Replication of polyoma DNA in isolated nuclei: analysis of replication fork movement.

VIROLOGY, 386-393 0022-538X/79/11-0386/08$02.00/0

Replication

of

Polyoma

DNA in

Isolated Nuclei:

Analysis of

Replication Fork Movement

GORAN MAGNUSSON* AND MAJ-GRETH NILSSON

MedicalNobel Institute,Departmentof Biochemistry,Karolinska Institute, Stockholm,Sweden

Received forpublication20March 1979

Themovementofreplication forks during polyoma DNA synthesis in isolated nuclei was analyzed by digesting newly synthesized DNA with the restriction

endonuclease HpaII which cleaves polyoma DNA intoeight unique fragments. Theterminus of in vitro DNA synthesis was identified by cleaving newly

com-pleted moleculeswithHpaLL.Thedistribution of labelintherestriction fragments showed that the in vitro DNA synthesis was bidirectional and had the normal

terminus of replication. Analysis of replicative intermediates pulse-labeled in vitrofurthersuggested that DNA synthesis in isolated nuclei isanorderedprocess

similartoreplication in intact cells. Replication forks moved withaconstant rate

from theorigin towards the terminus ofreplication. The nonlinear course of the

DNAsynthesis reaction in theisolated nucleiseems to resultfrom therandom inactivation of replication forks rather than a decrease in the rate of fork movement. During the in vitro synthesis a replication fork could maximally

synthesize a DNA chain about 1,000 nucleotides long. Theresults suggest that somereplication forks might be initiated in vitroatthe origin of replication.

The replication of polyoma DNA has been

studied in vitro with nuclei isolated from

in-fected cells. In this system adetailedpicture of the process for elongation of DNA chains has

beenobtained(6,15).Itwasinitially established

(13) that the nuclei supported the

semicon-servative replicationofsubstantial segments of

the viral genome. However, no DNA strands

werefoundthat had beensynthesizedfrom start

tofinish in theisolated nuclei.Furthermore,the

analyticalmethodsused in that and

subsequent

studies did not allow a determination of the

regionsof the viralgenome in which DNA was

synthesized.

Hereweanalyze newly

synthesized

DNA by the use of restriction endonuclease

cleavage. Cleavageof DNAwith restriction

en-donucleasesallows thestudyofeventsin

specific

partsofagenome.The restrictionendonuclease HpaII from Haemophilus parainfluenzae

cleaves polyoma DNA into eight unique frag-ments that can beseparated bygel

electropho-resis. The eight fragments have been ordered into a

physical

map by Griffin et al. (7). DNA

synthesis starts at auniquesitein thegenome,

and from there the

replication

forksproceed in both directions until the two forksmeet atthe terminus ofreplication, which is 1800 from the

originonthe circular map (3).

Tostudy the rate and extent of DNAsynthesis

inspecific regions of thepolyoma genome, viral

DNA waslabeled during the incubation of the isolated nuclei and then cleaved with HpalI.

The amount of radioactivity in the separated

restrictionfragments was then used to measure the numberofreplicationforks that had moved

throughrespective fragment duringthelabeling

period.

MATERIALS AND METHODS

For many of thedetailsconcerning cells, virus,and

generalmethodology,previouspublications should be

consulted(11,15, 19).

Cells andvirus.Growingcultures of 3T6 cellswere

infected atamultiplicityof20to50PFU/cell. Nuclei wereisolatedatabout26hpostinfection.Thepolyoma viruswas of the A2 type (7) of the Pasadena large-plaquestrain. Virusstockswereprepared byinfecting primarymousekidneycellsatamultiplicityof about

0.01 PFU/cell with repeatedly plaque-purified virus.

Chemicals. ['H]thymidine (20Ci/mmol) and [a-2P]dGTP (100 Ci/mmol) were obtained from New England Nuclear Corp.

Prelabeling of DNA with [3H]thymidine.

[3H]thymidinewasadded directly to the medium to a

final concentration of 1

MM

(20

pCi/ml).

After about

4 h, the labeling was terminated by removing the medium andrinsingthecellmonolayertwice with ice-coldTris-bufferedsaline. The cellswereimmediately used forpreparationof nuclei.

In vitro synthesis and purification of DNA. Nucleiwerepreparedexactlyasdescribedbefore (15).

After se(dimentation

the nuclei were suspended in 1 volume of isotonic buffer. For in vitro synthesis of DNA, four parts of the nuclei suspension were mixed withone part of buffer containing thestandard com-ponents of the reaction. Incubations were done at

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POLYOMA DNA REPLICATION 387

25°C and werestoppedbythe addition of5volumes of50mMTris-chloride (pH8.0)-10 mM EDTA. The nuclei were lysed bythe addition of sodiumdodecyl sulfate to lVi; and NaCl to 1 M. Viral DNA was selectively extracted and purified as described, includ-ing centrifugation through neutral sucrose gradients (9, 12). For purification of closed circular (form I) DNA,chromatographyonbenzoylated-naphthoylated DEAE-cellulosewasused(19).

Restriction endonuclease digestion of DNA. The restriction endonuclease from H.parainfluenzae (Hpall) was purified asdescribed by Sharp et al. (16). DNA was concentrated by ethanol precipitation be-foredigestion. The reactions were carried out at 37°C for2h in10mMTris-chloride(pH7.5)-10 mM MgCl2 and 1mMdithiothreitol. The reactionswerestopped by the addition of sodiumdodecylsulfateto0.5%. The sampleswerestored frozen andwereheatedto 50°C for15min beforegel electrophoresis.

Gel electrophoresis. Electrophoresis wascarried out incylindricalgels(6by150mm)consisting of 2.9% acrylamide,0.15%bisacrylamide,0.5%agarose in 0.09 MTris-borate buffer (pH8.3),0.0025MEDTA, 10% glycerol, and 0.5% sodium dodecyl sulfate (14). The gels were run at 110 V for 7 h. The gelswere then fractionated withaGilsongelslicer andanalyzedfor radioactivity.

RESULTS

Origin and

direction

of polyoma DNA

synthesis

in vitro. For the

analysis

of the

progression of

replication

forksthrough the

pol-yoma genomeduringDNAsynthesisin isolated

nuclei, it was necessaryto establish that DNA

replication in vitro had the same origin and

directionofsynthesisasthe invivoprocess. For

thispurposeweusedthe

technique

describedby

Danna and Nathans (4).

Replicating

DNA is

radioactively labeled,and onlymature

(form

I)

DNA molecules are examined. After a short

labelingperiod,the DNAwill

only

belabeledat

the terminus of replication. By

increasing

the

pulse

length,

replicating molecules,

whichatthe

start of the

labeling

period were further from

completion,

will have timetofinish

replication.

In thisway

radioactivity incorporated

into

com-pleted molecules willform a

gradient

from the

terminus towards the

origin

of

replication.

This

gradient can be determined by cleavageofthe

labeledDNAwith arestrictionenzyme and

mea-surement of the

radioactivity

in each

fragment.

Nuclei from

polyoma-infected cells,

prela-beled forseveral hourswith

[3H]thymidine,

were incubated with

[a-32P]dGTP

under standard conditions for 10 or 30 min. Total viral DNA

was applied to

benzoylated-naphthoylated

DEAE-cellulose, and form I was elutedwith 1

M NaCl and further

purified

by centrifugation

in

CsCl-propidium

diiodide

density

gradients.

From the nuclei incubated for 10 min, 1.5% of the total 2p

radioactivity

in viral DNA was

recovered as form I. The corresponding value for the material isolated after 30 min of

incuba-tionwas3.7%.

FormIDNAfromthe twosamples was then

cleaved with HpaII, and the digests were

ana-lyzed bygel electrophoresis. The 32P

radioactiv-ityof each DNAfragmentwasnormalizedtothe size ofthefragmentby using the

'H

radioactivity

asaninternalstandard. InFig. 1 the specific

:2P

radioactivityforeachfragmentisshown.Inthe

figurethe physicalmapofthe circularpolyoma

DNA molecule hasbeen linearized by opening it at the terminus of in vivo replication. The molecules which had completed a replication roundduringthe first 10min of incubation were

labeled exclusively in fragments2 and 6. After

30 min of incubation some radioactivity was present in fragments 1 and 7 in addition to

fragments2and 6. Itis clear that thetermination

site in vitrowasin thesameregion of the genome as invivo (3). Furthermore, the distribution of

radioactivity in the fragments was

symmetri-cally arranged around theterminus,suggesting

that the DNA synthesiswasbidirectional. Only

insignificant amounts of radioactivity were

foundinfragments 3 and 5attheorigin site for

DNA replication, located at the junction

be-tween the two fragments. Consequently, the

18

.

SH2I

0.

2

2 78 4 5 3 1 6

Fragment Order

FIG. 1. Distributionofradioactivityin newly com-pletedform I DNA molecules. Nuclei isolated from cellsprelabeled with[3H]thymidinewereincubated understandardconditions with[a-32P]dGTPfor10 or30min. Viral DNAwasextracted, andformIDNA was purified and digested with HpaII restriction endonuclease. The resulting restriction fragments wereseparated bygelelectrophoresis. The3H radio-activity from the in vivo prelabeling and the 32p radioactivity incorporated during the in vitro incu-bationweremeasured,andthe32P/3Hratioforeach fragmentwascalculated. This ratioisplottedversus respectivefragmenton alinearizedphysical mapas determined by Griffin et al. (7). Symbols: O---O, 10-min incubation; -, 30-min incuba-tion.

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datashow that DNAsynthesisin isolated nuclei isanorderedprocessinwhich replication forks progress inthe samedirection as inreplication

in intactcells.

Flow ofreplication forks duringinvitro

polyoma DNAsynthesis.In theprevious

sec-tion,form I DNA molecules that hadbeen

com-pleted in vitrowereanalyzed. A similaranalysis

wasdone withthe total viral DNAsynthesized

-in the nuclei. As mentioned above, more than

95%of the invitro-labeledDNAwasreplicative

intermediates; therefore, the contribution of la-belfrom form I DNAwasignoredinthis

exper-iment.

Cellswereprelabeledwith[3H]thymidine be-fore isolation of nuclei. The nuclei were then

incubatedfor 10, 20, or30min. The

non-radio-active nucleotidesdATP,dCTP,and dTTPwere present at 50 MM concentration, whereas [a-32P]dGTP waspresent at5,uM.Reactionswere

stopped by the addition of buffer containing EDTA and sodium dodecyl sulfate. The viral DNA was then selectively extracted and

puri-fied.

To analyze into what regions ofthe genome

[a-:2P]dGTPwasincorporated duringthecourse of the reaction, the purified viral DNA was

cleaved with HpaII restriction endonuclease. After the digestion, the DNA fragments were separatedby electrophoresis (Fig. 2A, B,andC). The ;3H label introduced in vivo, serving asan internal standard,wasrecoveredinsevenpeaks

corresponding to HpaII fragments 1 to 7

(frag-ment8ranoff theend of thegels).The amount ofradioactivityin each ofthesepeakswas

pro-portionalto the size of the restrictionfragment (7). The ;12P radioactivity showed a different

profile.Itwasrecoveredatpositions correspond-ingtoeach of theHpaIIfragments.Inaddition, 30to 40% of the 32P-labeledDNA remained at the top of thegels, havinga mobility less than that of HpaII-1. The relative amount of this material decreased during thecourse ofthe re-action. In otherexperiments theslowly migrat-ingmaterial could beeliminated by followinga

pulse-chase protocol (data not shown). These resultssuggest that the radioactivity atthetop of the gels and between the positions of the

J. VIROL.

restriction fragments consisted of branched DNA molecules containing replicationforks be-tween two restriction sites andtherefore had a

largermassthan the normallinearfragments. It

is also shown below (Fig. 5) that the DNA

re-coveredfrom thepeaks had full fragment length. To quantitate the synthesis of the different restriction fragments, 32P/3H ratioswere

calcu-lated for uncleaved DNA and for each of the

fragments after correction for the 32P

back-ground-levelpresentbetween the peaks of radio-activity (Table 1). The incorporation of:2P-label

into total DNAfollowedanonlinear timecourse

10 30 50

FradN

FIG. 2. HpaIIcleavagepatternofinvitro-labeled DNA. Viral DNAprelabeled with[3H]thymidinewas

further labeled in vitro with[a-32P]dGTPfor10(A),

20(B),or30min (C). The purified DNAwasdigested withHpaII, andtheresultingfragmentswere

sepa-ratedbyelectrophoresis in polyacrylamidegels.

Sym-bols:0--0,3H;* 32p.

TABLE 1. Distributionofradioactivityin HpaIIfragments of in vitrosynthesizedDNA" 12P/Hradioactivity (x 10) ratio

Labeling period

(min) Total HpaII-1 HpaII-2 HpaII-3 HpaIl-4 HpaII-5 HpaII-6 HpaII-7

0-10 2.78 0.61 0.51 0.85 0.95 1.80 0.79 1.16

0-20 4.40 1.28 1.13 2.24 2.19 2.65 2.01 3.24

0-30 5.00 2.00 1.83 2.75 3.25 2.83 2.79 4.22

"The32P/'Hratiosarecalculated from theexperimentshown in Fig: 2.

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POLYOMA DNA REPLICATION 389

as

previously

observed (19). The

32P/3H

ratios

of the restriction

fragments

were in all cases

lowerthan thecorresponding ratio for uncleaved

DNA. This result merely reflects the loss of

material containing forksfrom the positions of

the restriction fragments in the gels. That

HpaII-l,

for

example,

showedalowerrecovery thanthe much smallerHpaII-7 is also consistent

with thisnotion because alarger fragment has

ahigher

probability

of

containing

a

replication

fork than a small one. In general the

32P/3H

ratios increasedalmost linearly with time during

the incubation period. This increase probably

represented the composite effect of

incorpora-tion of labeled nucleotides into DNA and an

increaseof the

radioactivity

recoveredinlinear

restrictionfragments. One exceptiontothis

pat-tern was HpaII-5 that had by far the highest

:32P/^H

ratio afterthefirst 10 min ofincubation,

but then increased only about 1.5-fold during

the continued reaction. The resultsuggeststhat

early during the in vitro reaction, there was a

relative abundance ofreplication forks close to

theorigin ofreplication, locatedat thejunction

betweenHpaII-3 and-5,and thatthisregion of

the genome later was depleted of replication

forks. One temptinginterpretation of the result

is that initiation of DNA synthesis occurs in

vitro, but that the process is inactivated faster

than the

elongation

of DNAchains.

Toget a moresensitive deternination ofthe movement of replication forks, an experiment

similartothepreviousonewasdone. However,

inthisexperiment the radioactive labelwasonly

introducedduring the last10minof the reaction,

instead of

continuously

from thestart. The

re-actionswerestarted in thepresenceof unlabeled

nucleotides, dATP, dCTP, and dTTPpresent at

50

,uM

concentration,

and

dGTP

at5 ,uM

con-centration.

During

a

10-min

pulse (0

to10, 10 to

20, and20to30min,

respectively),

[a-YP]dGTP

was addedto afinal dGTP concentration of10

,uM.

Analysis

of the viral DNA

by

sedimentation

through

alkalinesucrose

gradients (Fig.

3Aand

B) showed that the3H

radioactivity

introduced

in vivosedimented intwo

peaks:

a

major

peak

at53S

(form

I

DNA)

andaminor

peak

at 16to

18S

(form

II

DNA).

The

3P

label from the0-to

10-min

pulse

(Fig. 3A)

appeared

as an

asymmet-ricpeak

extending

froma

position

corresponding

to

full-length

viral strands to the top of the

gradient. The

3P

label from the 20- to 30-min

pulse (Fig. 3B) sedimentedas a

relatively sharp

peakatabout 16S anda

second,

smaller

peak

at the top of the

gradient.

The material in this

second peak may have consisted of "Okazaki

fragments,"

newly

initiated DNA

chains,

or

sim-P

.X B

0

It

II

10 20 30

Fra onumber

FIG. 3. Sedimentation in alkaline sucrose gra-dients ofviral DNApulse-labeled in vitro. Nuclei isolated from cells prelabeled with [3H]thymidine

wereincubatedfor either10(A) or 30 min (B), with

[oa-32P]dGTP

presentfrom0 to 10(A)or20 to30min (B) after thestartof the incubation. Viral DNA was selectively extracted andpurified,andportionswere sedimentedthrough alkaline 5 to20% sucrose gra-dients.Symbols:O--_O,3H;H , 32 p.

ply

degradation products.

The

32P-labeled

viral

DNA from the 10- to20-min

pulse

hada

sedi-mentation

profile

intermediatebetween the

pro-files shown in

Fig.

3.Thesedimentation

profiles

show that the average chain

length

increased

during the reaction. The label in the chainswas

introduced

during

the last part ofthe

incuba-tions and thusreflected the

synthesis

of DNA

behind

replication

forks that remained active

during the reaction. Since

mainly long

DNA

chainswere

synthesized during

the laterpartof

the

reaction,

it is

again

clear that the isolated

nuclei

largely

support chain

elongation.

Asinthepreviousexperimentthe viral DNA

was cleaved with HpaII restriction endonucle-ase, and theresulting digestswere analyzed by

gelelectrophoresis.Weonlyshow thegel profiles

from the0- to 10-min and 20- to 30-minpulses

(Fig. 4A and B). They were similar to those

shown in Fig. 2. Ratios of

32P/3H

radioactivity

were calculated for uncleaved DNA and the VOL. 32,1979

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individual restriction fragments (Table 2). The results show the decreasing rate of the overall reaction. Therecovery ofHpaIIfragments 1 to 7, labeled during the 0- to 10-min pulse, was

similar towhatwasfound previously(Table 1).

Looking at the rates ofsynthesis of individual

fragments, they generally decreased in parallel

with the overall rateofsynthesis. One obvious

exception was the synthesis of HpaII-5. It startedatahighlevel,and then(during20 to 30

min) decreased toabout 10% of the initialrate. However,relativetothe otherfragments,

HpaII-5hadanormalrateofsynthesisduring20 to 30

minafter thestartof the incubation.

Does initiation ofnew replicationforksoccur

50 -A

306r

20 40 60

Fraction number

FicG. 4. HpaII cleavagepattern ofin vitro pulse-labeled DNA. Viral DNA synthesizedin t'itrofor 10

(A) or-30min (B)asdescribed in thelegendtoFig.3 uwas cleaved with HpaII restriction endonuclease. The cleaved DNA uwas electrophoresed through poly. acrylamide gels.

in isolated nuclei? The high rate of fragment

HpaII-5 synthesis early during the in vitro

re-actionssuggeststhat initiationmightoccur,but toanswerthequestionweneedtoknow whether

HpaII-5was completely synthesized during the in vitro reaction. In the previous section we

argued that the labeled DNA that contained

replication forks between two restriction sites

were not recovered in the positions of normal restriction fragments. If this notion is correct, labeled restriction fragmentsisolated from gels

should be of fulllength. Theextentofsynthesis

of thefragmentsin vitrocould be determinedby usingadensitylabel,in additiontoaradioactive label, duringthe in vitro incubation.

Anexperimentsimilartothepreviousone was

done. Viral DNA was density labeled with

bro-modeoxyuridine triphosphate instead of dTTP

throughout 10 or30 min ofincubation and

ra-dioactivelylabeled with[a_-12p]dGTPeither

dur-ingthe 0 to 10or20 to 30 minafter the startof the incubation. The DNA was cleaved with

Hpallrestriction endonuclease, and the

result-ing fragments were separated

electrophoreti-cally,locatedbyCerenkov radiation,eluted

elec-trophoretically from the gels,and concentrated

byethanolprecipitation.

Only the results of the analysis offragments

Hpall-3 and -5 are shown, but similar results

were obtained withHpaII-6and-7.

The fragments were first analyzed by sedi-mentationthroughalkalinesucrosegradients.In

Fig. 5 profilesofHpaII-3 (AandB) and

HpaII-5 (C and D) labeledduring 0 to 10 min (A and C) or 20 to30 min (B and D) after the start of nuclei incubationare shown. DNA labeled

dur-ing0 to 10 minsedimentedassharp peaks. The bulk of the:2p-labeledmaterial hadasomewhat increased sedimentation velocity because of its increased density resulting from bromouracil substitution (8).It is clear thatmostofthe:12p_ labeled DNA chains had the full length of the restriction fragments. However, inthe material labeled between20 and 30min(Fig. 5Band D) about 20% ofthe[32P]DNAwasshorter than full length.

The degree of bromouracil substitution in

HpaII-3 and -5was then measuredby

centrifu-T'ABLE, 2. DistributionofradioactiuityinHpaIIfragments ofDNApulse-labeledin vitro" .2p/3H radioactivity(x 10)ratio

Labelingperiod

(min)

TDNal

Hpall-I

HpaII-2

HpaHI-3

Hpall-4 Hpall-5 Hpall-6 Hpall-7

0-10 1.01 0.21 0.15 0.33 0.34 0.61 0.20 0.57

10-20 0.50 0.10 0.08 0.16 0.15 0.18 0.12 0.30

20-30 0.32 0.07 0.05 0.08 0.09 0.08 0.09 0.22

The 12P/H ratiosarecalculated from theexperimentshown inFig.4.

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POLYOMA DNA REPLICATION 391

Fracon nmber

FIG. 5. Sedimentation in alkaline sucrose gra-dientsof restrictionfragments HpaII-3andHpaII-5 fronm DNApulse-labeled in vitro. Isolated nuclei were incubatedasdescribed in thelegendtoFig.4for10 (A and C) or 30 min (B, D). Bromodeoxyuridine triphosphate waspresent throughout the reactions, and[a-32P]dGTPwasaddedduringthe last10min of the incubationas apulse-label. After digestion of viral DNA with HpaIIand separation ofthe DNA fragments,HpaII-3(A andB)andHpaII-5 (CandD) uere isolated andsedimentedthroughalkaline5to 20%/r sucrose gradients (6 h at 55,0f0 rpm at 4°C in a Beckman SW56 rotor). Symbols: O---O, 3H;

v v 32p

gation in alkaline cesium sulfate density

gra-dients (Fig. 6A, B,C, and D). The DNA labeled

with 3H invivoformedsymmetrical peaks

serv-ing as internal references of

buoyant

density.

HpaII-3 DNA(Fig.6AandB) formednarrower

peaks

than

HpaII-5

DNA

(Fig.

6C and

D),

as

expected

from the difference in molecular

weight.HpaII-3 (Fig. 6A)andHpaII-5 (Fig. 6C)

labeled with

12P

during the first 10min ofthe

reaction had a

higher buoyant density

and

formed somewhat broader

peaks

than the

'H-labeled DNA.Thepeak values of

buoyant

den-sity were increased

by

20

mg/cm:

for HpaII-3

and 37

mg/cm3

for

HpaII-5.

After 30 min of incubation the

"2P-labeled

DNA formed

sub-stantially broader peaks (Fig. 6B and D) with

peak valuesof

buoyant density

increased

by

58

mg/cm3forHpaII-3 and54

mg/cm'

for

HpaII-5. Knowing the

adenine-thymine

contentofthe

tworestriction

fragments (7),

the

buoyant

den-sityinalkaline cesium sulfategradientsofDNA

with complete substitution of thymine by

bro-mouracil can be calculated (1, 10, 13) to be 83 and 79

mg/cm',

respectively, for HpaII-3 and

HpaII-5.

The bromouracil substitution in

HpaII-3

and-5

synthesized during

0 to 10 min was

consequently

24and47%, respectively.The

corresponding

values for DNA labeledduring20

to 30min were 70and68%. We believethat 70% representfull substitution underour

experimen-talconditions, since no fragmentwe tested,

in-cluding the small HpaII-7, hadahigher degree

of bromouracil substitution.

From the length of HpaII-3 and -5 (890 and

410nucleotides, respectively), therateof chain

elongation can be estimated to about 300

nu-cleotidesduring the first10min.During thenext

20min,the number of newly synthesized

nucleo-tides inHpaII-3 had increased to890, whereas

the corresponding value for HpaII-5 was 400,

representing the full length of the fragments.

These values are corrected for a maximal

bro-mouracil substitution of 70%. Fromthese

consid-erationsitfollows that therateofDNAsynthesis

in the isolated nuclei is linear, when only the

replication forks that remain active are

meas-ured. These

replication

forksseem tobeableto

synthesizeaDNAchainatleast 900nucleotides

long which isin agreementwith theexperiment

presented inFig.1.In

addition,

the datasuggest

the

possibility

that some

replication

forks are

initiated invitro, sincetheyareactively

synthe-sizing

DNA within 400 nucleotides from the

origin of

replication

aslateas20 to30minafter

thestartofthereaction.

I

I

I

I

1020 3040 10 2030 40

FacUmmdm*

FIG. 6. Equilibriumcentrifugationinalkaline ce-siumsulfate gradientsofrestrictionfragments from DNApulse-labeledin vitro. Therestriction endonu-cleasefragmentsHpaII-3(AandB)andHpaII-5(C andD), generated fromDNA labeled with bromode-oxyuridinetriphosphate during 10(A and C) or30 min (B and D) incubation of isolatednuclei and radioactively labeled as described in the legend to Fig. 4, were centrifuged to equilibrium in alkaline cesium sulfate (1.45g/cm3). Centrifugation wasfor 120h at25,000 rpm at20°Cin aBeckman SW50.1

rotor. TheApvalues in thefiguredenotethe increase in buoyant density calculated from refractive index measurements.Symbols:0---0,3H;* 32P. VIOL. 32,1979

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392 MAGNUSSON AND NILSSON

DISCUSSION

DNAsynthesis ineucaryoticsystemshas been studied in detail with cells infected with papo-vaviruses. The viral genome whichconsists of a

double-stranded circular DNA molecule is

du-plicated via areplicative intermediate in which

DNA synthesisis initiatedat aspecific site and

then continuesbidirectionallyuntil thetwo rep-lication forks meet at a site opposite from the origin ofreplication (3,4). Theuseofsubcellular systems,isolated nuclei,andnucleoprotein prep-arations has ledtofurtherunderstandingof how DNA is synthesizedat eachreplication fork (5, 6, 15, 17). In the present study we have used isolated nuclei and

analyzed

whether the

repli-cation forks move in the same way as during

polyoma DNAsynthesis inintactcells.

We asked the following questions. (i) Does

DNA synthesis in vitro terminate at the same

site as the in vivo process? (ii) How are the replication forks distributed around the ge-nome? (iii) What is the maximal

length

of a DNA chain synthesized by an individual

repli-cation fork, and does the synthesis occur at a constant rate? (iv) Is there any indication of initiation ofnewrounds of DNAsynthesis?

(i) It is clear that replicative intermediates

that were successfully completed and

trans-formed into closed circular moleculeswere ter-minated atthenormal termination site (Fig. 1).

Thelabelingpatternalso shows thatreplication

forks approach the termination site from both directions. After 10 min ofincubation, in vitro radioactivelabelwasdetectedonlyverycloseto the terminationpoint. After30min ofreaction,

however, some molecules which at thestart of

thereaction had

replication

forksasfaras1,000

nucleotides from the terminationpointwere

suc-cessfullycompleted.SinceonlyformIDNA was

measured in this experiment, it ispossible that

even longer DNA chains were synthesized in

vitro,but that thosemolecules hadnotyetbeen

transformed into form I DNA.

(ii) The distribution and movement of

repli-cation forkswereanalyzed bylabelingthe DNA radioactively duringtheincubationof the nuclei and then isolatingthe total viral DNA. Of the radioactivitymorethan90%waspresentin rep-licative intermediates. The viral DNAwasthen

digested with the restriction endonuclease

HpaII, and the

radioactivity

was measured in

theseparatedrestrictionfragments. By knowing the size and thelocationofthefragments in the genome, a measure of the distribution of repli-cation forks shouldbeobtained.Itappears, how-ever, that when replicative intermediates are

digested with restriction enzyme, part of the

DNA does not behave as normal linear

frag-ments. By usingelectrophoresistoseparate the DNAfragments, those that contain a replication fork are lost. The quantitation in this type of experiment is therefore uncertain.Nevertheless, it is obvious from the resultspresented in Tables 1 and 2 that replication forksare ratherevenly

distributed around the genome. One exception

was the incorporation oflabel intoHpaIl-5 se-quences that was quite high early during the reaction, but later dropped to normal values. These results are in accord with earlier obser-vations (2; G. Bjursell, unpublished data) that replicative intermediates areevenly distributed between different stages ofreplication. However,

different conclusionswerereached from studies of simian virus 40 DNA replication, where a preponderance of "late" replicative intermedi-ates wasfound (18).

(iii) The movement and inactivation of repli-cation forks were analyzedin an experiment in which DNAwasdensitylabeled with bromode-oxyuridinetriphosphatethroughoutthe reaction

and,inaddition,radioactivelylabeledduring the

last part of the reaction. This protocolwas de-signed to analyze DNA synthesized at replica-tionpoints that remained active throughout the reaction. After digestion withHpaII, restriction fragments whichwereoffulllength (Fig. 5) were isolated and analyzedby centrifugation in alka-line cesium sulfate gradients. The result, de-picted in Fig. 6, shows that the density of the

radioactivelylabeled DNAchains increased

lin-earlywithtime,indicatingthatreplication forks

that remain active movewith aconstant speed

throughout the reaction. The presence of

lighter-than-average material in the gradients

(Fig. 6B and D), consisting offull-length DNA chains withonelightandoneheavypart,might

indicate that some replication forks either can pauseand thenresume synthesisormove quite

slowly. The finding that HpaII-3 (Fig. 6B) can

be completely synthesized in vitro again shows

thatreplicationforkscansynthesize chains ofat

least 1,000 nucleotides in the nuclei (compare Fig. 1).

(iv) Thelastquestionconcerninginitiation of

new rounds of replication remains essentially unanswered. Initiationmight occur,but thedata areinconclusive. Thehigh levelof DNA synthe-sis thatoccursclose to the origin ofreplication earlyduring thereaction (Table 2) could repre-sentsynthesisby replicationforksinitiated dur-ing theearly partofthe reaction. If this is true the rate ofinitiation then seems to droprather quickly during the in vitro reaction. The infor-mation presented in Fig. 6 and Table 2 shows thatevenaslateasbetween 20 and 30min after thestartof theincubation, sequences in HpaII-5aresynthesized. Fromthe sameexperiment it

J. VIROL.

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POLYOMA DNA REPLICATION 393

isclearthat thesynthesisof the DNA in

HpaII-5takes less than 20min,sothesynthesisseenin

HpaII-5laterthan 20minafter the start of the

reaction mightin fact havebeen initiatedduring

the first 10 to 15 min. A trivial explanation to this finding thatwe cannotruleoutis that the

replication forkswereinitiated in vivo and had

synthesizedDNA chainsafewnucleotideslong

atthe time the nucleiwereisolated. In thiscase,

however,wealso havetoassumethat the

repli-cation forkspauseforasubstantial time period

before they complete the synthesis of DNA in

HpaII-5.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish CancerSocietyandMagnusBergvall'sFoundation.

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