Proc. NatL Acad. Sci. USAVol. 80, pp. 105-109, January 1983Biochemistry

"Onion skin" replication of integrated polyoma virus DNA andflanking sequences in polyoma-transformed rat cells:Termination within a specific cellular DNA segment

(polyoma virus induction/LPT line/mitomycin C/Southern blotting/replicon terminus)

NAVA BARAN, ADA NEER, AND HAIM MANOR*Department of Biology, Technion-Israel Institute of Technology, Haffi&, Israel

Communicated by E. Peter Geiduschek, October 6, 1982

ABSTRACT Replication of integrated polyoma virus DNAand flanking cellular sequences was studied in an inducible lineof polyoma-transformed rat cells, designated the LPT line, thatcontains a single viral integration site. Chromosomal DNAs werepurified from LPT cells treated with the virus-inducing agentmitomycin C and from untreated cells and were digested with re-striction enyzmes. The digests were analyzed by the Southernblotting technique. The virus DNA and a recombinant plasmidcontaining flanking cell DNA were used as hybridization probes.The analysis showed that mitomycin C treatment caused a morethan 10-fold amplification of restriction fragments extending upto about 2.0 kilobase pairs into the cellular DNA flanking one endof the viral insertions, defined as the left joint. Fragments ex-tending beyond this region were not amplified. These resultsshowed that (i) integrated polyoma virus DNA undergoes multiplerounds ofreplication in mitomycin C-treated LPT cells and (ii) thereplication extends into the flanking sequences and is arrestedwithin a 0.40-kilobase-pair cellular DNA segment located about2.0 klobase pairs beyond the left joint. This segment may includea terminator of a normal cellular replicon.

The inducible LPT line of polyoma-transformed rat cells wasisolated from rat embryo muscle cells infected with polyomavirus (Py) (1). LPT cells contain Py DNA integrated into a singlesite in the chromosomal DNA (2, 3). The Py insertions vary inlength by virtue ofdifferent degrees oftandem duplications (3).The cells are heterozygous with respect to the insertions (4). Asmall fraction ofLPT cells (<0.20%) are spontaneously inducedto synthesize free Py DNA molecules and infectious polyomavirus (1, 5). The induction rate can be increased up to 300-foldby treating the cultures with physical and chemical carcinogens(1, 5-8) or agents that inhibit protein synthesis (9) or by fusingLPT cells with mouse cells (1).The high inducibility of the LPT line and the availability of

a detailed physical map of the Py integration site in LPT cells(3) makes this system suitable for studies of molecular eventsthat follow virus induction. Here we report a study of the rep-lication of integrated Py DNA and flanking sequences in LPTcells treated with the virus-inducing agent mitomycin C. Thiswork was inspired by the observation that virus activation leadsto enhanced replication of chromosome-associated viral DNAin Py and simian virus 40 transformants (10, 11) and by the"onion skin" model proposed by Botchan et aL (11) to accountfor this finding. According to this model, multiple rounds ofbidirectional replication are initiated at the normal origin oftheintegrated viral DNA and the replication forks move in oppositedirections into the flanking cellular sequences. Formation ofthese replicating intermediates may facilitate excision of the

viral DNA by homologous recombination events.In the present study, chromosomal DNAs were prepared

from untreated and mitomycin C-treated LPT cells and weredigested with restriction enzymes. The digests were analyzedby the Southern blotting technique (12). Py DNA and a recom-binant plasmid containing flanking cell DNA were used as hy-bridization probes. The effect ofmitomycin C treatment on theconcentration of restriction fragments containing both Py andflanking cell DNA sequences (junction fragments), or onlyflanking sequences, was studied. The results show that, as pre-dicted by the onion skin model, the chromosome-associated PyDNA and adjacent cellular sequences undergo multiple roundsofreplication in mitomycin C-treated LPT cells. A detailed anal-ysis of replication forks that move across one of the viral-cellDNA joints, defined as the left joint, showed that their move-ment is arrested within a 0.40-kilobase-pair (kb) cellular DNAsegment that maps about 2.0 kb to the left ofthe viral insertions.

MATERIALS AND METHODSCells and Virus. LPT cells were propagated as described (9).

To induce the polyoma virus, mitomycin C at 1.0 Ag/ml wasadded to the growth medium for 1 hr. The cells were furtherincubated for 23 hr in the absence of the drug and then har-vested. The methods used for growth ofpolyoma virus and pu-rification of Py DNA have been described (2, 9).

Fractionation of High and Low Molecular Weight DNA.Whole cell DNA was purified from untreated or mitomycin C-treated LPT cultures as described by Mendelsohn et aL (3). TheDNA was fractionated by a modified Hirt's procedure (3, 13).The Hirt's precipitate (the "chromosomal DNA fraction") andthe Hirt's supernatant (the "extrachromosomal DNA fraction")were further purified as described (3).

Analysis of Restriction Enzyme Digests by the SouthernBlotting Technique. The procedures used for digestion ofDNAwith restriction enzymes, gel electrophoresis, Southern blot-ting, DNA-DNA hybridization, and autoradiography have beendescribed by Mendelsohn et aL (3). DNA hybridization probeswere prepared by nick-translation (14) and had specific radioac-tivities of 2-5 X 108 cpm/,g. The probes were Py DNA anda recombinant plasmid containing a 0.90-kb flanking cell DNAsegment that maps next to the left boundary of the viral inser-tions (4).

RESULTSAmplification of Integrated Py DNA and Flanking Se-

quences in Mitomycin C-Treated LPT Cells. Our first aim wasto determine whether mitomycin C treatment induces repli-

Abbreviations: Py, polyoma virus; kb, kilobase pair(s).* To whom reprint requests should be addressed.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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~~~~~~~P 41-41,1 m -4

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FIG. 1. Physical map of the integration site of Py DNA in the LPT line and fragment amplification pattern in induced cells. (A) Physical mapof the Py insertion and flanking cellular DNA as constructed by Mendelsohn et al. (3) and Neer et al. (4). The distance between the BamHI siteat the left end of the map and the left viral-cell DNA joint is about 12 kb. Ori, origin of replication of the viral DNA. The "termination region"is defined in the last section of Results. -, Polyoma DNA; -, cell DNA;VJ\, boundary region. (B) m, Fragments that are not, or only slightly,amplified in mitomycin C-treated cells; -, fragments that are strongly amplified.

cation of chromosome-associated Py DNA in LPT cells. To thisend, we purified high molecular weight chromosomal DNAfrom untreated and mitomycin C-treated LPT cells. Samplesof these DNA preparations were digested with restriction en-zymes and the digests were analyzed by the Southern blottingtechnique. Py DNA was used as a hybridization probe in theseinitial experiments. The results were interpreted by using thepreviously derived physical map ofthe Py integration site shownin Fig. 1A. An analysis ofDNAs restricted with the enzyme BglI, which cleaves Py DNA once, is shown in Fig. 2. Track 1 showsthat the Bgl I digest of DNA from untreated cells contains twojunction fragments, as expected (Fig. 1A), and whole linear PyDNA molecules that originate from insertions including tandemrepeats of the viral DNA (3). The amount of chromosome-as-sociated Py DNA has greatly increased in the mitomycin C-treated cells (track 2). Most of the amplified viral DNA is in-cluded in a heterogeneous mixture of fragments that did notmigrate in the gel as specific bands. These heterogeneous frag-ments were probably generated by Bgl I cleavage ofreplicatingintermediates. The very wide range of mobilities of these frag-ments implies that the replicating DNA has a very complexstructure. The smaller (2. 1-kb) junction fragment can barely beidentified in track 2 and is more abundant than the correspond-ing fragment in track 1. The amount of DNA loaded into track

2 was one-fifth of the amount loaded into track 1. We thereforeconclude that the 2. 1-kb fragment was amplified more than 5-fold in the mitomycin C-treated cells. This result and the resultsobtained in similar analyses of other enzyme digests imply thatmitomycin C treatment induces more than two rounds of rep-lication of integrated Py DNA and flanking sequences in LPTcells.To examine the replication ofthe flanking cell DNA directly,

we carried out analogous experiments in which a plasmid con-taining the flanking cell DNA segment whose -map position isindicated in Fig. 1A was used as a hybridization probe. An anal-ysis of DNAs from untreated and mitomycin C-treated cellsdigested with the enzymes Bgl I, Hpa II, and EcoRI is shownin Fig. 3. The Bgl I digests (tracks 1 and 2) contain two fragmentsthat hybridize with the cell DNA probe, one ofwhich is the 2.1-kb junction fragment previously identified by hybridizationwith Py DNA [because of the short exposure time the 2. 1-kbfragment is visible here only in track 2, which contains DNAfrom the mitomycin C-treated cells; it has also been observedin Bgl I digests of DNA from untreated cells after longer ex-posures (4)]. The other fragment contains only cell DNA se-quences and maps next to the 2. 1-kb junction fragment (Fig.

| 2 34 56

FIG. 2. Amplification of integrated Py DNA in mi-tomycin C-treated LPT cells. Ten micrograms of chro-mosomal DNA from untreated LPT cells (track 1) and2 pg of chromosomal DNA from mitomycin C-treatedcells (track 2) were separately digested with Bgl I. Thedigests were electrophoresed in separate lanes of a single1% agarose gel. A blot prepared from this gel was hy-bridized with 32P-labeled Py DNA and exposed for au-toradiography. Details of these procedures have beenpresented elsewhere (3). 4, Junction fragments. Num-

4 bers on the left are fragment sizes in kb.

2 10

0771.00 as0.90 x3

4;

FIG. 3. Amplification of junctionfragments and fragments containingonly flanking cellular DNA in mito-mycin C-treated LPT cells. Equalamounts (10 ,ug) of chromosomal DNAsfrom untreated (tracks 1, 3, and 5) andfrom mitomycin C-treated (tracks 2, 4,and 6) LPT cells were digested withBgl I (tracks 1 and 2), Hpa II (tracks 3and 4), and EcoRI (tracks 5 and 6). Thedigests were electrophoresed in 1.4%agarose gels. Blots prepared from thesegels were hybridized with the recom-binant plasmid probe described in thetext. 4, Junction fragments; <, frag-ments containing only flanking cellDNA. Numbers on the left of the gelsare fragment sizes in kb.

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106 Biochemistry: Baran et al.

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1A). Clearly, the concentrations of both fragments are consid-erably higher in the DNA prepared from mitomycin C-treatedcells. The corresponding Hpa II digests (Fig. 3, tracks 3 and 4)display similar patterns: A junction fragment and a fragmentcontaining only flanking cell DNA have both been amplifiedin mitomycin C-treated cells. The EcoRI blots (tracks 5 and 6)display one band that contains the cloned cell DNA segmentitself (4). It has also been amplified after mitomycin C treat-ment. Quantitative estimations, in which the intensities of thebands were compared with those of bands containing knownamounts of plasmid DNA, indicate that the concentrations ofall the fragments observed in Fig. 3 are increased at least 10-foldin the mitomycin C-treated cells. These results support the con-clusion that mitomycin C treatment induces multiple roundsofreplication ofcovalently linked Py DNA and flanking cellularsequences.

It should be noted that junction fragments that sometimescould not be detected in blots hybridized with Py DNA, becauseof masking by heterogeneous material, could easily be identi-fied and quantitated in blots hybridized with the recombinantplasmid, which displayed much smaller amounts of heteroge-neous material and was of better quality. We believe that thiscontrast reflects the structure of the replicating DNA, whichis more complex within the region ofthe tandemly repeated Pyinsertions than within the flanking cellular sequences.The Amplified Restriction Fragments Detected by Hybrid-

ization with the Recombinant Plasmid Probe Are Derived fromChromosomal DNA and Not from Contaminating Extrachro-mosomal DNA Species. Although we purified the chromosomalDNA used for these experiments by a method that selectivelyremoves small extrachromosomal DNA molecules, some smallermolecules could remain in our preparations. The potential con-taminants include not only free Py DNA but also moleculescontaining both Py and flanking cell DNA that might be excisedfrom the chromosomal integration site and replicate autono-mously. The fragments shown to be amplified in Figs. 2 and 3could be generated from such contaminants and not from au-thentic chromosomal DNA. This possibility was examined asfollows. Samples of the large chromosomal and the smaller ex-trachromosomal DNA fractions, obtained by our purificationprocedure from mitomycin C-treated LPT cells, were electro-phoresed in an alkaline agarose gel. The alkali dissociated allDNA molecules that were not covalently linked (3). A blot pre-pared from this gel was hybridized with the plasmid containingthe flanking cell DNA segment. An autoradiogram of this blot(Fig. 4) shows that most of the sequences that are complemen-tary to the probe are included in a heterogeneous mixture of

1 2

4,0

FIG. 4. Distribution of sequences complementary to therecombinant plasmid probe between chromosomal and ex-trachromosomal DNA fractions. Equal amounts (10 dig) ofthe chromosomal DNA fraction (track 1) and of the extra-chromosomal DNA fraction (track 2), prepared by the mod-ified Hirt's technique (3), were electrophoresed in alkali ina 1% agarose gel, as described by Favaloro et al. (15). A blot

0.9 prepared from this gel was hybridized with the recombinantplasmid probe. Numbers on the left indicate positions ofmarker fragments electrophoresed in the same gel.

A b aT

7 i ~ r-l '7

B Ia

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FIG. 5. Schematic illustration of the strategy used to map a ter-mination site for replication in the cell DNA flanking the Py inser-tions. -, Py insertions; =, flanking cell DNA sequences. The clonedflanking cell DNA segment is designated by a box at the left side ofthe insertion. T, termination site for replication; v, restriction enzymecleavage sites; thin bars, fragments generated by digestion with thisenzyme.

high molecular weight chromosomal DNA fragments (track 1).The rather small amount ofcomplementary sequences presentin the extrachromosomal DNA fraction is included in fragmentsthat probably represent degraded chromosomal DNA (track 2).Neither fraction contains any discrete species. These results andthe results of other control experiments, in which restrictionenzyme digests of the chromosomal and extrachromosomalDNA fractions were compared, indicate that mitomycin C-treated LPT cells do not synthesize extrachromosomal DNAspecies that include sequences complementary to the clonedcell DNA segment. Therefore, we conclude that the amplifiedfragments are derived from a region of authentic chromosomalDNA including the viral insertions and flanking cellular se-quences that has replicated in situ.The Induced Replication Is Terminated Within a Discrete

Region of the Flanking Cellular DNA. Our next goal was todetermine how far into the flanking cellular DNA the replica-tion induced by mitomycin C treatment proceeds. Our experi-mental approach to this problem is illustrated in Fig. 5. A sche-matic representation ofthe left portion ofa Py insertion and the

169

FIG. 6. Demonstration that frag-ments whose ends map at relativelylong distances from the viral insertionsare not amplified in mitomycin C-

2 3 4 5 6 treated LPT cells. Ten-microgramI samples of chromosomal DNAs from

untreated (tracks 1, 3, and 5) and frommitomycin C-treated (tracks 2, 4, and6) LPT cells were digested withBamHI

+8 6 (tracks 1 and 2), Xba I (tracks 3 and 4),< andBgl II (tracks 5 and 6). The digests

were electrophoresed in a 0.70% agar-13.7 -$ose gel. Blots prepared from this gel

were hybridized with the recombinant24 E plasmid probe. 4, Junction fragments;

I <-, fragments derived from the homol-ogous chromosome that does not con-tain Py DNA.

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FIG. 7. Mapping of the replication terminus in the flanking cellDNA. Ten-microgram samples of the chromosomal DNAs from un-treated (tracks 1, 3, 5 and 7) and from mitomycin C-treated (tracks 2,4, 6, and 8) LPT cells were digested with HindEI/Kpn I (tracks 1 and2), HindlH/Xho I (tracks 3 and 4), Hindl/Bgl II (tracks 5 and 6), andHindu (tracks 7 and 8). The digests were electrophoresed in 2% agar-ose gels (tracks 1, 2, 3, and 4) or in 1.4% agarose gels (tracks 5, 6, 7,and 8). Blots prepared from these gels were hybridized with the re-combinant plasmid probe. 4, Junction fragments; <, fragments con-taining only flanking cell DNA; A-, fragments derived from the ho-mologous chromosome that does not contain Py DNA.

adjacent cellular sequences, including the cell DNA segmentcloned into the plasmid probe, is shown in Fig. 5A. It is assumedthat replication is initiated within the viral insertions and endsat the site designated T in the flanking DNA. Fig. 5 B and Cillustrates the DNA shown in Fig. 5A undergoing one and twocycles ofreplication, respectively. Digestion ofthe unreplicatedDNA with a restriction enzyme yields fragments a and b (Fig.5A), which can be detected in a blot analysis by hybridizationwith the cloned cell DNA segment. The replicating structuresyield two and four copies, respectively, offragment a; they alsoyield fragments b' and b" instead of fragment b (Fig. 5 B andC). DNA from normally growing LPT cultures should yield ap-proximately equal concentrations of fragments a and b becausemore than 99% of the cells in these cultures contain unrepli-cated DNA. DNA from LPT cells treated with an inducing agentcontains many replicating intermediates of the types shown inFig. 5 B and C. Therefore, a blot prepared from an induced cellDNA digest should display amplification offragment a. On theother hand, fragment b is not expected to be amplified in thisdigest, because it should have a different electrophoretic mo-bility from that of fragments such as b' and b". In general, afragment whose left end maps between the cell-viral DNAjointand the termination site would be amplified whereas a fragmentwhose left end maps beyond the termination site would not beamplified. If, however, the replication is terminated at varioussites in different cells, rather than at one fixed site, a steadydecrease in the degree of amplification of various fragmentswould be expected, depending on how far these fragments ex-tend into the flanking cell DNA.We have tested the model shown in Fig. 5 by blot analysis

ofDNAs from untreated and mitomycin C-treated cells digestedwith BamHI, Xba I, and Bgl II, which cleave the flanking cel-lular DNA at relatively distant positions (Fig. 1A), again usingthe recombinant plasmid containing the cloned cell DNA as ahybridization probe. As shown in Fig. 6, the junction fragmentsgenerated by these enzymes were not, or only slightly, ampli-fied in the mitomycin C-treated cells. This result implies thatreplication ends between the cleavage sites of these enzymesand the cleavage sites of the enzymes used in the experiment

shown in Fig. 3. In addition to discrete bands, tracks 2, 4, and6 in Fig. 6 also display a heterogeneous population ofmoleculesspread throughout a large portion of the blots. These are pre-sumably fragments derived from replicating structures such asb' and b" in Fig. 5.A similar analysis of various restriction enzyme digests de-

signed to locate more precisely the region in which replicationis terminated is shown in Fig. 7. Fig. 1B illustrates the dataobtained in this analysis and the results of the previous exper-iments graphically. One example ofthe results obtained in Fig.7 and illustrated in Fig. 1B is the observation that one HindIII/Bgl II junction fragment is strongly amplified after mitomycinC treatment whereas another HindIII/Bgl II fragment, whichmaps further left, is only slightly amplified (compare tracks 5and 6 in Fig. 7). This and the other results illustrated in Fig.1B indicate that replication ofthe flanking cell DNA ends about2.0 kb beyond the left viral-cell DNA joint within the 0.40-kbsegment designated on the physical map (Fig. 1A) as "termi-nation region."

Another interesting result shown in Fig. 7 (tracks 5 and 6)is that the corresponding fragment, derived from the homolo-gous chromosome that does not harbor a viral insertion, has notbeen amplified in the mitomycin C-treated cells. We have alsofound that mitomycin C-treatment of normal rat cells does notcause amplification of the corresponding sequences (unpub-lished data).

DISCUSSIONWe used the Southern blotting technique to compare restrictionenzyme digests ofchromosomal DNAs purified from untreatedand mitomycin C-treated LPT cells. These experiments showedthat mitomycin C treatment causes multiple rounds of repli-cation of chromosome-associated Py DNA in LPT cells. Wehave also provided direct evidence for enhanced replication ofcell DNA flanking the Py insertions on their left side by usingnot only Py DNA but also the recombinant plasmid containingthe 0.90-kb flanking cell DNA segment as a hybridizationprobe. Furthermore, our demonstration of amplification ofjunction fragments by hybridization with both probes provedthat replication forks move continuously across the left viral-cellDNA joint. Experiments in which only Py DNA was used as ahybridization probe indicated that junction fragments derivedfrom the right side of the viral insertions are also amplified inmitomycin C-treated LPT cells (unpublished data). Thus, rep-lication forks appear to cross both viral-cell DNA joints in thesecells.A comparison between Figs. 2 and 3 indicates that the in-

tegrated Py DNA sequences in mitomycin C-treated LPT cellsare amplified to a greater extent than the cellular DNA flankingthe Py insertions. This quantitative difference implies that thenumber of replication forks in the viral insertions is larger thanthe number of forks in the flanking cell DNA, as might be ex-pected if replication is initiated in the viral insertions and pro-ceeds into the flanking DNA. The assumption that replicationis initiated in the viral insertions, and not in the adjacent cellDNA, is also supported by the observation that sequencesflanking the integration sites that do not contain viral DNA inboth LPT and normal rat cells are not induced to replicate (Fig.7 and unpublished data). Thus, it appears that in mitomycin C-treated LPT cells the integrated Py DNA and flanking se-quences undergo onion skin replication (11).t

t We have discounted and omitted more complicated models becausethey required numerous complicated additional assumptions.

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The fragment amplification pattern shown in Fig. 1B indi-cates that the replication induced by mitomycin C ends withinthe 0.40-kb segment designated as the termination region. Itmight be argued that fork progression is arrested in this regionbecause it includes sequences that preferentially bind mito-mycin C molecules. However, we have recently observed asimilar pattern of fragment amplification in LPT cells exposedto bromodeoxyuridine and UV light, which are almost as effec-tive as mitomycin C as inducing agents in. this system (7). It isbelieved that mitomycin C preferentially binds to guanosineresidues in DNA (16) whereas exposure to bromodeoxyuridineand UV light affects primarily thymidine residues (17). There-fore, these agents are not expected to interact preferentiallywith the same sites in DNA. Because replication induced byeither of these agents ends within the same 0.40-kb segment,this segment may include an authentic termination signal forchromatin replication.

It has been reported that the completion and merging ofDNA replicons represents a rate-limiting step in chromatin rep-lication (18-20). Studies of simian virus 40 minichromosomereplication have indicated that the two replication forks of thisvirus, which proceed from a common origin toward a commontermination region, stop at preferred DNA sequences locatedin this region (21). The retardation of replicon merging can beaccounted for by the presence of similar sequences in mam-malian chromatin at the junctions between adjacent replicons.Sequences that block movement of replication forks have alsobeen identified in the Escherichia coli chromosome and in the.plasmid R6K (22-25). The integrated Py DNA and flanking cel-lular DNA in LPT cells can be regarded as an artificially con-structed replicon that can be induced to replicate indepen-dently of other neighboring replicons. Thus, the replicationforks proceeding from the viral insertions into the flanking cel-lular. DNA probably do not encounter other forks proceedingin opposite directions, as observed in normal replication ofmammalian chromatin (26). This. property of the LPT systemmade it more suitable for detection of a termination signal thanreplicating replicons in normal cells, in which it is difficult to

find out whether termination occurs when the forks meet orwhen they-encounter a specific signal.We thank Yaffa Botfor technical assistance. Financial support for this

project was provided by the United States-Israel Binational ScienceFoundation.

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