Replication of polyoma DNA in isolated nuclei. V. Complementation of in vitro DNA replication.

Copyright@1975 American Society for Microbiology Printedin U.S.A.

Replication

of

Polyoma

DNA

in

Isolated

Nuclei

V.

Complementation

of In Vitro DNA

Replication

BERND OTTO AND PETER REICHARD*

Medical NobelInstitute, Departmentof Biochemistry, KarolinskaInstitute, S-10401Stockholm, Sweden Received forpublication4October1974

Nuclei from polyoma-infected 3T6 fibroblasts elongate in vitro the progeny

strandsofthe replicative intermediatesofpolyoma DNA. Whenhigh

concentra-tions of such nuclei were incubated, short DNA fragments were formed and

subsequently addedontogrowingprogenystrands. When nucleiwererepeatedly

washed with buffercontainingdetergent and then incubated at low

concentra-tions, DNA synthesis was decreased. In particular, the joining process was

reduced, resulting inanaccumulation of short DNA fragments.Allaspectsofthe

syntheticcapacity ofthe nucleiwererestoredby additionof cytoplasmicextract.

Additions of purified enzymes (polynucleotide ligase from calf thymus or

Escherichia coli togetherwith E. coli DNA polymerase I) increased thejoining

function of the nuclei. The system can be used for the identification of the enzymaticsteps concernedwith polyoma DNA replication.

Wedescribed earlier an in vitro system for the study of polyomaDNAreplication,consisting of

isolated nuclei from 3T6 mouse fibroblasts in-fected withpolyoma virus (15). During

incuba-tion, the nuclei are able tocontinue and

com-plete the elongation of progeny strands which

were initiated in vivo (9). This process was

shown to proceed in a stepwise fashion and to involve the intermediate formation of Okazaki typefragments (11)about 150nucleotides long, which are initiated at their 5' end by short

stretches of ribonucleotides (2, 8).

Experiments by Mueller and co-workers dem-onstrated that the in vitrosynthesis of chromo-somal DNA in nuclei from HeLa cells could be stimulated by cytoplasmic factors (4, 10). Here

we explore the possibility of influencing the synthesis of aviral DNA inisolated cell nuclei

by addition ofcytoplasmic extracts orpurified enzymes. With this system it is possible to

demonstrate effectsondiscretesteps

participat-ingin the overall process, and our experiments shouldprovide a complementation system

sim-ilar to what has been described for

microorga-nisms (7).

MATERIALS AND METHODS

[3H ]thymidine ([3H]TdR; 6.7 Ci/mmol) and [a-32PJdGTPwereobtainedfromNewEngland Nuclear Corp.;

["4C]dATP

was purchased from Amersham. Escherichia coli DNA polymerase I (fraction VII. Jovin et al. [6]) was a giftfrom Lambert Skoog; E. colipolynucleotide ligasewaspreparedessentially by the method of Olivera and Lehman (12) with some minor modifications (B. Heyden, Dipl. Arbeit,

Uni-versity ofTiibingen, Tiubingen, Germany, 1970) was

agift from Heinz Schaller. Calf thymus DNA ligase I, purified 1,000-fold by the method of Soderhall and Lindahl(13) and then furtherpurifiedby chroma-tography onphosphocellulose, was a gift from Stefan Soderhall. Most of the methodology including the handling of cells and virus has been described pre-viously (15).

Invivolabeling of DNA.Immediately before the preparation ofnuclei, cellswere incubated for6to7 minwith [3H]TdR (1.0

MM)

intheculture medium. Labeling wasterminated byremoval of the medium and addition of5 ml ofice-cold Tris-buffered saline (15). Under theseconditions,about 90% ofthe radio-activity was recovered in the progeny strands of replicativeintermediates.

Preparationand incubation of nuclei. The prepa-ration ofnuclei from fivepetri dishes(15 cm), each containing approximately 107 cells, is described. All manipulations were performed at 4C. (i) Normal nuclei: the Tris-buffered salinewasremoved,and the cellmonolayers were washedtwotimes with5 ml of isotonic

N-2-hydroxyethyl-piperazine-N'-2'-ethane-sulfonic acid (HEPES) (15). Cellswere

scraped

and diluted into one 15-ml centrifugetube with 5ml of isotonic HEPES, and Nonidet P-40 was added to a

final concentration of 0.5%. The suspension was

vortexedat2-minintervals,firstfor 90sand then five

times for 30 s on a Lab Line Super Mixer. Isotonic HEPES (10 ml) wasadded, and the suspension was

centrifugedfor10minat800x g. The

pellet

contain-ing the nuclei (about 0.5 ml) was

resuspended

with

1.5 ml of isotonic HEPES and used either

directly

forincubationorstoredat -70C after

quick

freezing

in an ethanol-dry ice-bath. (ii) Depleted nuclei:

fro-zensuspensionsofnormal nucleipreparedfromabout

5 x 107cells werethawed and diluted into 20 ml of 259

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isotonic HEPES. Nonidet P-40was addedtoa final

concentration of 0.5%, and the suspension was vor-texed five times for 30 s as described above. After

centrifugation for 10 min at 800 x g, the nuclear

pelletwasresuspended in 1.5 ml of isotonic HEPES. The depleted nuclei were then frozen and storedat

-70C.

Incubation conditions.The conditions previously

described (15)weremodified asfollows:

[a-"32P]deox-ynucleoside triphosphate (4,000 to 8,000 counts per minperpmole)or ["4C ]dATP (700countsperminper

pmole) were used at saturating concentrations (10

MM), and ribonucleoside triphosphates were always includedataconcentration of 60MM (exceptforATP,

whichwaspresent at2mM). Further variations of the incubation conditionsaregivenforindividual experi-ments.All incubationswereat25C. Selective

extrac-tion ofviralDNAaccordingtoHirt(5)wasperformed

asdescribed previously.

Preparations of cytoplasmicextracts.All

manip-ulationswereperformedat4C. Between 26 and 28 h afterinfection,the cellmonolayerswerewashed twice

with5ml ofice-cold Tris-buffered salineperdish and

then covered with 5 ml of isotonic HEPES. After 5 min, buffer was removed and plates were left in a

vertical position foranother5mintodrain. The cells

were scraped off and treated five times at 2-min

intervals with three strokes in aloose-fitting Dounce

homogenizer. This highly concentrated lysate was

centrifugedfor 40 minat25,000 x g.NaClwasadded

to the supernatant to give a final concentration of

0.1 M. Thesupernatantwasdialyzed for24h against

isotonic HEPES supplemented with0.1 MNaCland

was then frozen at -20 C. About 0.5 to 1.0 ml of

cytoplasmicextractwasobtained from5x 107cells.

Determination of DNA synthesis. The in vivo

['H]TdR prelabel described abovewasusedto

stan-dardize the amount of nuclei used in the in vitro

incubations. For each preparation of nuclei, the

specific activity (3H counts permin perAgofDNA)

wasdetermined first. From this value and from the

amount of 'H label in a given experiment we could thencalculate theamountofnuclei used. This value is expressed as Hg ofDNA per 100

Al

ofincubation mixture. The in vitro DNA synthesisiscorrelatedto the in vivoprelabel and expressedasthe ratio of "2Por

4Clabelto 'Hlabel.

Alkalinesucrosegradientcentrifugation. These were all run in a Beckman SW 56 rotor for 3 h at

55,000 rpm at 4C. An internal marker (16S) of labeled, linear polyoma DNA obtained by cleavageof

form Iwith E. coli R, restrictionenzyme(1)was usu-allyincluded.

RESULTS

Stimulation of DNA synthesis of depleted nuclei by ribonucleoside triphosphates. Al-though RNA synthesis appears to be required for the elongation of polyoma DNA in isolated nuclei (8), the addition of ribonucleoside

tri-phosphatesother than ATPto incubation

mix-tures stimulated incorporation of radioactive deoxynucleoside triphosphates into DNA only marginally and under special conditions (8). Probably the normal isolated nuclei retain

con-siderablepools of ribonucleosidetriphosphates. When ribonucleoside triphosphates other than ATP were added to depleted nuclei, both the

rate and extent ofDNA synthesis were

stimu-lated (Fig. 1). Incubation mixtures in all further experiments contained ribonucleoside triphos-phates at saturating concentrations unless stated otherwise.

Dependence of DNA synthesis on

concen-tration of nuclei and stimulation by addition ofcytoplasmicextract.Inthefollowing

experi-ment, the concentration of nuclei (normal or

depleted) in the incubation mixturewasvaried,

and the amount of DNA synthesis was

deter-mined. As described above, the replicative in-termediates in nucleiwere prelabeled with

trit-ium, while the in vitro incorporation utilized [a-32P]dGTP. The 32P to 3H ratio is thus a measure of in vitro DNA synthesis. This ratio was drastically decreased at low concentrations of nuclei. The effect was apparent with both types of nuclei, even though it was somewhat morepronounced with the depleted nuclei (Fig.

2). Addition ofhigher concentrations of dithio-threitol (up to 10 mM) had no effect. The

results suggest that at low concentrations of nuclei the activity of the system is limited by

somefactorswhicharelost from the nuclei. The

effectwasless pronounced during short

incuba-tion times (5 min), indicating that factorswere

eluted from nuclei during DNA synthesis in

1.0

I

-.

t

0.5

U)-10 20 30

MINUTES

FIG. 1. Effect of ribonucleoside triphosphates on

DNAsynthesis of depleted nuclei. Depleted nuclei (27

ug ofDNA) were incubatedfor differenttimes with and without 60 MM each of CTP, GTP, and UTP.

Standard conditions weremodified asfollows:

deox-yribonucleoside triphosphates were used at 10 MM,

ATP at 0.4 mM final concentrations. Symbols: 0,

with 60 MM each of CTP, GTP and UTP; and *, without CTP, GTP, and UTP.

260

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1.5

20 40 60 s0

CONCENTRATION OF NUCLEI(jigDNA/loojA FIG. 2. Dependence of viral DNA synthesis on

concentration of nuclei. The indicated amounts of

nuclei (micrograms ofDNA/100-microliter volumes) wereincubated under standardconditions for30min. Symbols: 0,depletednuclei; and*,normal nuclei. vitro as well as during preparation of nuclei.

Thisinterpretationisalsofavouredbyan

exper-iment (data not shown) in which depleted

nuclei were first incubated for 4 min under

standard conditions but without labeled

deox-ynucleoside triphosphates. After pelleting, dif-ferent amounts of nuclei were resuspended in

standard incubation mixtures containing

"2P-labeled dGTP and DNA synthesis was

mea-sured for 25 min. In such experiments, DNA synthesis (32P to 3H) was low and showed less

dependence ontheconcentration of nuclei.

These results suggested that DNA synthesis of diluted nuclei could be restored by the addition of factors lost from them, and that such factors might be present in cytoplasmic extracts.This approachassumesthat nucleican

exchange proteins with the incubation medium. The effect of cytoplasmic extracts on in vitro

DNA synthesis is shown in Fig. 3. Two

concen-trations of nuclei were incubated for 30 min

with increasing amounts of extract. In both

cases DNA synthesis was stimulated, but the

degree of stimulationwasmuch larger withthe

lower concentration of nuceli. The extent of DNA synthesis obtained with the largest amount of extract was thesame, however, and

wasidenticaltothatobservedwithhigh

concen-trations of nuclei intheabsence of extract(Fig. 2). The amount of extract required to obtain half maximal stimulationwasabout threetimes

larger with the lower concentration of nuclei. Cytoplasmic extracts used in this and all fur-ther experiments describedwerepreparedfrom polyoma-infected 3T6 cells, but similar effects

wereobtained withextractsfromgrowing, unin-fectedcells. Extracts heatedfor5minat 100 C lost their stimulating capacity. Furthermore,

the stimulating capacitycould be fractionated byammonium sulfateprecipitationand precip-itated between 30 and 60% saturation. Both of

these results suggest that the stimulating fac-tors areproteins.

The polyoma-specific nature of the DNA synthesizedindepleted nuclei in the presence of

extracts was

demonstrated

by reannealing

ex-periments (Table 1). Equal relative amountsof in vitro 32P-labeled DNA were annealed to

polyoma

DNA

independent

of whether DNA

was synthesized in the absence of extracts

(42%)

or in thepresenceof extracts(39%).

Sim-ilar results were obtained with thein vivo

3H-labeledDNAwhichhybridizedto 37% and32%, respectively. Inaparallel experiment, authentic polyoma DNA reassociated to about the same extent

(43%).

Sedimentation properties of in vitro syn-thesized DNA.In thefollowingsections we will show that the effect of extract also is of a

qualitativenatureand inparticular reflectsthe

ability

ofthe nuclei tojoinsmall DNApiecesto

longer chains. In Fig. 4, two alkaline sucrose

gradient centrifugationsofthe products

synthe-sized by normal and depleted nuclei, respec-tively, arecompared.After a30-min incubation

withnormalnuclei, mostof theincorporated in vitro label wasfoundinlong chainssedimenting

close to 16S while with depleted nuclei about

5 10 15 20

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CYTOPLASMIC EXTRACT (i's)

FIG. 3. Effect of cytoplasmic protein extract on

DNA synthesis of depleted nuclei. Prelabeled, de-pleted nucleiwereincubatedat twoconcentrationsof nuclei withincreasingamountsof cytoplasmic protein

extracts from polyoma-infected cells (microliters). Incubations were carried out under standard condi-tionsfor30min.Inparallelincubations,DNA synthe-sis of indicated amounts of extracts per se was

measured. DNA synthesis (32P/3H) of nuclei was correctedfor theincorporationvaluesofextractalone. Thelargest correction (20 uliters of extract) amounted

to15%. Theproteinconcentrationoftheextract was

about 10 mg/ml. Symbols: 0, 27 Ag of DNA/100

Alitersfinalvolume;and*, 13 lAgofDNA/100

Mliters

final volume.

,I

Ic

,'

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TABLE 1. ReannealingtopolyomaDNA bound tofiltersa

DNA synthesis Inputradioactivity

(counts/min)

Reannealing(%)b

Sample 32P/3H 32p 3 14C 32P 3H "C

Depleted nuclei 0.55 180 324 42 37 Depleted nuclei + 10Mlitersofextract 1.03 462 438 39 32 Depleted nuclei + 15 ulitersofextract 1.42 522 366 39 32

PolyomaDNA 910 43

aThe conditionsforreannealing were the same aspreviously described (15). 'Filters contained 0.5ggofpolyomaDNA.

half of the radioactivity appeared as 4 to 5S

fragments. These short fragments were earlier shown tobe precursors of

long

strands (8). The

results (Fig. 4) suggestthat depleted nuclei are

lacking inthe capacitytojoin thefragments. A second point concerns the amount of 3H

sedi-menting in the position ofform I DNA (53S). When the values for the fraction of prelabeled DNAsedimenting at53Swere corrected for the amountofprelabelinform IDNA

by

theinvivo pulse (12%), an additional 12% of the in vivo

labeled viral DNA was transformed to form I DNA after a 30-min incubation. The

corre-sponding valuefordepleted nucleiwasonly2%.

Additionofcytoplasmicextracts restored the

capacity of depleted nuclei to join 4 to 5S

fragments. Figure5shows theeffect of two con-centrations of extract on the sedimentation

profile in alkaline sucrose gradients. In both cases a considerable stimulation of total DNA

synthesis had occurred, and is expressed byan

increased amount of in vitro label

(321p)

appear-ing as long single-stranded chains sedimenting

close to 16S and as form I DNA (53S). In the

absence of nuclei, the extract alone showed a

limited capacity to synthesize DNA (Fig. 5B), most ofwhich sedimented asshortchains. This

activity probably explains the shoulder at the 4to 5Sposition inFig. 5Cand D. Theaddition

of extract also considerably increased the amount of prelabel in form I DNA, which was

stimulated from 2%T (Fig. 5A) to 11% (C) and 19%(D).

Effects of some purified enzymes on DNA

synthesisofdepletednuclei. It appearedtobe ofinteresttoinvestigatewhetherthe additionof certain purified enzymes, either alone or in

combination, could mimic the effect of crude

cytoplasmic extracts. Depleted nuclei clearly are deficient in the ability to join short DNA

chains. Since this deficiency might in turn be the cause of the general inhibition of strand

elongation,

we first investigated whether

poly-nucleotide ligase might substitute forthe cyto-plasmic extract. Addition of highly purified

ligasefrom either calfthymusorE. coli did not

increase the total amount of in vitro label

incorporated

into

polyoma

DNA. However,

there was a

slight

effect on the distribution of

isotope

inalkalinesucrose

gradients

involvinga

shift of some ofthe material

present

as short

DNA

fragments

to

longer

chains

(Fig.

6B and

C).

Addition of more

ligase

did not increase theseeffects.

However,

addition of DNA

polym-erase I from E. coli

together

with ligase in-creased this shift

(Fig.

7D and E). There was

nowalso a

slight

stimulation of total amount of DNA

synthesis

(up

to 125% of the control) but the amount of stimulation observed with

cyto-plasmic

extracts

(Fig.

5) was definitely not

at-tained.

Similarly,

there was only aslight stim-ulation of the amount of

isotope

appearing in form I. Our results demonstrate that the

proc-essof

polyoma

DNA

synthesis

in isolated nuclei

canbeinfluenced

by purified

enzymes and that

depletion

of

ligase

and DNA polymerase are

involved in the loss ofsynthetic capacity ofthe nuclei. Since E. coli DNA polymerase I has both

polymerizing

and exonucleolyticactivities

(14)

both

might

be involved in the observed

stimulatory

effect. However, even

though

these enzymes were added in

large

excess,

they

could

by

no means completely restore the

ac-tivity

ofthenuclei, suggesting that

cytoplasmic

extracts

supply

otherfactorsaswell.

The stimulatory effects described above

sug-gestthat

purified

enzymes andeffectorspresent in

cytoplasmic

extracts can penetrate into iso-lated nuclei and participate in the

replication

process inside the nuclei. Alternatively, DNA and enzymes maydiffuse out from the

nuclei,

and thestimulationsobservedinthepresenceof extractsmaythus represent processesoccurring outside thenuclei. Todistinguishbetweenthese

two alternatives the following experiment was done. Depleted nuclei were incubated either alone or in the presence of an excess of DNA

ligase

from calfthymus orcytoplasmicextract. At the end of theincubation, DNA synthesized

outsidethe nucleiwasseparated from theDNA

synthesizedinsidebycentrifugation. About10% of the total in vitro incorporated

radioactivity

OTTO ANDREICHARD _J.VIROL.

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100

1I

U

1 1000

I

I

I

-1500

0

I

I_ Io

Il

I

I

I

-t500

20

40

[image:5.503.105.388.67.477.2]

FRACTION NUMBER FROM BOTTOM

FIG. 4. Characterizationof viralDNA synthesized in normal and depleted nuclei.Prelabelednuclei (36 ,g of

DNAof normal nucleiand27,g of DNA of depletednuclei) wereincubated under standard conditions with

14C-labeled dATPfor 30 min. Portions (200 Mliters) of Hirtsupernatant fluidwere centrifuged through an

alkalinesucrosegradientasdescribedintext.Panel A, normalnuclei; panel B, depleted nuclei. Symbols:0,

3H;and*, "C.

was found outside the nuclei when

they

were

incubated aloneorinthepresence ofligase.The amount increased to 27% in the presence of extract. Figure 7shows the sedimentation pro-files in alkaline sucrose gradients of the DNA

synthesized in the nuclei. It is clear that sim-ilar effects to those observed in Fig. 5 and 6 were found. A comparison of Fig. 7C and 5D suggests that the radioactive material present as a shoulder in the position of4 to 5S DNA fragments in Fig. 5D was outside the nuclei. In conclusion, the addition of cytoplasmic

extract indeed stimulated the overall

elonga-tionprocess ofDNAreplication insidedepleted

nuclei.

DISCUSSION

The capacity ofnuclei to complete the elon-gation of progeny strands during incubation

depended on the concentration of nuclei. The relative amount ofpolyomaDNA

synthesis

was

greatly decreased when low concentrations of nuclei were used. This effect was more pro-nounced with nuclei which had been

prepared

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264 OTTO AND REICHARD

A

B

200

-0

0

C

100

,*

0~~~~~~~~~_I~~~~~~

*0

0~~~~~~~~

600~~~~~~~~

*16 S

20

40

0

20

40

FRACTION

NUMBER FROM BOTTOM

FIG. 5. Effect of cytoplasmic protein extracts onDNA synthesis of depleted nuclei (product analysis). Prelabeled,depletednuclei(13,ugof DNA)wereincubated eitherintheabsence(A)orpresenceof20Mliters(C)

or50 Mliters (D) of cytoplasmic proteinextractfrompolyoma-infected3T6 cells. A50-gliteramountofextract was also incubated without nuclei (B). All incubations were for 30 min under standard conditions with [32P]-labeleddGTP. The datawereobtainedfromidenticalportions (200,litersofHirtsupematantfluid with

5 .liters of "'C-labeled linearpolyomaDNA as a marker)run inparallelonalkaline sucrosegradients (see

text).Thearrowsgivetheposition ofthemarker(16S). Symbols:0, 3H;ando, S2p.

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I,

X

I

20

400

20

40

FRkCTION NR

FROM WTT

FIG. 6. Effect of somepurified enzymes on DNA synthesis of depleted nuclei. Prelabeled, depleted nuclei (13 ggof DNA)wereincubated without added enzymes(A);with E. colipolynucleotide ligase (B)orcalfthymus DNA ligase I (C);orwith E. coli DNA polymerase I in the presence of either calf thymus DNA ligase I (D) or E. colipolynucleotideligase(E).Incubationswerefor30min.Thestandard incubation mixture with [82P]-labeled dGTPwassupplemented with NAD+ (10-I M final). Portions (200 /uliters) ofHirtsupernatant fluid together with 5 /Lliters of "4C-labeled linearpolyoma DNA as a marker were centrifuged through alkaline sucrose gradients (see text). Thearrowsgive the position of the marker (16 S). Symbols: 0, 3H;and o, 32P.

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I

I

m

B16S

u B~~~~~~~

50 * 50' * -'50

20 40 20 40

FRACTION NUMBER FROM BOTTOM

FIG. 7. Effect of ligaseorcytoplasmic proteinextractonDNAsynthesis inside depleted nuclei. Prelabeled depleted nuclei (13 ugofDNA) wereincubated under standard conditions with

[32P]-labeled

dGTPfor30min: (A)alone, (B) with DNA ligaseIfromcalf thymus, or(C) with50

,liters

of cytoplasmic protein extract from polyoma-infected mouse fibroblast 3T6 cells. DNA synthesis was stopped by adding 100

Mlliters

ofice-cold isotonic HEPEStotheincubation mixture.Aftercentrifugation for10minat 4Cat 800 xg, the nuclearpellet

wassuspended in 100gliters of isotonic HEPES and viral DNAfromboth the supernatant and the nuclei

sus-pensionwasextracted by the Hirt procedure (5). Identicalportions (200Ilitersof Hirt supernatant fluid from the nucleifraction and5/.liters of'4C-labeledlinearpolyomaDNAas amarker) wereruninparallel in alkaline

sucrosegradients (see text). Thearrowsgivetheposition ofthemarker(16S). Symbols:0, 3H;and ,S2P.

by repeated washing with buffers containing

Nonidet P-40 and which were therefore less contaminated with cytoplasm. The decrease

wasmainlyapparent inthe capacityofnucleito sustain DNA synthesis and did not so much affect initial rates. From our data, it seems likely that part of the enzymes are washed out during preparation ofnuclei and part leave the nuclei during DNA synthesis. Nevertheless,

depleted nuclei incubated at a high concentra-tion retained their capacity to continue the

elongation process.

Earlier results showed that polyoma DNA synthesisinnucleiinitially involved the forma-tion ofshort DNAfragments, 100 to 150 nucleo-tides long, and that these fragments subse-quently were joined to form longer progeny strands (8). With depleted nuclei we now find anaccumulation ofshortfragments also during

prolonged incubation indicating that some of the components being removed are involved in thejoining process.

The synthetic capacity can be fully restored

by addition of cytoplasmic extracts, and the active factors in the extracts appear to be proteins. Restored DNA synthesis leads to the formation oflong progeny strands. The extracts thussupply enzymes required forthejoining of the short DNA fragments as well as for other steps of the elongation process. Extracts also increase the rate oftransformation ofreplicative intermediate-DNA into mature viral DNA (form I DNA), but it is uncertain whether extractsalso supply enzymes for the segregation of completely replicated DNA molecules. For-mation of formI was,however, never as efficient as was reported for crude lysates of polyoma-infected 3T3 cells (3).

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[image:8.503.123.401.59.360.2]

About equal activities were found with

ex-tracts from infectedoruninfected growing 3T6

cells. This result indicates that at least all enzymatic activities which can replace

func-tions in the elongation process are of cellular

rather than of viralorigin.

Polynucleotide ligase is an enzyme which

would be expectedtoparticipate inthe joining

process. Neither purified calf thymus ligasenor

E. coli ligase stimulated the overall capacity of the nuclearsystemtosynthesize polyoma DNA. However, ashift of isotope from short fragments to long progeny strands was observed. This

result indicates that ligase activity probably

wasoneof thedepleted nuclear factors, but that

the cytoplasmic extract clearly supplied other additional missing fractions. E. coli DNA

po-lymerase I, together with ligase, gave only a

small stimulation of the overall synthesis, but

increased the joining to a larger extent than ligasealone.Both the polymerizing activity and the exonuclease activity of this enzyme might

be responsible for the observed effect.

Theexperiments involving addition of highly purified enzymes indicate that proteins with

molecular weights of about 100,000 (6, 13) can

influence a synthetic processwhich apparently

occurs inside nuclei. It seems reasonable to

assumethat thisoccursafterpenetrationofthe

enzyme intothe nuclei.

The nuclearsystemwas originally developed

with the hope that it would be useful in the dissection of intermediate steps of polyoma DNA synthesis. The effects described in this

paperopen upexperimentalpossibilitiesinthat

direction. The demonstration of stimulatory effects on a depleted system must be inter-preted with great caution since clearly neither E. coli ligase norE. coli DNApolymeraseI per se participate in polyoma DNA synthesis. The

demonstrated permeability for proteins of the nuclear in vitro system appears promising,

however,and itnowseemsexperimentally feasi-ble to identify enzymes participating in the intermediate steps ofDNA synthesis either by fractionation ofcytoplasmic extracts orby

im-munological techniques.

ACKNOWLEDGMENTS We thankGunilla S6dermanforexpertassistance. The studywassupported byapostdoctoral fellowshipfrom

theDeutscheForschungsgemeinschafttoB.0.andbygrants

from the Swedish Cancer Society and Magnus Bergvalls StiftelsetoP.R.

LITERATURE CITED

1. Crawford, L. V., C. Syrett, and A. Wilde. 1973. The replication of polyoma DNA. J. Gen. Virol. 21:515-521. 2. Eliasson,R., R. Martin, and P.Reichard. 1974.

Charac-terization of the RNA initiating the discontinuous synthesis of polyoma DNA. Biochem. Biophys. Res. Commun.59:307-313.

3. Francke, B., and T. Hunter.1974.InvitropolyomaDNA synthesis: discontinuous chain growth. J. Mol. Biol. 83:99-121.

4. Hershey, H. V., J. F. Stieber,andG. C. Mueller. 1973. DNAsynthesis in isolated HeLa nuclei. Eur. J. Bio-chem.34:383-394.

5. Hirt, B.1967.Selectiveextractionofpolyoma DNA from infectedmousecell cultures.J. Mol.Biol. 26:365-369. 6. Jovin, T. M., P.T. Englund, and L. L. Bertsch. 1969. Enzymatic synthesis of DNA. XXVI. Physical and chemicalstudies ofahomogenous DNApolymerase.J.

Biol. Chem. 244:2996-3008.

7. Klein, A., and F. Bonhoeffer. 1972. DNA replication. Annu.Rev. Biochem.41:301-332.

8.Magnusson, G., V. Pigiet, E.-L. Winnacker,R.Abrams, and P. Reichard. 1973. RNA linked shortDNA

frag-mentsduring polyoma replication. Proc. Nat. Acad. Sci.U.S.A.70:412-415.

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