Proc. NatL Acad. Sci. USAVol. 78, No. 12, pp. 7261-7265, December 1981Biochemistry

Yeast 2-,um plasmid DNA replication in vitro: Origin and direction(Saccharomyces cerevfiiae/DNA initiation/electron microscopy/chimeric plasmids/cdc mutants)

HITOSHI KOJo*t, BARRY D. GREENBERG**, AND AKIO SUGINo**§*Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709; andtCurriculum in Genetics, The University of North Carolina, Chapel Hill, North Carolina 27514

Communicated by jerard Hurwitz, May 29, 1981

ABSTRACT Most yeast strains harbor extrachromosomal 2-pm DNA, and this DNA synthesis, like nuclear DNA replication,is strictly under cell cycle control. A soluble extract of yeast Sac-charomyces cerevisiae carries out semiconservative replication ofadded 2-pIm DNA and Escherichia coli chimeric plasmids con-taining the 2-jum DNA. Replication is initiated on 10% ofthe DNA,and one round; of replication is completed. The major products inearly stages of replication are 0 ("eye") forms which originate 140± 50 nucleotides within one of the 599-base-pair inverted repeatsof 2-pum DNA. Their replication is bidirectional and discontin-uous. Extracts prepared from the cell division cycle mutant cdc8show temperature-sensitive 2-pm DNA synthesis in vitro, sug-gesting that this in vitro system resembles in vivo 2-pum plasmidDNA replication. This system should provide a useful assay for thepurification and characterization ofyeast'DNA replication proteins.

Yeast has one of the best understood eukaryotic genetic sys-tems, and many DNA replication and cell division cycle (cdc)mutants have been isolated (1). Nevertheless, the only knowncorrespondence between a gene and its encoded product is theDNA ligase gene, cdc9 (2).

Most yeast strains contain extrachromosomal DNAs, and thebest known is 2-,um plasmid DNA. This DNA provides a prom-ising model system for the study of eukaryotic DNA replication(3-5), which, like chromosomal replication, is under strict cellcycle control and depends on the same nuclear gene products(6-8). Its small size and abundance make it a more convenientsystem for replication studies than chromosomal DNA. Theplasmid DNA has been completely sequenced (9).An in vitro DNA replication system employing the yeast 2-

,um plasmid, which appears to initiate in vivo DNA replication,has been described recently (4). It is not known, however,whether replication was initiated at the in vivo origin or pro-ceeded with bidirectionality as it does in the cell. Furthermore,the activity was extremely low, disallowing the isolation ofDNAreplication proteins by complementation assays.

In this paper we describe an in vitro 2-pm plasmid DNAreplication system that has at least 1 order ofmagnitude greateractivity than the previously described system (4). The origin anddirection of replication have been determined and are similarto those in vivo. Hence, this system can be readily exploited forthe purification of DNA replication proteins and for the eluci-dation of DNA replication in a eukaryotic cell.

MATERIALS AND METHODSStrains. Saccharomyces cerevisiae A364A (a adel,2 ural

his7 lys2 tyri cir+), NCYC74-CB11 (a adel MAL6 cir0) andNCYC74-CBl1' (a adel MAL6 cir+) (10) were provided by D.M. Livingston. S. cerevisiae cdc4, -7, -8, -9, and -28 (1) were

obtained from the Yeast. Stock Center, Berkeley. Escherichiacoli ED8767 (recA56 metB hsdS supE supF) containing the chi-meric plasmids pJDB36 and -41 (3) were from J. D. Beggs. E.coli containing the plasmid YRp7 (11) was from J. Carbon.

Radioactive Chemicals. [methyl-3H]dTTP (75.2 Ci/mmol;1 Ci = 3.7 x 10O' becquerels) was from New England Nuclear;[a-32P]dTTP (2000 Ci/mmol) was from Amersham.

Other Chemicals. NAD' and all ribo- and deoxyribonucleo-tides were purchased from P-L Biochemicals (Milwaukee, WI).Agarose, nalidixic acid, novobiocin, spermidine, Trizma base,bovine serum albumin, phenylmethylsulfonyl fluoride (Ph-MeSO2F), and (-glucuronidase type H-I (Glusulase) were fromSigma. Aphidicolin was provided by A. H. Todd. Zymolyase60,000 was from Kirin Brewery, Japan; endonuclease EcoRI wasfrom Bethesda Research Laboratories.

Plasmid DNAs. The 2-,um DNA was isolated from S. cere-visiae A364A cells as described (10). -E. coli containing chimericplasmids CV03, CV3, CV7, CV17, and CV18 (12) were providedby J. Broach, and all E. coli plasmid DNAs were prepared asdescribed (13).

Media. Medium for plasmid preparation (medium A + Cas-amino acids) has been described (14). YPD broth for yeastgrowth was 2% (wt/vol) peptone (Difco)/1% yeast extract(Difco)/2% (wt/vol) glucose.Crude Extracts for in Vitro DNAReplication. Exponentially

growing yeast (5 x 107 cells per ml) were harvested, resus-pended in 0.02 vol of 10% (wt/vol) sucrose/50 mM Tris-HCI,pH 7.5/1 mM EDTA, frozen dropwise in liquid N2, and storedat -80°C until used, when they were thawed at room temper-ature and chilled to 0°C. To this suspension were added 4 MKCl to a final concentration of 0.25 M, 0.1 M spermidine (pH8.0) to 5 mM, 0.5 M EDTA to 10 mM, and either Glusulase orZymolyase 60,000 to 2 mg/ml or 0.5 mg/ml, respectively. After30 min at 0°C, PhMeSO2F was added to 0.1 mM to reduce pro-teolysis, and the spheroplasts were collected at 3000 rpm fbr5 min in a Sorvall HB4 rotor. Pellets were resuspended in 50mM Tris'HCl, pH 7.5/1 mM EDTA/0.25 M KCl/0.1 mMPhMeSO2F and incubated at 0°C for 30 min. Cell lysates werecentrifuged at 40,000 rpm at 2-4°C for 30 min in a Spinco SW41rotor, and an equal vol of saturated (NH4)2SO4 solution wasadded to the supernatant over 20 min with stirring at 4°C. Afteran additional 20 min at 40C, the solutions were divided into 1-ml aliquots, which were centrifuged for 5 min in a Brinkmanminicentrifuge at 4°C, and the pellets were frozen at -80°Cuntil used. Just prior to use, each pellet was suspended in 0.5

Abbreviations: cdc mutant, cell division cycle mutant; PhMeSO2F,phenylmethylsulfonyl fluoride; cir+, yeast harboring 2-,um plasmidDNA; cir°, yeast containing no 2-,m plasmid DNA.t On leave from The Research Laboratories, Fijisawa PharmaceuticalCo., Ltd., Osaka, Japan (permanent address).

§ To whom reprint requests should be addressed.7261

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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ml of 50 mM Hepes, pH 7.8/10% (wt/vol) glycerol/i mMEDTA/1 mM dithiothreitol/0.1 mM PhMeSO2F.

Assay for 2-,um DNA Replication. The 0. 1-ml reaction mix-ture contained 35 mM Hepes (pH 7.8), 10 mM MgCl2, 1 mMdithiothreitol, 2 mM spermidine (pH 8.0), 5 mM ATP, each ofthe other three rNTPs at 200 ,M, all four dNTPs at 10 ,uM(dTTP was labeled with either 3H or 32p, 200-1000 cpm/pmol), bovine serum albumin (100l ,g/ml), 1 mM NAD+, 25%glycerol, 1-5 Ag of either native 2-,um DNA or the chimericplasmid containing 2-pum DNA, and protein (1-5 mg/ml) fromthe crude extracts. Incubation was at 30°C for the indicatedperiods. The reaction was terminated as described (4).

Electron Microscopy of in Vitro DNA Replication Products.In vitro replication products were treated with proteinase K(100 pug/ml) at 37C for 30 min in the presence of 0.5% Na-DodSO4, extracted with phenol, and dialyzed against 10 mMTris HCl, pH 8.0/1 mM EDTA. The DNA samples were ex-

amined by the method of Kleinschmidt (15) either with or with-out prior cleavage with EcoRI. DNA lengths were measuredfrom photographic enlargements with a map measurer.

Other Methods. All sucrose density gradient centrifugationswere performed as described (14) with a Spinco SW27 rotor.All DNA manipulations, including agarose gel electrophoresis,DNA transfer to nitrocellulose filters, and DNA-DNA hybrid-izations were performed as described (13).

RESULTSCharacterization of in Vitro 2-,um DNA Replication. In-

cubation of 2-pum DNA with a crude extract of yeast led to in-corporation of labeled dNTPs into acid-insoluble material. Thisincorporation depended totally on exogenous DNA (Fig. 1A).Further requirements for DNA synthesis are detailed in Table1. The reaction was essentially completed within 30 min.

Fig. 1B shows the optimal concentration of crude extract inthe reaction. Between 0 and 3 mg of protein per ml, there was

a linear increase in dTTP incorporation, whereas at higher con-

centrations, a gradual decrease in incorporation was observed.This decrease was not due to degradation of the template, how-ever, because only 10-20% ofthe DNA was converted to linearforms at this concentration of crude extract.

Table 2 shows the template specificity for in vitro DNA rep-

lication with crude extracts from isogenic cells either containing(cir+) or lacking (cir°) the 2-pum plasmid. Native 2-pAm DNA(Fig. 2) and the chimeric plasmids containing either the entire2-pum DNA (pJDB36 and pJDB41) or the EcoRI fragment con-

taining the in vivo replication origin (CV17 and CV7) (12) werethe most efficient templates. Conversely, the plasmids con-

150

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0

P-

Ea

0 10 20 30 60 0

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10 20Crude extract,mg protein/ml

FIG. 1. Kinetics and optimal protein concentration of yeast 2-MmDNA synthesis- in vitro. incorp, Incorporated. (A) The reaction mix-tures contained 3 mg of protein per ml from S. cerevisiae A364A (cir+)crude extracts without (o) or with (0) 0.2 pmol of native 2-,m plasmidDNA. After incubation at 30°C, acid-insoluble radioactivity was mea-

sured. (B) The optimal concentration of crude extract was determinedas in A. The reaction was carried out at 30°C for 30 min.

Table 1. Requirements for DNA replication in vitro

Omissions and dTMP incorporatedadditions pmol %

Complete 10.4 100-ATP 1.1 10-CTP, GTP, UTP 9.3 89-dCTP or dATP 2.1 20-NAD', dithiothreitol, orspermidine 10.3-10.7 100

-DNA 0.6 5+KCl, 100mM 1.2 12+Glycerol, 25% 15.4 148

DNA synthesis activity of the extract from S. cerevisiae A364A wasmeasured in the standard assay containing 0.12 pmol of native 2-JhmDNA and 3 mg of protein per ml of the -extract for 30 min at 30TC.

taining the other EcoRI fragment (CV18 and CV3) supportedsynthesis at levels no higher than their parental plasmid (CV03)did. pMB9 and YRp7 also supported appreciable synthesis andat very similar levels, but CV03 and pBR322 did not.

There are moderate differences in DNA synthesis betweencir' and cir0 extracts. However, whereas no 2-,um-DNA-en-coded functions are required for in vitro DNA synthesis, ex-tracts from cir+ cells may contain undefined functions that en-hance this capability.

The observed synthesis was not DNA repair synthesis, asdetermined by experiments replacing dTTP with BrdUTP. Re-action products with native 2-,um DNA were subjected to equi-librium alkaline density gradient centrifugation, and all of thenewly synthesized DNA banded at the heavy position (data notshown). Furthermore, after brief incubation, virtually all in-corporated radioactivity sedimented at 4-5 S in alkaline sucrosegradients. Because the template DNA was not significantly de-graded, these data are consistent with de novo discontinuoussynthesis on both template strands in contrast with continuousstrand extension as expected in repair synthesis.Among the inhibitors, only N-ethylmaleimide and aphidi-

colin inhibited in vitro DNA synthesis (Table 3). The sensitivityof the in vitro system to aphidicolin parallels the sensitivitiesof purified yeast polymerases I and II, implying that either en-zyme (or both) participates in 2-,um DNA synthesis in vitro.. Ofthe other antibiotics, only novobiocin and coumermycin Al ap-

Table 2. Template specificity of DNA synthesis in vitro

DNA synthesis,pmol of dTMP incorporated

DNA source cir+ cir°

None 0.4 0.3Native 2-Mm plasmid 10.5 5.1CV03, CV18, CV3, or pBR322 1.2-2.2 <0.5CV17 10.2 8.2CV7 8.4 6.0pMB9 7.2 4.6pJDB36 or pJDB41 24.2-25.9 14.2-15.6YRp7 5.1 4.8

DNA synthesis activity of the extract from either S. cerevisiaeNCYC74-CB11' (cir+) or.NCYC74-CB11 (cir°) cells was measured asin Table 1, except that 0.12 pmol of the indicated plasmid DNAs wasadded. CV03 consists of E. coli plasmid pBR322 and yeastLEU2 DNA.CV18, 17, 3, and 7 consist of CV03 and, respectively, the small or largeEcoRI fiagment of 2-,um form A DNA or the large orsmallEcoRrfrag-ment of 2-pum form B DNA. pJDB36 and 41 consist of pMB9 and eitherform A or B of 2-Mm DNA, respectively. YRp7 consists of pBR322 andyeast TRYl gene and possibly one of the yeast nuclearDNA replicons.

A < -B I-

I ~

l,,j! P 4 l ||I I I

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Form A Form B

FIG. 2. Schematic diagram of the two forms of 2-pum plasmid DNA.Included in the figure are the two perfect 599-base-pair inverted re-

peats (IR) and recognition sites for the restriction endonucleasesEcoRI(-), HindIII (v), and Hpa I (A). L and S are the large and small uniqueportions of the 2-pm DNA, respectively.

preciably inhibited DNA synthesis, and then only at relativelyhigh concentrations.

Analyses of in Vitro Products. The reaction products aftershort incubation times sedimented at about 3-4 S in alkalinegradients (data not shown). Upon further incubation, the S valueof the slowly sedimenting material increased slightly to about5 S, with a simultaneous increase in the amount ofmore rapidlysedimenting material. This behavior parallels the ligation of invivo replication intermediates in other eukaryotes (16). How-ever, because >70% of the total radioactivity remained in the5S region after 20 min of incubation, the ligation of small DNAspecies appeared to be inefficient.

In neutral sucrose gradients, some newly synthesized DNAlabeled with 32P cosedimented with the 3H-labeled templateDNA, but most sedimented more slowly. These slowly sedi-menting molecules were not degradation products because no

template DNA was degraded to slowly sedimenting forms; nei-ther were they ofchromosomal origin. Hence, they were prob-ably replication intermediates that separated from the templateDNA during analysis.

Electron microscopic analysis of in vitro products (Fig. 3)

l\

FIG. 3. Electron micrographs of 2-pm plasmid DNA moleculesreplicating (arrowhead) in vitro. Representative molecules are shownfrom the 5-min synthesis reactions (Table 4). (a and d) Nonreplicatingsupercoiled and relaxed DNA molecules, respectively. (b and c) Rep-licating supercoiled molecules. (e and f) Replicating relaxed DNAmolecules. (Bar = 0.5 ,um.)

Table 3. Sensitivity of in vitro DNA replication tovarious inhibitors

dTMPincorporated

Inhibitors pmol %

Control (no inhibitor) 10.4 100N-Ethylmaleimide, 1 mM 0.3 3araCTP, 50 pM 7.0 67ddTTP, 50 ,uM 8.7 84Aphidicolin, 5 gg/ml 1.1 11Coumermycin Al, 50 ,g/ml 2.1 20Novobiocin, 500 pg/ml 3.3 32Nalidixic acid, 500 /Ag/ml 8.3 80a-Amanitin, 100 /.g/ml 10.7 103

DNA synthesis in the presence of various inhibitors was measuredas in Table 1 with native 2-pm plasmid DNA as template. araCTP,arabinofuranosylcytosine 5'-triphosphate; ddTTP, 2',3'-dideoxythy-midine 5'-triphosphate.

showed both supercoiled and open circular DNA moleculescontaining an eye (0-form) structure, presumably a replicationintermediate. Table 4 summarizes the analysis of products ob-tained from 5- and 20-min incubations. Within 5 min, 7% ofthetotal DNA molecules contained replication bubbles. Most ofthese were 0.05-0.3 Am long, but some molecules were rep-licated over 50% ofthe genome. The replicated portions ofDNAwere mainly double-stranded, eliminating the possibility of R-loop structures. The percentage of replicating molecules wasunchanged by 20 min; however, there was a decrease in thepercentage of early replicating forms with a commensurate in-crease in late forms. This was also accompanied by an increasein open circular and linear forms.The Origin and Direction of Replication. Native 2-gm DNA

contains two identical 599-base inverted repeats (9) (Fig. 2). Itexists in two equimolar isomeric forms (forms A and B, Fig. 2),and this was expected to introduce some complications in thefollowing assays. Hence, the chimeric plasmid pJDB36, whichconsists ofpMB9 plus form A ofnative 2-gm DNA (3), was usedas template.The replication products, after a 5-min incubation, were ana-

lyzed by electron microscopy after EcoRI digestion. Replicationbubbles were observed in all three fragments; however, thefrequency was highest in the second largest fragment (Fig. 4B),which contains the in vivo origin of2-pum DNA replication (12).Moreover, the locations of these bubbles were strikingly non-random. A center at a unique site was greatly preferred, andthe bubble expanded bidirectionally. Particularly, the site cor-responded to the presumed in vivo origin (12) within the in-verted repeat region. The presence ofreplication bubbles in thesmallest fragment (Fig. 4C), which contains the other invertedrepeat, was statistically insignificant because of its low fre-quency. Interestingly, the replication bubble in the pMB9moiety was equally specific but did not coincide with the in vivoorigin. Furthermore, when pMB9 DNA was incubated withcrude extracts, DNA synthesis was initiated at the same site asin Fig. 4C (data not shown). Because each replicating moleculecontained only one bubble (Fig. 3), in vitro DNA replicationwas principally initiated at either the inverted repeat region ofthe larger EcoRI fragment of form-A 2-jim DNA or near theEcoRI site in pMB9 and progressed bidirectionally.

Specific initiation of in vitro DNA replication was also sup-ported by pulse-labeling experiments (Fig. 5). After a 1-minincubation, the region between bands A (pMB9) and B (EcoRIlarge fragment of form-A 2-p.m DNA) was primarily labeled onagarose gels. With increasing incubation times, the region be-

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08r B

0.60A

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0.2

04 C

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FIG. 4. Relationship between the centers of replication bubblesand the sizes of bubbles in in vitro replicating pJDB36 plasmid mol-ecules treated with EcoRI. The length of the EcoRI-treated pJDB36products in the 5-min synthesis reactions were measured and classifiedinto three distinctive groups. (A) pMB9 (1.61 ± 0.15 tim). (B) TheEcoRIlarge fragment of 2-Mm form A (1.16 ± 0.06 ,tm). (C) The EcoRI smallfragment of 2-jum form A (0.67 ± 0.07 ,nm). The centers (b) and widths(a) of the replication bubbles were measured as shown at the top of thefigure. ----, Average distance of the replication bubbles from one endof the molecule, corresponding to 0.13 ± 0.07 j.m, 0.41 ± 0.15 Sm, and0.27 ± 0.10 ,m in A, B, and C, respectively. In A, four molecules hadthe replication bubbles significantly further from the others and werenot included in the average. The bars in the ordinate axes indicate thelocation of the 599-base-pair inverted repeats of 2-gm DNA in B andC and the in vivo origin of pMB9 DNA replication in E. coli in A.

tween bands B and C (EcoRI small fragment of form-A 2-pumDNA) became labeled. Because replication forms migrate moreslowly than the native DNA fragments, these data are indicativeof preferential initiation within the B fragment. After a 20- to30-min pulse, radioactivity was detected in all three fragmentsas expected for completely replicated forms. However, this evi-dence was not conclusive because the ligation ofshort fragmentsinto longer forms was inefficient, and such fragments were eas-ily separable from the template. To further investigate thispoint, EcoRI-digested pJDB36 DNA was transferred from agar-ose gels to nitrocellulose, and in vitro replication products were

FIG. 5. Identification of initiation site of in vitroDNA replication.pJDB36 DNA was labeled with [a-32P]dTTP for 20 sec (a), 1 min (b),5 min (c), 10 min (d), and 20 min (e), purified, and analyzed by agarose

gel electrophoresis after EcoRI digestion. The figure shows the scan-ning of the autoradiogram by E.-C. densitometer. Migration was fromleft to right, and the position of each DNAband was identified by stain-ing with ethidium bromide.

used as hybridization probes. Preferential hybridization to theB fragment was observed with replication products from shortincubation times, in support of the above conclusions.

Effect of cdc Mutations on in Vitro DNA Replication. cdc4,-7, and -28 are thought to effect the initiation ofDNA replication(17). The cdc8 gene is required for elongation of DNA strands(7, 8, 18). Table 5 shows temperature inactivation ofthe in vitro2-,um DNA synthesizing activity in crude extracts preparedfrom some of these cdc mutants at 42TC. Contrary to the in vivoobservations, higher temperatures only inactivated the cdc8gene product, whereas the other extracts paralleled the wild-type control. However, if these cells were heated to the re-strictive temperature prior to harvesting, DNA synthesis activ-ity in their subsequent crude extracts was considerably lowereven at permissive temperatures (such as 250C) as reported (4).The only exception was cdc9, the DNA ligase gene, whichshowed no decrease in either case (data not shown).

DISCUSSIONWe have described an in vitro replication system ofyeast 2-pumplasmid DNA. The activity of this system is at least 1 order ofmagnitude higher than the system previously described (4)

Table 4. Electron microscopic analysis of the DNA molecules in vitro replication of2-pm plasmid

Number of molecules*Reaction Super-time, twisted Open-circle ReplicatingDNA Linearmin Total DNA DNA Early Late DNA0 558 503 (90) 49 ( 9) 1 (0.2) 2 (0.4) 3 ( 0.5)5 1343 978 (73) 169 (13) 86 (6) 7 (0.5) 103 ( 8)

20 1719 922 (54) 466 (27) 21 (1) 88 (5) 212 (12)The products of in vitro 2-pum DNA synthesis were purified and analyzed by electron microscopy. After

taking pictures from randomly selected fields, the various DNA species were scored as indicated. Early-replicating molecules were those in which <50% of the 2-;zm plasmid was replicated, whereas late-rep-licating molecules were those in which >50% was replicated.* Percentage is shown in parentheses.

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Table 5. Effect of preincubation on DNA synthesisDNA synthesisactivity at 30(C,(pmol of dTMPincorporated) Ratio,

Crude extract Without With (with/without)Wild type 25.3 20.1 0.79cdc28 15.6 14.8 0.95cdc4 18.3 17.0 0.93cdc7 18.9 17.2 0.91cdc8 13.1 1.8 0.14cdc9 24.9 19.8 0.80

Crude extracts were prepared from the various cdc mutants grownat 230C and preincubated either at 300C or at 42"C for 10 min; theirin vitro DNA synthesis activities were measured at 300C for 30 min inthe presence of pJDB36 as a template.

when a chimeric plasmid consisting ofpMB9 DNA plus the 2-,um DNA is used as template. This system exhibits strict tem-plate specificity, initiates DNA synthesis at the same site usedin vivo (12), and proceeds bidirectionally. The main productswere 4-5S pieces of DNA that were inefficiently ligated, evenafter a 20-min incubation at 30'C. Notably, the crude extractcontains no appreciable DNA ligase activity. When partiallypurified yeast DNA ligase was supplemented into the in vitrosystem, the main products were unit-length linear 2-pum DNAmolecules, although the additional ligase did not stimulate invitro DNA synthesis.The in vitro 2-,um DNA replication origin is within one of

the inverted repeats, 140 ± 50 nucleotides from the end of theinverted repeat proximal to the endonuclease Xba I site (9).However, no particular structural features based on the avail-able sequence data are sufficient to explain this specificity be-cause only one of the two inverted repeats is used as an origin(Fig. 4). Hence, some other unique sequence must confer thenecessary characteristics for preferential initiation at this site.It has been shown that efficient transformation of 2-,um DNAinto yeast cells requires about 100 base pairs adjacent to theinverted repeat, in addition to the inverted repeat containingthe in vivo origin (12).

In addition to the 2-,um DNA and chimeric plasmids con-taining the in vivo 2-pum origin, the E. coli plasmid pMB9 wasan active template for in vitro synthesis (Table 3). Several ob-servations argue against the results being artefactual. Electronmicrographs of in vitro products showed the same type of eyestructure observed in native 2-pum DNA. This structure ap-peared at one specific region of the molecule (Fig. 4), whichdoes not coincide with in vivopMB9 origin in E. coli. However,the nucleotide sequence ofpMB9 does not show extensive ho-mology to the inverted repeats of 2-pum DNA (unpublisheddata).Our crude extracts also supported DNA synthesis in the pres-

ence of YRp7 plasmid DNA (Table 2), which contains a yeastnuclear DNA replicon (11, 19). Electron micrographs show theappearance of an eye structure after brief DNA synthesis. Be-cause pBR322 DNA is poorly replicated by our crude extracts,it can be assumed that this plasmid does not replicate from thepBR322 origin. Hence, in vitro product analysis may provideuseful information regarding a natural chromosomal origin.

Chan and Tye (20) have cloned a number ofDNA segmentsfrom yeast DNA which permit autonomous replication of theyeast LEU2 gene. These inserts represent both unique and re-

petitive segments ofthe yeast genome. Due to the large numberof yeast chromosomal replicons, it is unlikely that each shouldbe recognized by a unique replication complex; hence, somesequence ambiguity is likely at origins recognized by specificreplication complexes. This may explain why our crude extractsreplicate both 2-pm and pMB9 DNAs.The in vitro 2-pm DNA synthesis described here is very sen-

sitive to aphidicolin, a specific inhibitor of eukaryotic DNApolymerase a and yeast DNA polymerases I and II (21). Wehave successfully isolated an aphidicolin-resistant DNA polym-erase I mutant from an aphidicolin-sensitive yeast (unpublisheddata). In crude extracts from this mutant, 2-,um DNA replica-tion was 20-40 times more resistant to aphidicolin than in wild-type extracts, suggesting that the 2-pum DNA replicase is DNApolymerase I.

Although in vivo DNA replication is temperature-sensitivein all the cdc mutants used in this study, only the cdc8 crudeextract was temperature-sensitive in in vitro 2-pum DNA rep-lication. Several explanations are plausible. Residual cdc geneproduct may be sufficient for the relatively low level of in vitrosynthesis, or the mutants may be leaky. Nevertheless, the pos-sitilityrthat our in vitro replication system does not resemblein vivo replication cannot be eliminated.

Finally, in vitro replication products of 2-pum DNA werecharacterized by electron microscopy, and the origin and di-rection of 2-pum DNA replication were determined. The pos-sibility of reiterative initiations at the origin is excluded byDNA-DNA hybridization experiments (Fig. 5), which showthat the in vitro products hybridize to the entire DNA template,rather than just to the region containing the origin.

We are indebted to Drs. N. R. Cozzarelli, L. B. Rothman-Denis, andJ. W. Drake for critical reading of the manuscript; and to Dr. P. 0.Brown for his help on the electron microscopic study.

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860-864.10. Livingston, D. M. (1977) Genetics 86, 73-84.11. Tschumper, G. & Carbon, J. (1980) Gene 10, 157-166.12. Broach, J. R. & Hicks, J. B. (1980) Cell 21, 501-508.13. Davis, R. W., Botstein, D. & Roth, J. R. (1980) Advanced Bac-

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