Proc. Natl. Acad. Sci. USAVol. 90, pp. 10514-10518, November 1993Biochemistry

Identification of barriers to rotation of DNA segments in yeastfrom the topology of DNA rings excised by an induciblesite-specific recombinase

(transriptional supercoiling/plasmid partition/Zygosaccharomyces rouxu R-recombinase/DNA topoisomerases/intraceliular DNA)

MARC R. GARTENBERG* AND JAMES C. WANGDepartment of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138

Contributed by James C. Wang, August 2, 1993

ABSTRACT Controlled excision ofDNA segments to yieldintracellularDNA rings ofwell-defined sequences was utilized tostudy the determinants of trancriptional supercoiling of closedcircular DNA in the yeast Saccharomyces cerevwsiae. In Atop]top2ts strains of S. cerevisiae expressing Escherichia coli DNAtopoisomerase I, accumulation of positive supercoils in intracel-lular DNA normally occurs upon thermal inactivation of DNAtopoisomerase H because of the simultaneous generation ofpositively and negatively supercoiled domains by transcriptionand the preferential relaxation of the latter by the bacterialenzyme. Positive supercoil accumulation in DNA rings is shownto depend on the presence of specific sequence elements; oneikely cause of this dependence is that the persistence of oppo-sitely supercoiled domains in an intracellular DNA ring requiresthe presence of barriers to rotation of the DNA segmentsconnecting the domains. Analysis of the S. cerevsiae 2-!mplasmid partition system by this approach suggests that theplasmid-encoded REP] and REP2 proteins are involved informing such a barrier in DNA containing the REP3 sequence.

Interphase chromosomes are generally thought to be intri-cately organized inside cells. In situ hybridization and immu-nofluorescence experiments show that entire chromosomes,as well as unique chromosomal elements such as telomeresand centromeres, may occupy specific positions within theinterphase nucleus of eukaryotic cells (1-4). In electron mi-crographs of isolated eukaryotic chromosomes or bacterial"6nucleoids," supercoiled loops ofDNA 1-300 kilobase pairs(kb) in length appear to emanate radially from amorphousstructures that have survived the preparation procedures (5,6). These results and other physicochemical measurementshave led to the suggestion that intracellular DNA is organizedinto discrete topological domains through attachment to chro-mosomal scaffolds or nuclear matrices, the remnants ofwhichare presumably the amorphous structures seen in the electronmicrographs of the isolated materials (7-9). The presence ofmultiple topological domains is supported by studies of pho-toadduct formation between psoralen, an intercalating agent,and intact and radiation-nicked intracellular bacterial DNA:nicking of intracellular DNA results in progressive reductionofpsoralen photobinding, as expected for the relaxation ofonetopological domain by a single nicking event within it (10, 11).In addition, attachment ofchromosomes to cellular structureshas also been implicated in studies of the Escherichia colireplication origin (12, 13), active replication centers in strippednuclei (14, 15), and nuclear reconstitution (16, 17).Whereas the ordering of intracellular chromosomes is likely

to be of functional importance, few methods are available todissect the molecular architecture of the ordered structure inits native state. For eukaryotic cells, a distinct class ofDNA

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fragments rich in AT-base pairs, termed SARs or MARs (forscaffold attachment regions or matrix association regions,respectively), is associated with cellular remnants that havesurvived the salt or detergent extraction procedures used intheir preparation; these DNA elements are thought to serve asthe sites for anchoring chromosomes in vivo (8, 18). Directevidence is lacking, however, about whether the SARs orMARs serve as the boundaries of topological domains. Thepresence of about 50-100 topological domains in an E. colichromosome, for example, was inferred from the psoralenphotobinding results (11). The binding of psoralen to intracel-lular DNA is dependent on the nucleoprotein architecture aswell as the degree of supercoiling of intracellular DNA, andthus interpretation of the binding data is not straightforward.

In this communication, we describe an approach capable ofidentifying specific DNA sequences and their associated mac-romolecules that may retard or prevent the rotation of intra-cellular DNA about its helical axis. This approach utilizes theinduction of a site-specific recombinase to generate well-defined DNA rings in yeast cells. According to the twin-domain model of transcriptional supercoiling, positive super-coils would form in front of a transcribing polymerase, andnegative supercoils would form behind it, ifthe transcriptionalmachinery is prevented from circling around the DNA (19). Ona circular template, however, the two oppositely supercoileddomains are connected by two DNA segments; supercoils ofopposite signs would annihilate each other rapidly throughrotational diffusion of the connecting DNA segments unlessboth segments encounter barriers to their rotational motion. Inyeast cells lacking active DNA topoisomerases I and II butexpressing E. coli DNA topoisomerase I, an enzyme thatspecifically removes negative supercoils (20), the accumula-tion of positive supercoils in aDNA ring is therefore expectedto depend on the presence of such barriers. Assuming that thetranscriptional machinery provides a barrier on one of theconnecting segments, it should be possible to identify barrierson the other connecting segment by monitoring positive su-percoil accumulation under these conditions. We present herea specific example of this approach. Our analysis of the REPsystem of the Saccharomyces cerevisiae 2-gm plasmid sug-gests that the plasmid-encoded REP] and REP2 proteins,which are involved in plasmid partition (21, 22), may anchorintracellular DNA containing their cognitive site, the 2-,umplasmid REP3 sequence.

MATERIALS AND METHODSYeast Strains and Pamids. Strain DMY201 [a ade2-101 cir°

Ahis3-200 leu2-AJ lys2-801 Atrpl(gal3) ura3-52] was kindly

Abbreviation: RS, recognition sequence(s).*Present address: Department of Pharmacology, University ofMed-icine and Dentistry of New Jersey-Robert Wood Johnson MedicalSchool, 675 Hoes Lane, Piscataway, NJ 08854.

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provided by D. Mahoney (Whitehead Institute). Strain CMS3[a ade2-101 cir° Ahis3-200 leu2-Al lys2-801 Atop:: URA3top24 Atrp)(gal3) ura3-52] was derived from DMY201 bymultiple steps ofgene transplacements (23). Strains CMS4andJCW831-1 were derived, respectively, from CMS3 and JCW8[a ade2-101 leu2-Al lys2-801 Atop)::URA3 top24 Atrpl(gal3)ura3-52] by the introduction of a GALI promoter-linked R-re-combinase gene into the chromosome (see description below).

Plasmids pHM153 and pHM401, CV20 and CV20ts, andRC3 were kindly provided by Y. Oshima (Osaka University),B. Veit and W. Fangman (University of Washington), and D.Hamer (National Institutes of Health), respectively. Fortargeted insertion of the GAL) promoter-linked R-recombi-nase coding sequences into the chromosome, a 2615-bpEcoRI-Sal I fragment from pHM153 (24) was first inserted inbetween the EcoRV and Stu I site within the URA3 insert inpUC18-URA3 (R. A. Kim and J.C.W., unpublished results),yielding pCAS03. A Sfi I fragment of pCAS03, in which theGAL) promoter-linked R-recombinase gene is flanked byURA3 sequences, was inserted into the URA3 marker em-bedded in the chromosomal Atop) locus of CMS4 andJCW831-1, converting the strains to ura3-.Plasmid pKWD50, the parent recombination substrate

used in the excision experiments, was constructed by routinecloning procedures and is illustrated in Fig. 1. The excisioncassette is composed of a yeast L YS2 fragment (Bgl II-XhoI, 2480 bp) flanked by directly repeated 58-bp recognitionsequence (RS) sites, obtained from plasmid pHM401 (25).The cassette was inserted between the Pvu II and Sma I sitesof YEp24 (26). Several derivatives were constructed frompKWD50 by inserting various DNA fragments in betweentwo closely spaced Sac II and Xho I sites on the lys2 segment:pKWD51 contains a CUP) promoter fragment (EcoRI-BamHI, 435 bp) from RC3 (27), oriented to direct transcrip-tion toward the proximal RS; pKWD54C contains the REP3locus (Pst I-Ava I, 606 bp) from YEp24; pKWD54B wasderived from pKWD51 by the addition of the REP3 locusimmediately upstream of the CUP) promoter. In addition,pKWD73C was constructed from pKWD50 by the addition ofthree direct repeats of a 123-bp REP3 fragment spanning nt3135-3258 of the A-form 2-,um plasmid (28). In this construc-tion, the REP3 fragment was obtained by the application ofthe PCR, and the endpoints of the fragment were chosen tocorrespond to an Ava I site and a deletion endpoint in aplasmid Xho4 (29). Sequencing of the cloned PCR productshowed that one of the three inserted copies contains a G -)

A transition at nt 3158. The plasmid pKWD70A was derivedfrom pKWD54C by the insertion of a URA3 promoter frag-ment (EcoRI-BspMI, 234 bp) between REP3 locus and itsproximal RS; transcription from this promoter is directedtoward the proximal RS. In pKWD54B, pKWD54C,pKWD70A, and pKWD73C, the region spanning the Hpa Iand SnaBI sites in the original .REP3 locus of the parentYEp24 vector had been deleted. Plasmids CV20.rlr andCV20.r2 were derived from CV20 by deleting, respectively,an 870-bp Stu I fragment and a 2410-bp Ban II fragment.YEptopA-PGDP contains the E. coli topA gene downstreamof the promoter of the yeast GPD gene (encoding glyceral-dehyde-3-phosphate dehydrogenase) as well as a TRPImarker (30).

Excision of Intracellular DNA Segments and the Inactivationof Yeast DNA Topoisomerase H. Yeast cells harboring variousplasmids were typically grown at 26°C to logarithmic phase inthe appropriate drop-out medium containing 2% (2 g/100 ml)each of dextrose and raffinose. Cultures were diluted by 50-to 100-fold into the same drop-out medium containing 2%raffinose and grown to midlogarithmic phase; galactose wasthen added to 2% to induce synthesis of the R-recombinase.After 5 hr the cultures were shifted to 35°C, a nonpermissive

temperature for top24, for 90 min. Minor variations inprocedures are described in the text and figure legends.

Two-Dimensional Agarose Gel Electrophoresis. Cell growthwas terminated by the addition of, in rapid succession, EDTA(to 40 mM final concentration) and an equal volume of anice-cold cocktail of ethanol/toluene/1 M Tris HCl, pH 8.0(95:3:2 by volume) (31). Nucleic acid isolated from 25 ml ofeach culture by the spheroplasting procedure (32) was resus-pended in 20 ,ul of 10 mM Tris HCl, pH 8.0/1 mM EDTA.Each sample (5 Al) was treated with 10 ,g of DNase-freeRNase A for 15 min at 37°C and then loaded directly into asample well of a 0.7% agarose gel slab (20 cm x 10 cm x 0.5cm) containing 0.1 M Tris-borate (pH 8.3), 2 mM EDTA, and0.6 ,ug of chloroquine diphosphate per ml. After electropho-resis in the first dimension (top to bottom), the gel slab wasequilibrated with the same buffer containing 3 ,g of chloro-quine diphosphate per ml. Electrophoresis in the seconddimension (left to right) was then performed. DNA wasdetected by blot hybridization using appropriate 32P-labeledprobes.

Primer-Extension Analysis of Transcripts. Total nucleicacid was isolated from each culture containing about 108cells, as described (33). CUP) transcripts from pKWD51were monitored by primer extension using the primer 5'-GAACAACTGAGGGGTCCTTTC-3' (34, 35).

RESULTSUse of an Inducible Site-Specific Recombinase to Form DNA

Rings in Vivo. We have employed the R-recombinase encodedby the pSRl plasmid of the yeast Zygosaccharomyces rouxii(36) to form DNA rings in the budding yeast S. cerevisiae.This recombinase, similar to the well-characterized FLPenzyme encoded by the 2-,um plasmid of S. cerevisiae (37),excises and religates the intervening DNA between twosimilarly oriented RS to form a closed circle (36). The Z.rouxii recombinase is active in S. cerevisiae but does notcross-react with the FLP system (38); thus controlled exci-sion ofDNA sequences flanked by RS can be carried out bythe induction of the Z. rouxii enzyme in S. cerevisiae with orwithout the 2-gm plasmid.

Fig. 1 depicts the construction of the parent plasmidpKWD50 and various derivatives of it used in the studiesreported here. The plasmid pKWD50 was used to transforma S. cerevisiae strain JCW831-1 (Atop) top24), which lacksDNA topoisomerase I and expresses a temperature-sensitiveDNA topoisomerase II (35). This strain also contains achromosomally located R-recombinase gene under the con-trol ofan inducible yeast GAL) gene promoter and a plasmid-borne E. coli topA gene under the control of a constitutiveyeast promoter. As shown in the leftmost panel in Fig. 2, asignificant fraction of the recombination substrate plasmidwas converted to the product rings following the induction ofthe recombinase at 26°C by galactose.

State ofSupercoiling ofthe Excised DNA Ring in Yeast Aopltop2-4 Cells Expressing E. coliDNA Topoisomerase I. At 26°C,a permissive temperature forDNA topoisomerase II encodedby the top24 allele, the parent pKWD50 and the excised2.5-kb lys2 ring are resolved into arcs of negatively super-coiled topoisomers following two-dimensional gel electro-phoresis (Fig. 2, lane 1). Presumably, these DNA ringsexisted as minichromosomes inside the cells, and the pres-ence of active yeastDNA topoisomerase II maintained theseintracellular minichromosomes in a relaxed state; negativelysupercoiled DNA rings were then formed upon deproteina-tion of the isolated minichromosomes (39).Upon incubation of the induced cell culture at 35°C, a large

fraction of the parent plasmid pKWD50 migrated as posi-tively supercoiled rings (Fig. 2, lane 2). This finding isexpected from previous studies: inactivation of yeast DNA

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a IYS2noI

a

| ~~PKWDSO

URA3 HpaI AMPrSnaBI

REP3

b pKWD50

pKWD51 r __

REP3pKWD54C = - l

PCUPl REP3

pKWD54B _

3x REP3ApKWD73C/

PURA3 REp3pKWD70A = _

FIG. 1. Plasmids used in the controlled formation of intracellularDNA rings. (a) Construction of the R-recombinase substratepKWD50. Half-filled rectangles represent recognition sites of therecombinase (RS); the directly repeated orientation of the RS sitesprograms the recombination reaction for excision. Recombinase-catalyzedDNA breakage and rejoining occur within the RS sites (36).The segment denoted by lys2 was derived from the S. cerevisiaeLYS2 coding region; Xho I and Sac II are two unique restrictionendonuclease sites within the 2.5-kb segment and were utilized in theconstruction ofthe various derivatives. The remainder ofthe plasmidis from the S. cerevisiae/E. coli multicopy shuttle vector YEp24. (b)Sequence elements in the excision cassettes ofplasmids derived frompKWD50. Promoters are labeled and represented by arrows pointingin the direction of transcription. The REP3 locus or fragmentsderived from it are represented by hatched boxes. In some of thederivatives theREP3 region is deleted from the YEp24 backbone (seetext).

topoisomerase II in the Atop] top24 strain at this tempera-ture would leave E. coli DNA topoisomerase I as the onlysignificant DNA relaxation activity, and the specific removalof negative supercoils by the bacterial enzyme would lead tothe accumulation of positive supercoils (30, 35).

In contrast to the positive supercoiling of the parentplasmid pKWD50 in JCW831-1 cells at 35°C, the bulk of theexcised 2.5-kb ring remained as negatively supercoiled topoi-somers under the same conditions (Fig. 2, lane 2). The stateof supercoiling ofthe excised ring is not significantly changedby the inclusion of a promoter on this ring. In pKWD51, thecopper-inducible CUP) gene promoter was incorporated intothe lys2 DNA segment in pKWD50. The presence of theCUP) promoter has little effect on ring topoisomer distribu-tion at 35°C (Fig. 2, lane 3).

Analysis of CUP) message by primer-extension demon-strates that the CUP) promoter on the excised lys2 ring istranscriptionally active (Fig. 3). In this experiment, a primerwas chosen to yield products unique to CUP) transcripts ofthe excised lIys2 ng and distinct from those of the unreactedpKWD51: the former would yield extension products 195 and204 bp in length, corresponding to CUP) transcripts from two

1 2 335°C - + +CuS04 - +

Plasmid pKWD5OpKWD5OpKWD51Recombiniation

substrate

4.j.:..X.

ExcisedDNA ring

FIG. 2. Analysis of excision substrates and products by two-dimensional agarose gel electrophoresis. Expression of R-recombi-nase gene in strain JCW831-1 was induced with galactose for 5 hr at26°C. The excision substrate plasmid and induction protocol areindicated above each diagonal lane: CUSO4, + indicates the additionof CUSO4 to 1 mM, 3.5 hr after the addition of galactose; 35°C, +indicates a shift of growth temperature from 26°C to 35°C. DNA wasrecovered from cells immediately after the addition of an ethanol/toluene/Tris buffer mixture to cultures 90 min after the temperatureshift. Topoisomers of the excised DNA ring and recombinationsubstrate were resolved by two-dimensional gel electrophoresis intoarcs; highly positively supercoiled topoisomers coalesce into a singlespot at the clockwise end of each arc under the gel electrophoresisconditions employed.

closely spaced transcription start sites (40), and the latterwould yield extension products several kilonucleotides inlength. As shown in Fig. 3, a doublet of primer-extensionproducts =200 nt in length was detected in cells harboringpKWD51 after induction of R-recombinase (lane 1), but notin the same cells before recombinase induction (lane 2) or ininduced cells harboringpKWD50, which does not contain theCUP) promoter (data not shown). When CUSO4 was addedto 0.8 mM final concentration 3.5 hr following the inductionof the recombinase in cells harboring pKWD51, the intensityof the 200-bp primer-extension products was significantlyincreased (compare the intensities of the doublets in lanes 1and 3), indicating that the CUP) promoter on the excised lys2ring remains inducible by copper ions.

Effect of the REP System for the Partition of Yeast 2-pmPlasmids on Positive Supercoil Accumulation in DNA Rings.The above results show that DNA rings that do not accumu-late positive supercoils can be readily generated in yeast cellsexpressing only a negative supercoil-specific DNA relaxationactivity. The ability to form such intracellular DNA rings ofwell-defined sequences makes it possible to identify elementsthat enable the accumulation of positive supercoils in the

1 2 3Gal + - +CuS04 - + +

FIG. 3. Analysis of transcripts from the CUP)33 promoter that were produced in strain JCW831-1

I ~ w cells harboring pKWD51. An oligonucleotideprimer was selected such that excision of the lys2

w * ring would move the primer binding site to -200nt downstream of the CUP) promoter; prior toexcision of the lys2 ring, the primer would detect

* '~ * transcripts originating from CUP) promoter onlyif they are longer than 5000 nt. The arrow marksthe positions of the 195- and 204-nt-long primer-extension products, corresponding to those ex-pected from the two known starts of the CUP)promoter in the excised lys2 ring. The induction

jil protocol used is indicated above each lane: Gal, +indicates the addition of galactose to 2% for 5 hrbefore harvesting cells; CUSO4, + indicates theaddition of CUSO4 to 0.8 mM final concentration90 min before harvesting of cells.

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excised rings in similarly treated cells. We show below thatthe addition of the REP3 sequence of the yeast 2-gm plasmidto the 2.5-kb lys2 ring, in the presence of the 2-,um plasmid-encoded REP) and REP2 gene products, can alter the topol-ogy of the ring when the negative supercoil-specific E. coliDNA topoisomerase becomes the only cellular DNA relax-ation activity.A DNA ring that contains three tandem copies of a 123-bp

segment from the 2-,um plasmid REP3 region was generatedusing the excision substrate pKWD73C (Fig. 4). The experi-ment was performed in two S. cerevisiae strains that differprimarily by the presence and absence of the 2-,um plasmid,denoted respectively by cir+ and cir°. Both of the strains bearAtop] and top24 mutations as well as a GAL) promoter-linkedR-recombinase gene and a plasmid expressing E. coli DNAtopoisomerase I constitutively. In the cir+ strain, JCW831-1,the excised Iys2 DNA containing REP3 sequences accumu-lated positive supercoils readily: on a two-dimensional gel, thepositively supercoiled topoisomers had coalesced into a singlespot (lane 1). The bulk of the same excision product remainednegatively supercoiled in the ciro strain, CMS4 (lane 2).Positive supercoiling of the excision product was observedwhen CMS4 cells were transformed with plasmid CV20, whichcontains the entire 2-,um plasmid, a LEU2 marker, andpBR322 sequences (lane 3). These results suggest that REP3-dependent accumulation of positive supercoils requires 2-,um-encoded gene products.To show more specifically that the REP) and REP2 gene

products are required for the REP3-dependent changes inring topology, additional experiments were performed withCMS4 transformants of three CV20 derivatives: CV20ts,which carries a temperature-sensitive rep) mutation (41);CV20.r2, which carries a deletion in rep2; and CV20.rlr,which carries a deletion in the RAF and REP) region of the2-,um plasmid sequences. As shown by the gel electropho-resis patterns in Fig. 4, lanes 4-6, transformation with any ofthe three CV20 derivatives rather than CV20 itself fails torestore the positive supercoiling response at 35°C.Experiments were also carried out with plasmids that

yielded excised lys2 DNA rings containing a REP3 sequenceas well as a CUP) or URA3 promoter. As shown in Fig. 5, thepositive supercoiling response of the excision product in aREP) + REP2+ genetic background is enhanced by the pres-ence of a REP3 sequence in the excised ring (compare thetopoisomer distributions in lanes 1 and 3 of Fig. 5) and evenmore so by the presence of a REP3 sequence and a CUP)promoter (Fig. 5, lane 2). Similarly, when the excised lys2

1 2 3 4 5 6

1 2 3

FIG. 5. Two-dimensional analysis of excision substrates andproducts that carry a promoter and/or the 2-ILm REP3 partitioninglocus. JCW831-1 (cir+) was cotransformed with YEptopA-PGPD andan additional plasmid as described below. CuS04 was added to allcultures to 1 mM final concentration. Lanes: 1, DNA from cellsharboring pKWD51, which contains a CUP) promoter in the lys2DNA segment; 2, DNA from cells harboring pKWD54B, whichcontains the CUP) promoter and REP3 locus; 3, DNA from cellsharboring pKWD54C, which contains the REP3 locus only.

ring contained a REP3 and a URA3 promoter, the topoiso-mers had coalesced into a single positively supercoiled spoton the two-dimensional gel after 90 min at 35°C (Fig. 6).

DISCUSSIONThe induction of the Z. rouxii R-recombinase in S. cerevisiaeprovides a convenient method of forming intracellular DNArings ofwell-defined sequences. Even though the induction ofthe GAL) promoter-linked recombinase by the addition ofgalactose is suboptimal in the particular strains used becauseofthe presence ofa gal3 mutation, a significant fraction ofthelys2 fragment was excised in all cases 5 hr after the inductionof the recombinase. In a GAL3 double topoisomerase mutantyeast strain, nearly quantitative excision was observed 3 hrafter recombinase induction (unpublished data).By designing the proper substrates for the recombinase-

mediated excision reaction, information on the organizationand dynamics of intracellular DNA can be gleaned from thetopology of the excision products under various conditions.As illustrated by the results depicted in Fig. 2, for a 2.5-kbexcision product containing internal coding sequences of theyeast LYS2 gene, the accumulation of positive supercoils isbarely detectable in the presence of E. coli DNA topoisom-erase I and in the absence ofyeastDNA topoisomerases I andII. The excised DNA migrated in the two-dimensional gel asnegatively supercoiled topoisomers, which is expected fortopoisomers derived from a relaxed intracellular DNA ringcontaining a string of nucleosomes. Furthermore, the pres-

Time at 035C.mma

N& , ~~~~- ,-~~%r

20

S60 90

iA

&bI6t

FIG. 4. Influence of S. cerevisiae 2-,m plasmid partition systemon the accumulation ofpositive supercoils in intracellularDNA rings.Strains JCW831-1 (cir+) and CMS4 (cirP) were cotransformed withplasmids pKWD73C and YEptopA-PGPD and a third plasmid asspecified below. Induction of R-recombinase, inactivation of yeasttopoisomerase II encoded by the top2-4 allele, and cell harvestingand DNA recovery were carried out as described before. Lanes: 1,JCW831-1, no third plasmid; 2, CMS4, no third plasmid; 3, CMS4with plasmid CV20 containing the entire 2-pxm sequences; 4, CMS4with plasmid CV20ts, which carries a repits allele; 5, CMS4 withplasmid CV20.r2, which carries a Arep2 allele; 6, CMS4 with plasmidCV20.rlr, which carries Arepl and Araf alleles.

FIG. 6. Two-dimensional agarose gel analysis of the accumula-tion of positive supercoils in the lys2 DNA ring excised frompKWD70A. The excised ring contains the 2-pm REP3 locus and aURA3 promoter. Yeast strain JCW831-1 (cir+) was cotransformedwith YEptopA-PGPD and the excision substrate pKWD70A; theresident 2-;&m plasmid provided the REP] and REP2 proteins. Thenumber above each diagonal lane denotes the time in minutes aftershifting the growth temperature to 35°C to inactivate the top24encoded yeast DNA topoisomerase II.

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ence of a functional CUP] promoter in the lys2 ring does notenhance significantly the accumulation of positive super-coils. There are two plausible interpretations of the CUP]result. First, the transcriptional machinery initiating at theCUP] promoter may not provide a significant barrier torotational diffusion of the DNA around its helical axis.Alternatively, the transcriptional machinery may form asignificant barrier, but one barrier on a circular DNA isinsufficient to prevent the cancellation of two oppositelysupercoiled domains through rotational diffusion of the twoconnecting DNA segments. We favor the second interpreta-tion, as the presence of a CUP] promoter on an excised lys2DNA ring containing an oppositely oriented URA3 promoterenhances the positive supercoiling response (unpublishedresults). How the spiraling motion of a transcriptional ma-chinery along its template is retarded in eukaryotic cells is notknown; cytological experiments suggest that nascent tran-scripts may be localized through specific interactions withcellular entities (42, 43).A major motivation of the work reported here is the

identification of DNA sequences that are involved in theorganization of intracellular DNA. Our approach relies onmonitoring topological changes in intracellular DNA rings ofwell-defined sequences, as illustrated in the study ofthe yeast2-,um plasmid REP system. It is clear that under our exper-imental conditions, the accumulation of positive supercoils inthe 2.5-kb lys2 DNA ring is enhanced by the presence of theREP3 sequence in cis, and only when both REP] and REP2gene products are provided in trans. The simplest interpre-tation of these results is that REP) and REP2 proteins, bythemselves or in combination with other chromosomal geneproducts, can anchor a REP3 site to a large cellular structure.There are two types of models of the mechanism of

REP-mediated partition of 2-,um plasmids (22). Becauseplasmids larger than several kilobase pairs are preferentiallyretained in the mother cells of budding yeast in the absenceof a partitioning system, the larger plasmids presumably donot diffuse freely in the cellular milieu. Thus one modelpostulates that the REP system promotes plasmid diffusion,for example, by initiating plasmid compaction, and the othermodel postulates that the REP system actively drives thedispersion of the plasmid molecules, for example, by formingan intranuclear structure to provide a dispersed set of at-tachment sites for REP3-containing plasmids. Our resultsfavor the latter possibility and also provide evidence that theREP proteins mediate a physical association with REP3sequences.

In the presence ofREP) andREP2 proteins, the 2.5-kb lys2DNA ring bearing REP3 but lacking a known promoter canaccumulate positive supercoils in yeast cells with E. coliDNA topoisomerase I as the only effective DNA-relaxationactivity. We are uncertain whether this accumulation reflectsthe presence ofcryptic transcripts on the REP3-bearing DNAring or whether macromolecular assemblies other than tran-scriptional machineries might be involved in the generation ofoppositely supercoiled domains. In either case, the studypresented for the REP system is exemplary of the applica-bility of the experimental strategy described; it should befeasible to apply this approach to the identification anddissection of other elements that may retard or prevent therotation of intracellular DNA around its helical axis, includ-ing those for the anchoring of intracellular DNA to nuclearmembrane or other cellular structures.

We thank those who kindly provided yeast strains and plasmids tous. This work was supported by grants from the National Institutesof Health, the National Science Foundation, and the LeukemiaSociety of America.

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