS AND METHODS

Virus and cells.Asingle-plaqueisolate of a large-plaquestrain ofpolyomaviruswaskindlyprovided by

M. Martin andpropagated atlowmultiplicity (10-3)

onprimarybabymousekidneycells.3T6mouse fibro-blasts were seeded at 2.5 x 106 cells per 150-mm

t Publication 1087 from the McCollum-Pratt Institute.

culture plate (Falcon Plastics) inDulbecco modified Eagle medium (GIBCO Laboratories) supplemented with 5% calfserum (GIBCO)and grown at 38'Cina 5% CO2 atmosphere. Cells were infectedon the 2nd

day after seeding (approximately80% confluency) at amultiplicity of infection of approximately15.

Reagents. HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid), dithiothreitol, phenylmeth-ylsulfonyl fluoride, ethylene glycol-bis(B-aminoethyl

ether)-N,N-tetraacetic acid, and unlabeled deoxyri-bonucleoside 5'-triphosphates were obtained from Sigma Chemical Co. Pyruvate kinase was obtained

from Calbiochem. [3H]thymidine (55 Ci/mmol) and

[a-32P]dTTP(317Ci/mmol)wereobtained from ICN.

[a-32P]dGTPwasobtained fromAmershamCorp. The

restrictionenzymeBglIwasobtainedfrom Bethesda Research Laboratories.

Preparation of polyoma nuclearextracts(23). Twenty-sevenhours afterinfection, cell cultureswere incubatedin vivo either with 20,uCiof[3H]thymidine

(55 Ci/mmol) in 10 ml of Dulbecco modified Eagle

medium for 90 minorwith 100 LCiof[3H]thymidine

in 2ml for5min.Theplates (nowcontaining4x 107 cells andapproximately20 LgofpolyomaDNA)were

rinsed three times with 5 ml of ice-cold hypotonic bufferA(10mMHEPES, pH7.8,5mMKCI,0.5mM

MgCl2,0.5mMdithiothreitol,and1mM phenylmeth-ylsulfonyl fluoride) and drained. The cells were

re-moved with arubber scraper, transferred to an ice-cold Dounce homogenizer, and disrupted with 10

strokes oftight-fittingpestle.Nucleiwerepelletedat

3,000 xg inaSorvall typeSS-34rotorfor 5 min. The supernatantwasremoved,and the nuclearpelletwas suspended with hypotonic extraction buffer (0.5 ml perplate) and extracted at0°Cfor2 h. Nucleiwere

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removed bycentrifugation (6,800xg) for15min, and the supernatant solution of the nuclear extract was

used for further study.

In vitro DNAsynthesis (30).ViralDNAsynthesis wasassayed in a total volume of100,llin the presence of: 40 mMNaCl,5mMMgCl2,5mMethylene glycol-bis(B-aminoethyl ether-N,N'-tetra-acetic acid, 4 mM phosphoenol pyruvate,2 mMATP,3.0IUof pyruvate kinase per ml, and 100,uM each dATP, dCTP, and dGTP.Experiments using [3H]- and [32P]dTTPwere

performedat25ytCi/ml(18 and300Ci/mmol, respec-tively). Nucleiornuclearextract wasadded ina vol-umeof90,dl,and incubationswereperformedat30°C. Duplicateportions of10 or 20 ulwereremoved and precipitated with2ml of cold 10% trichloroacetic acid containing10mMsodiumpyrophosphate. Acid-insol-uble materials were collected on glass fiber filters (WhatmanGF/C) and washed four times with2mlof cold 5%trichloroacetic acid-5 mM sodium pyrophos-phateand oncewith cold95% ethanol. Radioactivity wasdetermined on the dried filters byliquid scintil-lationcounting in4mlof Beckman EP fluor.

Analysis of viral chromatin by sucrose gra-dient sedimentation. Nuclearextracts(0.5ml)were

sedimentedthrough linearsucrosegradients(10ml,5 to20%,wt/vol) prepared in buffer B (10mMHEPES, pH 7.8,5 mMKCI, and0.5mMMgCl2) for90minat

255,000xg inaBeckmanSW41rotor.Fractionswere

collected from thebottom, andportions of each frac-tionwereanalyzed for acid-insoluble radioactivityas

described above. Relative sedimentation velocity of minichromosomes was determined relative to the sed-imentationof form IpolyomaDNA(20S)on aparallel but separatesucrosegradient inhigh-salt buffer C (0.5

MNaCl, 10mMEDTA, and 10mMsodium acetate, pH 6.0).

Analysis of viral DNA by sedimentation on

neutral and alkaline sucrose gradients.After in vitroincubation, viralDNA wasprepared from viral chromatin by the method of Hirt (11).Samples (100 y1)weresedimentedthrough neutral sucrose gradients (3.7 ml, 5 to 20%, wt/vol) in buffer C above a 70%

sucrosecushion (100,l)inbuffer Coralkalinesucrose

gradients which also contained 0.05% sodium

sarcosi-nateand0.3MNaOH. Gradientswerecentrifuged in

a Beckman SW60 rotor at350,000 x g for 110 min (neutral) or 200 min (alkaline). Fractions were col-lected from thebottom, and acid-precipitable radio-activitywasdeterminedas above.

Agarose gel electrophoresis of viral DNA (27). Electrophoresis was performed by using 1.4% (wt/vol) agaroseslabgels (18cmby 19 cm by 3 mm) in 40 mM Tris-hydrochloride,5 mMsodiumacetate, and 1 mM EDTA, pH 7.6. Samples of nuclear extract were

treated with0.6% sodium dodecyl sulfate, 10 mM Tris-hydrochloride, and 10 mM EDTA (pH 7.6) for 20 min

at30°C andelectrophoresed for12to 18 h at 2.7 V/

cmalongwithpolyoma form I and II DNA markers. The gelwasstained with ethidiumbromide (0.5,ug/ ml, 30min), and the fluorescentbands were photo-graphed under UV illumination (254 nm) through a Wratten no. 25 (red) filteronPolaroid type 55 P/N film. Autoradiography wasperformed at 4°C on wet gels, using Kodak X-Omat R film. BglI restriction enzyme digestionofdeproteinized DNA synthesized

bythenuclearextract invitrowasperformedas de-scribed by thesupplier.

Electron microscope analysis of viral DNA formsseparated byagarosegelelectrophoresis.

Sections of 1.4% agarose gels corresponding to the form I, RI-a and RI-b bandswereexcised and dissolved in 5 volumes of saturated potassium iodide, 10 mM

sodiumphosphate (pH 7.0), and 1 mMsodium thio-sulfate.Sampleswerespreadfor electronmicroscopy without furtherprocessing, usingthe aqueousmethod of Davis et al. (la). Every DNA molecule seen was photographedat x20,000magnification withaJEOL JEM 100Belectronmicroscope,projectedwitha

Ni-kon Shadowgraph comparator (total magnification, approximately x200,000), and traced with a Keufel and Esser map measurer. Photographswere printed

onIlfobrom 5.1M resin-coated paper without further

contrastenhancement.

RESULTS

Kinetics of in vitroDNA

synthesis by

the nuclear extract.

Polyoma

minichromosomes

were extracted fromvirus-infected 3T6 cells at a time corresponding to maximal viral DNA

synthesis (27 h

postinfection

in our system),

using a hypotonic extraction procedure

origi-nally described forisolation of simian virus40

(SV40)minichromosomes (22).Thenuclear

ex-tract, preparedfrom cells labeled foreither5 or

90minwith

[3H]thymidine

beforeharvest,

con-tained 10 to20%of thetotallabeledviral DNA

as determinedby theHirt procedure (11). This protocol allowed

preferential

labeling of either

the RI (5-min pulse) or mature (90-min pulse) formIspecies.The efficiencyofextraction was

comparable

to results obtained with SV40-in-fected cells (5, 22), but was less efficient than methods usingTritonandhigh salt (9).

Theability ofnuclearextracts toincorporate

[32P]dTMP was determined by using reaction

conditionsoptimizedforpolyomaDNA

synthe-sis in isolated nuclei (30).

[32P]dTMP

incorpo-rationinto DNA was rapid during the first 5 min and reached a plateau after 10 min (Fig. 1).

Maximalinvitro DNAsynthesisby the nuclear extract (both initial rate andplateau) was

ob-tainedafter extraction ofnuclei for2 h at0°C.

EndogenousDNA synthesis in the nuclear

ex-tract wasstable for at least 8 h and decreased

only 20% after storage overnight at 0°C. The

rateof DNAsynthesis in the nuclear extract was

compared with the rate of synthesis by nuclei

isolated by an isotonic procedure (20). Both

reactions reached a plateau after 10 min. The

initialrateof dTMP incorporation was 0.10 and

1.14pmol/min per plate for the nuclear extract

and the intact nuclear preparations,

respec-tively. Since the nuclear extract contained 10 to 20% of the total polyoma DNA in the cell (based on the efficiency ofextracting DNA labeled in

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E

O 10

z 0

0.

Os

0 5 10 20 30 60

INCUBATION TIME (min)

FIG. 1. Kinetics ofDNA synthesis bythenuclear extract. Nuclearextract(correspondingto3.5x JOr

cells) in 500 ,Il was assayed for endogenous DNA synthesis in thepresenceof [a-32P]dTTP (158x 103 cpm/pmol) asdescribed in Materials andMethods.

At the indicatedtimes, 10-,ld portionswereremoved,

and the amount ofacid-precipitable [a-32P]dTMP wasdeternined.

vivo), the initialrateof DNA synthesis in both

preparationswasroughly comparable, assuming

equal extraction ofarepresentative population

of functionalreplicating complexes (see

Discus-sion). The extent ofdenovo synthesis in vitro

after 10 min canbe calculated fromthe initial

rate (see above) and the amount ofreplicative

intermediates(0.6pmol/plate)asapproximately

120 pmol of nucleotide per mol ofreplicative

intermediate. This calculation isanaverageand

does not discriminate between forks that may

varyintheir relativeefficiency. After hypotonic

extraction ofminichromosomes,nuclei still

con-tained 60 to 80% of the viral DNA labeled in

vivo, but less than 5% of the DNA synthetic

capacity of either the nuclear extract or the

intactnuclei. Thecytoplasmicsupernatant

con-tained the remainderof the totalpolyomaDNA

in thecell (10to20%).

Characterization of the reaction

prod-uctsby sedimentation analysis. The

32P-la-beled productsof in vitro DNA synthesis were

characterized either as viral chromatin or as

deproteinized DNA. The 32P-labeled products

synthesized bythe nuclear extractduringa

30-min incubation sedimented between 50S and

90S, withamajor peakat84Sandashoulderat

56S relative to polyoma form I DNA (Fig. 2).

For comparison, mature polyoma

minichromo-somes (labeledwith[3H]thymidinefor 90minin

vivo) sedimentedatapproximately56S,whereas

minichromosomes labeled in vivo foronly5min

(predominantly RI minichromosomes)

sedi-mented atapproximately 84S (Fig. 3). This

sed-imentation behavior was comparable to results obtained by others using different extraction

conditions for polyoma virus (29) or for SV40

minichromosomes (5, 23).

The 32P-labeled products, deproteinized by

treatmentwith0.6%sodiumdodecyl sulfateand

3

E

4

in

22

z

0

to

0 5 10 15 20

[image:3.495.157.446.76.307.2]

FRACTION

FIG. 2. Characterization of the chromatin product of DNA synthesis in the nuclear extract. After incu-bation ofthe nuclear extract for 30 min with

[a-32P]dTTP (0)asdescribed in Fig. 1, the sample was

sedimented withoutfurther treatment through a neu-tral sucrosegradient as described in Materials and Methods. Radioactivity was determined after acid precipitation of 10 ul of each fraction. The viral DNA

was labeled in vivo with 11H]thymidine (0) for 30 minimmediately before harvest. The arrow indicates thepeakofform Ipolyoma DNA (20S) sedimented on

a separate 5 to 20% sucrose gradient in high-salt buffer C.

E

0

4-0

[image:3.495.259.453.156.304.2]

FRACTION

FIG. 3. Sedimentation of mature (form I) and RI viral chromosomes. Infected cells (3.5 x 107 cells/ plate) were labeled either with 20,uCi of 19H]thymi-dine(55 Ci/mmol) in 10 ml for90min (0)or with 100

,iCi of/5H]thymidine in 2 mlfor 5mmn (0) before

harvest. Portions (500,ul)of thenuclear extracts were sedimented asdescribed in Materials and Methods. Radioactivity was determined after acid precipita-tion of10O,ufor thesample labeled for 90minand the entire 0.5-ml fraction for the sample labeled for5mmi.

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808 GOURLIE ET AL.

1 MNaCl (11), were characterizedby

sedimen-tationonbothneutral and alkaline sucrose

gra-dients. The in vitro DNA productssedimented

in aneutralsucrosegradient slightlyfasterthan polyoma form I DNA (20S) (Fig. 4), which is

consistent with the sedimentation behavior of RI DNA (21). In contrast to the SV40 system

(23), the majority of the invitro-labeled DNA

remained associatedwith the parental genome

whenanalyzed eitheras intact chromatin (Fig.

2) or as deproteinized DNA (Fig. 4).

Sedimen-tation under alkaline conditions of the

depro-teinized, 32P-labeled product of a 30-min

incu-bationinvitrodemonstrated thatthedaughter strandsrangedinsizefrom 16S (full-length lin-earmolecules) to4S (Okazaki fragments) (Fig.

5). Significantamountsof mature form I DNA

(53S) were notobserved.

Characterization

of the reaction

prod-ucts by agarose gel electrophoresis. The

results of agarose gel electrophoresis of

32P-la-beledmaterialsynthesizedin vitro are shown in

theautoradiograminFig.6.Themajorproducts

ofvitrosynthesis appearedas twobands

migrat-ingslower than relaxed circular polyoma DNA

(form II). We refer to these bands as RI-a and RI-b, based on their cosedimentation with RI minichromosomes labeled in vivo (Fig. 3). Su-percoiled Cairns-type replicative intermediates would be expected to migrate as a spectrum

from form I (earlyreplicativeintermediates) to

6

I

0

-4

z 03

0.

0 10 20 30 40 50 60

FRACTION

FIG. 4. Characterization of the deproteinized product ofDNAsynthesisbysedimentation inneutral sucrose.Afterincubation of the nuclearextractfor30

min with[a-32P]dTTP(0)asdescribed in Fig.1,the

sample was treated according to the procedure of

Hirt(11). The extracted DNA wasthen sedimented

on aneutralsucrosegradientasdescribed in

Mate-rials andMethods. Fractionswerecollectedfrom the

bottom, and acid-precipitableradioactivity was de-terminedasdescribedabove. Viral DNAwaslabeled in vivofor30min immediately before harvest with [3H]thymidine (0).

E

Iy

,ss

60

60

z

0.

Ni

40-0

20-0 (0 20 30 40

FRACTION

FIG. 5. Characterization of the deproteinized

productof DNAsynthesis bysedimentationin alka-linesucrose.Aportionofthesamesamplelabeled in vitro and in vivo as described in Fig. 4 was sedi-mentedonanalkalinesucrosegradientasdescribed inMaterials and Methods. Fractionswerecollected from the tube bottom andanalyzedfor32P in vitro

(0)

or3Hinvivo

(0)

label.

a position slowerthan form II

(late

replicative

intermediates)

(16,

25).

Thesame

species

in the

same relative amountswere present in vivo as

determined

by

pulse-labeling

with

[3H]thymi-dine. Therelative

labelings

ofbothRI-aand

RI-b in vitro were also

comparable

when intact

nucleiwereused

(Gourlie

and

Pigiet, manuscript

in

preparation).

Since variations in agarose

concentration,

electricfield, and temperature

differentially

af-fectthe

migration

of

linear,

circular,

and

super-coiled DNA ofthe same molecular

weight (4),

we

investigated

the effect of agarose

concentra-tions from 0.8 to

1.4%

(wt/vol).

The RI-a and RI-bbands maintainedthesamerelative mobil-itiestothe

polyoma

formII

marker,

but became broaderandmorediffuseatlowergel

concentra-tions, indicating thatthere may be further

het-erogeneity withinthebands.

Labeling of the RI-a band wasrapid and

es-sentially

complete after 5 min (Fig. 6). In

con-trast,labeling of the RI-b band increased from

about 20% of the totalincorporationat 5minto

about 40%of the total at60min.Thus,

substan-tiallabelingofRI-b continuedaftertotal

incor-poration had reached a plateau (10 min).

La-beled

form

I DNA accumulated to

approxi-mately3% of the total

incorporation by

60min,

consistent withresultsof sucrosegradient

sedi-mentation (Fig. 5).

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[image:4.495.256.447.65.277.2] [image:4.495.51.246.399.655.2]

Incubation Time

(min)

5 10 30 60

-RI-b

-_

[image:5.495.58.240.69.304.2]

-I

FIG. 6. Characterizationof the products of in vitro DNA synthesis by agarosegel electrophoresis. Nu-clear extract was incubated asdescribed in Fig. 1

with[a-32P]dTTP.Attheindicatedtimes,

50-,ullpor-tionswereremovedfromthe incubation,treated with 0.6% sodium dodecyl sulfate, 10 mM EDTA, and 10 mM Tris-hydrochloride (pH 7.6) for 1 h at 20°C, mixedwith 1/10volumeof 0.05%bromophenol blue, 25 mMEDTA, and 50% glycerol, andapplied to a 1.4%agarosegel. Electrophoresiswascarriedoutas

describedinMaterials andMethods. Autoradiogra-phywasperformedat4°C for14 h.Migration ofform

I and form II DNA is indicated.

Tocomparethedistributionof RI-aand RI-b

DNAs as a function of sedimentation of the

chromatinproducts,agarosegelelectrophoresis

was performed on the DNA from individual

fractions of thesucrosegradient showninFig.2.

Theautoradiogram of this gel (Fig. 7A) showed

the distribution of label incorporated into the

RI-a andRI-bbands.Directquantitation ofthe

radioactivity in the excised bands (Fig. 7B)

showed that RI-a sedimented as a single peak

with anaveragerate of 84S (fraction 9),

corre-sponding to the fraction which contained the

maximum in vitro label. The RI-b DNA, in

contrast, wasdistributed into twopeaks of

ap-proximately equal magnitudeatfractions 9(84S)

and 11 (68S). The ratio of label incorporated

into the RI-aandRI-b bands(Fig. 7C)increased

as afunctionofgreatersedimentationrate, with

RI-abeingthe dominantcomponentin the

sed-imentationregion shown tocontain replicative

intermediates (50to 90S) (see Fig. 3and

refer-ence13).

Characterization of the reaction

prod-ucts by electron microscopy. To determine

thecomposition of the RI-a and RI-b bands,we

excised theseregions directly from a 1.4%

aga-rosegelrun asdescribedin Fig. 7and prepared

theDNA for electron microscopy (see Materials

-RI-i and

Methods).

Of the 22 DNA molecules

ob-served from the RI-aband, 78%were

unambig-I 5 7 9 10 11 12 1316 19

f~

-RI-a

-RI-b

-m

-M

I

B

601

40(

c

0

E

cil

0.

-0

204

E

z

0

FRACTION

FIG. 7. Gel electrophoretic analysis ofDNA in

fractions from aminichromosome sucrosegradient.

Portions (20ul) takenfrom thepreparative sucrose

gradientdescribed inFig.3wereanalyzed byagarose

gel electrophoresisasdescribed inFig.6. Electropho-resiswascarriedoutfor 16 h at 2.5 V/cm.

Autora-diography ofthegel (A)wascarried outfor 4°C for

12days. Bandscorrespondingto RI-a(0) andRI-b

(0)werecut outofthegeland melted with 100,ulof

water for 10 min at 100°C, andradioactivity was

quantified by usingBeckman HPfluor (B). The

dot-ted line represents [3H]thymidine incorporated in

vivofrom Fig. 3. The ratiosof isotope incorporated

into RI-a andRI-bareplottedin C.

0

0

-4

0

3~~~~~~;1

I 2~~~~~~~~~~~I

- - -.---

I~~~~~%-~o ClI\

0 5 10 15

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810 GOURLIE ET AL.

uous9structures. Otherstructuresobserved

in-cluded "connected" monomers (two monomers

joined by a double-stranded linker of variable

length) (6%) and a structures (3%). Figure 8

showsrepresentative micrographs ofthe DNA

moleculesfound in theRI-aband. The putative

replicated regions of the 9 structures ranged

from 46 to87%ofthe total genomelength.Ten

of the 18 unambiguous 9 structures were

clus-tered at 67 +4%replicated. Of the 28 molecules

observed from the RI-bband, 48%were

unam-biguousa structures and 18% wereastructures.

Other structures observedinthe RI-b band

in-cludedlinkedandmultiplylinkeddimers and9

structures.Figure9showsrepresentative

micro-graphs ofmoleculesfound in the RI-b band. As

a control, DNA molecules extracted from the

form I band were monomeric, with fewif any

complexstructures.

Characterization of the reaction prod-ucts by

BglI

restriction enzyme digestion.

We further characterized the 32P-labeled

reac-tionproducts afterdigestionwiththerestriction

endonuclease BglI. Cleavageofpolyoma DNA

occurs once near the origin ofreplication (10)

andwould convertreplicativeintermediatesinto

H-likestructures.Restrictiondigestionwas

per-formed on the DNA from a sample ofnuclear

extractincubated in vitro with[a-32P]dGTPfor

30 min. Theautoradiogram of this material after

electrophoresis on a 1.2% agarose gel (Fig. 10) shows a gradation of radioactivity extending

fromthe migration position of late Cairns species

totheearliest Cairnsstructuresmigratingclose

to form III. A second spectrum ofreplicative

intermediates wasapparentmigratingin a range

intermediate between the late Cairns species

andformIII. Thisspectrum represents

replica-tive intermediates that hadreplicatedina

uni-directed manner and assumed aY-type

config-uration after digestion with BglI

(Buckler-White, Krauss, Benbow, and Pigiet, submitted for publication). Molecules comigrating with

form IIIincluded all double-stranded linear

mol-ecules produced from cleavage of form I, form

II,oranyconcatenated dimers. Thispatternof

isotope incorporation revealed a spectrum of

replicativeintermediates and was comparable to

that obtained for DNA labeled in vitro with

[ax32P]dGTP

bynuclei prepared by an isotonic

method(15) or DNAlabeled in vivo with

32Pi.

DISCUSSION

We have demonstrated that

minichromo-somespresent insoluble nuclear extracts

incor-porate deoxynucleotide monophosphates into

polyoma DNA in vitro. The distinctive advan-tage of the soluble system lies in the ability to

isolate mature and replicating

minichromo-somes (50 to90S) as intactnucleoprotein

com-plexes and toidentifyindividual DNA molecules

and enzymeactivitiesassociated with these

com-plexes.

Interpretation of studies usingisolated

mini-chromosomesdependsonunderstandingthe

na-tureof thematerialsextracted and whetherthey

arerepresentativeof the minichromosomes

pres-ent in the intact cell. The lower rate ofDNA

synthesis by the nuclearextractrelativeto

iso-latednuclei(about10%)raises several

questions

about the

synthetic capacity

of the

minichro-mosomes. Thesimplest interpretationis that the

low

incorporation

of

deoxynucleotide

mono-phosphates by the nuclear extract reflects the

low yieldof extraction ofreplicating

minichro-mosomecomplexes. This argument issupported

bytheinitialrateofdeoxynucleotide

monophos-phateincorporation,which wasroughly

propor-tionaltotheefficiency (10to20%)of extraction. Thesyntheticrateofnuclearextracts was

com-parabletothesyntheticrateobserved innuclei afteradjusting for this extraction

efficiency.

The

validityofthisargument rests on theassumption

thattheextracted minichromosomesare

repre-sentative of the total

population

of functional complexesinnuclei.

The retention of labeled viral DNA in the

nucleus, even after extensive extraction with

hypotonicorhypertonic detergent buffers (1), is

consistent with the finding that substantial

amountsofviral DNA aretenaciouslybound to

the nuclear matrix. If the matrix is the site of

some stages of viral replication,

minichromo-somes may represent a stage with no

require-mentfor matrixattachment.

Alternatively,

ifall

stages of replication take place on the matrix

(19), minichromosomes may represent species released from cellsbyourmanipulations. Itwas

ofinterest that

approximately

10%ofthe

mini-chromosomes were associated with amorphous material (possibly matrix components) even

afterhigh-saltextraction (13). In any event, the

isolated minichromosomes are likely to prove

especiallyvaluable for identification of the

pro-teins involved in replication even if the DNA

synthesiscarried out invitro is not

fully

repre-sentative of events inside the nucleus.

Sedimentation analysisof the products of in

vitro DNA synthesis by the nuclear extract

showed that themajority of labeled DNA

sedi-mented with the same distribution

(approxi-mately 84S) as the viral minichromosomes

la-beledduringashort (5-min) pulsein vivo (Fig.

2 and 3). The smaller amount of in vitro label

which sedimented with mature

minichromo-somes (Fig. 2) may correspond either to early

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FIG. 8. Electron microscopeanalysis of DNA molecules comigrating with the RI-a band on agarose gels. TheRI-aband was excised from an agarose gel, dissolved in potassium iodide, and prepared for electron microscopy asdescribed in Materials and Methods. (A) B Structure where the putative unreplicated region measures 0.90 um andthe replicated arms measure 0.76 and 0.76

tum.

(B) BStructure where the putative unreplicated region measures 0.80pm and the replicated armsmeasure 0.90 and 0.85

tum.

(C) B Structure

wheretheputative unreplicatedregion measures 0.60pmand thereplicatedarmsmeasure 1.03and 0.99 ,um. (D) BStructurewhere theputativeunreplicated region measures 0.59pmand thereplicated arms measure 1.10 and 1.06

tum.

(E) B Structure where theputative unreplicated region measures 0.57pmand thereplicatedarms

measure 1.17 and 1.16pm.(F) BStructure where the putative unreplicated region measures 0.49pumand the replicated arms measure 1.20 and

1.19ptm.

Molecules A, B, C, D, and F were printed at the samemagnification;

molecule E wasprintedat25% higher magnification. Themarkerscorrespondto 0.5pm.

811

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812 GOURLIE ET AL.

FIG. 9. Electronmicroscopeanalysisof DNA comigrating with theRI-bbandonagarosegels. The RI-b

band was excisedfromanagarosegel andpreparedforelectron microscopy as described inFig. 8. (A) a

Structure where the circlemeasures 1.70

Aim

and thetailmeasures 0.66,um. (B)aStructure wherethecircle

measures 1.56,Im and the tail measures 0.99jim.(C)aStructurewhere the circle measures 1.64jimand thetail

measures 1.87jim. (D)aStructure where the circle measures 1.70jimandthetails measure 0.51and0.22jim. (E) Linked dimerstructurewherethe two circles measure 1.74 and 1.62jim.(F)Multiplylinked dimer structure where the circles measure 1.61 and 1.59 jim. Molecules A, B, C, D, and E were printed at the same

magnification;molecule Fwasprintedat25%highermagnification. The markerscorrespondto0.5jum.

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FIG. 10. BglI digestion oftheproducts ofin vitro DNA synthesis. Nuclear extract, incubated in vitro

for60mmnwith[a_3'P]dGTP,wasdeproteinizedand

digested with BglI as described in Materials and Methods. Electrophoresis in 1.2% agarose was

per-formnedat5 V/cmfor18hat4i C.Migration of form

IIIand late Cairns(LC)structuresisindicated.

replicativeintermediatesasdetectedbyelectron

microscopy (13) or to termination products

re-semblingmatureform I DNA(14, 17, 24).

Com-parisonofFig. 3and4showsthatsedimnentation

of viral minichromosomes resolved the

replica-tive intermediates from the mature forms far betterthan sedimentation ofdeproteinizedDNA

(Fig.4).

Agarosegelelectrophoresis of theproductsof DNA synthesis in vitro demonstrated that the

majority oflabel appeared astwo bands (RI-a

and RI-b), each migrating slower than form II

polyoma DNA (25), and that these formswere

also present in RI DNA pulse-labeled in vivo. Thecomplexcontributions of molecularweight, superhelicity, and topology to the behavior of DNAsonagarosegels(4)complicateanycertain identification of these bands at this timne. Evi-dence that they are members of different

con-formnational families (24) is supported by the increased band width observedatloweragarose

concentrations and is consistent with the spec-trum of intermediates seen in the BgllI digest

pattern (Fig. 10) and the electron microscope

evidence discussed below. The kinetics of

label-ingof the RI-a and RI-b bands is consistent with RI-a being an initial product, rather than the

result of conversion from RI-b. In contrast to

the rapid labeling of RI-a, the slower rate of

labeling of RI-b is consistentwithconversionof some RI-a into RI-b and/or conversion from

early replicative intermediates migrating as a

continuum from formI tothe late Cairns posi-tion. The appearanceof RI-b in tworegions of

the sucrose minichromosome gradient (Fig. 7)

mayindicate thatbothof thesepossibilitiesare

true.

The results obtained byelectronmicroscopy

(Fig. 8 and 9) suggest that the RI-a band

con-sisted almost entirely ofunambiguous 6

struc-turesrangingfrom46 to 87% replicated with a

modal value of 67 + 4%

replicated.

The RI-b

band was more complex, consistingof a and a

structures which either could have arisen by

breakage of 9 structures or could have been

generated by a

rolling

circle mechanism and branchmigration. In addition, linked and mul-tiply linked

dimeric

species reminiscent of the

conformational families described by Sundin

andVarshavsky (24) werealsoseen,althoughat

lowerfrequencies. Althoughwecannot

unequiv-ocally

ruleoutthepossibility that RI-aand

RI-b correspond to the 80 and 90% late Cairns species observedinSV40-infectedcells(25), the

kinetics oflabeling and sedimentation properties ofRI-aandRI-b,aswellasthe electron

micros-copy, render this possibility extremely unlikely

for polyoma-infected cells. The results of this

paper areconsistentwith, butdo not prove,the

hypothesis that incorporation into RI-b is due to either breakage ofRI-a or maturation into topologically linked speciesasdescribedby

Sun-din and

Varshavsky

(24), orboth.

Mature form I polyoma DNA was not

pro-ducedinvitroto anappreciableextent,

amount-ingtoless than 5%of the total incorporation by

60 min (Fig. 7A). This lack of conversion into

form

I

molecules,

as observed

by

others for

intactnuclei (2,6,25) andsoluble

minichromo-somesystems (5, 22) maybe due tothe release

of factors essential for termination intothe

cy-toplasm during nuclearisolation.

ACKNOWLEDGMENTS

We acknowledge the contributions of Glen Humphrey, CarolBreaux, and MarvinBaynefortheircritical evaluation of ourworkandmanuscript.We alsoacknowledgethe contri-butionsofMitchellK.Hobish,who initiated thisstudy.

This research was supported by Public Health Service grantsGM-23813 and GM-23970 andpostdoctoral fellowship

CA-06304toB.B.GfromNationalInstitutesofHealth.

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