Proc. Nat. Acad. Sci. USAVol. 69, No. 9, pp. 2518-2522, September 1972

Isolation of Supercoiled Colicinogenic Factor E1 DNASensitive to Ribonuclease and Alkali

(chloramphenicol/Escherichia coli/supercoiled DNA/rifampicin)

D. G. BLAIR*, D. J. SHERRATTt, D. B. CLEWELLT, AND D. R. HELINSKI

Department of Biology, University of California, San Diego, Revelle College (La Jolla) 92037

Communicated by S. J. Singer, June 19, 1972

ABSTRACT The synthesis of the covalently-closed,circular DNA form of colicinogenic factor El (ColEl) con-tinues in Escherichia coli cells after the addition of chlor-amphenicol. A large portion of the purified supercoiledColE, DNA molecules made in the presence of chloram-phenidol are converted to the open circular DNA form aftertreatment with alkali (pH 13), RNase A, or RNase H. Thesetreatments do not significantly affect the covalently-closedform of ColE, DNA isolated from normally growing E. colicells. The open circular product resulting from treatmentof supercoiled ColE, DNA with RNase A possesses a singlebreak in one strand of the circular duplex. The site sensi-tive to RNase A occurs with equal probability in either ofthe complementary strands. Both synthesis of ColE, DNAand the formation of supercoiled ColE, DNA sensitive toRNase A or alkali are prevented by the inhibitor of RNAsynthesis, rifampicin. These results indicate that cova-lently-closed ColE, DNA containing one or more ribo-nucleotides accumulates during ColE, replication in thepresence of chloramphenicol. It is proposed that this in-corporated RNA served as a primer during the initiation ofsynthesis of ColE, DNA and that its removal from thecircular DNA is inhibited in cells incubated in the presenceof chloramphenicol.

The colicinogenic factor El (ColEl) is one of a group of relatedbacterial plasmids found in Escherichia coli that code for theproduction of extracellular, antibiotic proteins. The ColElplasmid is a covalently-closed, circular duplex DNA moleculewith a molecular weight of 4.2 X 106. About 20-30 copies percell are present under conditions of normal logarithmic growth(1, 2). A fraction of these molecules can be isolated in theform of a relaxation complex of supercolled DNA and protein(3). Synthesis of ColE1 DNA continues when protein syn-thesis is inhibited by the addition of chloramphenicol (CM)(4). While synthesis of the bacterial chromosome ceases within1-2 hr after addition of CM, plasmid synthesis is maintainedfor up to 10-20 hr, resulting in the accumulation of as many as1000-3000 copies of supercoiled ColEl DNA per cell (4, 5).Under these conditions, virtually all of the plasmid DNAcan be isolated as covalently closed, protein-free molecules(4). In this report the sensitivity of these molecules to highpH and to certain ribonucleases is described, properties that

Abbreviations: ColE,, colicinogenic factor El; CM, chloram-phenicol.* Department of Chemistry, U.C.S.D.t Present address: Microbial Genetics Group, School of Biology,University of Sussex, Falmer, Brighton BN1 9QF, England.I Present address: Departments of Oral Biology and Micro-biology, University of Michigan, Schools of Dentistry andMedicine, Ann Arbor, Mich. 48104.

2518

indicate that they contain one or more ribonucleotides aspart of their covalently-closed, double-stranded structure.A model will be presented for the formation of these ribonu-cleotide-containing DNA molecules as the result of a re-quirement for an RNA primer for the initiation of ColE,DNA synthesis. An RNA primer was first suggested by Brut-lag et al. (6) to explain the requirement for RNA synthesis inthe conversion of single-stranded M13 bacteriophage DNAto the RFII form. A similar dependence of DNA synthesis onRNA synthesis has also been demonstrated in several othersystems including the plasmid ColEj (5-8).

MATERIALS AND METHODS

Strains and Media. The E. coli K-12 strains, JC411 (ColE,)and CR34 (ColE,), and the Tris HCl and phosphate-bufferedmedia used in these experiments have been described (i, 9).The ColE, plasmid in these strains was derived from E. coliK-30.

Growth and Labeling Conditions. Cells were routinely grownfrom a 2% inoculum to a cell density of 3 to 5 X 108 cells perml. In the absence of chloramphenicol, cells were labeled with10-20 /Ci/ml of ['H]thymine (40-60 Ci/mmol) in the pres-ence of 1 /Ag/ml of unlabeled thymine. Chloramphenicol treat-ment involved the addition of solid chloramphenicol to aconcentration of 150 MAg/ml to cells growing logarithmicallyat a density of 3-5 X 108 cells per ml. Label was added eitherimmediately after addition of CM, or 1-2 hr later, whenchromosomal DNA synthesis had ceased. For labeling with[I4C]thymine, the final concentration was 0.3 MCi/ml in atotal thymine concentration of 2.7 ,ug/ml.For 32p labeling, 200-500 mCi of carrier-free H3 2PO4 was

added per 30 ml of culture. The media contained about 1mmol/liter of unlabeled inorganic phosphate.

Isolation and Purification of DNA. Labeled cells were lysedand the ColEj DNA was partially purified from the bulk ofthe chromosomal DNA by the lysozyme-EDTA-Triton-X100procedure (3, 9). Supercoiled ColE, DNA was isolated by thedye-bouyant density procedure of Radloff et al. (10). Theethidium bromide was removed by 1 or 2 extractions at 40with CsCl-saturated isopropanol (11). CsCl was then removedby dialysis at 4° against TESP [50 mM Tris HCl (pH 8)-50mM NaCl-5mM EDTA-50 mM K2HPO4].

Analysis of Alkali-induced Breakdown of Supercoiled ColE,DNA. ColE, DNA (0.1-1 ,ug) was incubated at pH 13 and370 in a total volume of 200 Mul consisting of 20 ,ul of

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RNase-Sensitive ColE, DNA 2519

purified DNA in TESP plus 180 Ml of 0.2 M K2HPO4 (pH13). After incubation, the reaction mixture was neutralizedby addition of 200 jl of 1.0 M Tris*HCl (pH 7.5). 300 ulof the neutralized mixture were then layered on a 5-ml 5-20% sucrose gradient containing 50 mM Tris-1.05 M NaCl-5 mM EDTA and centrifuged 110 min at 45,000 rpm and 150in a Spinco SW50.1 rotor in a Beckman model L2 or IA prepar-ative ultracentrifuge. Under these conditions, irreversiblydenatured, covalently-closed, circular DNA sediments to aposition near the bottom of the tube, while denatured strandsof ColE1 DNA and any undenatured or renatured ColE1 DNAare found near the center of the tube as two clearly resolvedpeaks. The denatured material is found in the leading peak inthis region, ahead of the position of native supercoiled ColE1DNA.

RNase Treatment of Supercoiled ColE1 DNA. PancreaticRNase A at a concentration of 3 mg/ml in 9 mM Tris HCl(pH 7:5) was first treated by heating for 5 min in a boilingH20 bath. The solution was then quickly cooled on ice andkept at 40 until use. Incubations were performed at 370 in afinal reaction volume of 300 Al. The reaction mixtures con-tained 0.1-1ug of ColE1DNA and 300 Ag of heat-treated RNaseA in the following buffer: 36 mM Tris*HCl (pH 8.0)-33 mMNaCl-3mM EDTA-13 mM K2HPO4. After RNase incubationfor the desired time, 100 Ml of a solution of 5 mg/ml of Pronasein TES [50 mM Tris HCl (pH 8.0)-50 mM NaCl-5 mMEDTA] was added to each reaction mixture and incubationwas continued for 10 min at 37°. The Pronase had been di-gested for 30 min at 370 to eliminate possible nuclease con-tamination. After Pronase treatment, a 0.3-ml portion of eachmixture was analyzed by centrifugation in a 5-ml, 5-20%sucrose gradient for 165 min in the Spinco SW50.1 rotor. Thesucrose gradient contained 50 mM Tris. HCl (pH 8)-0.55 MNaCl-5mM EDTA. The Pronase treatment was necessary toinsure good recoveries of DNA from the sucrose gradients.Without this treatment, the ColE1 DNA pelleted to the bottomof the centrifuge tube, apparently as a result of binding toRNase molecules.RNase T1 and RNase H incubations were performed in a

similar fashion. RNase T, reaction mixtures (200,gl) contained0.1-1 Ag ColE1 DNA and 300-3000 units of T, ribonuclease in55 mM Tris HCl-20 mM EDTA-5 mM NaCl at pH 7.5.Pronase treatment was as described above. Both heated andunheated T, RNase were tested. For RNase H, the buffer was2 mM Mn++-20 mM (NH4)2S04-36 mM Tris * HCl-1 mM 2-mercaptoethanol-6 mM NaCl, at pH 7.9. The heat-labileRNase H was not heated before use.

Alkaline CsCl Centrifugation. DNA was centrifuged in aSpinco SW56-Ti rotor for 36-40 hr at 40,000 rpm in a 1.5-mlgradient containing 1.38 g CsCl, 30 Ag bovine-serum albumin,and 0.02% Sarkosyl, in 0.12 M Na3PO4. The uncorrected pHof this solution was 12.6-12.7 (Radiometer type B electrode-GX2301B standardized with Beckman pH 12.45 buffer solu-tion no. 3010).

Reagents and Enzymes. The sources of most of the reagentsused have been described (3, 9). Ribonuclease A (beef pan-creas, 3000 units/ml; code RASE) and ribonuclease T1 (3 X105 units/mg; code RT,) were obtained from WorthingtonBiochemical Corp., Freehold, N.J. Pronase CB and chlor-amphenicol were obtained from Calbiochem., San Diego,Calif. Ribonuclease H was generously provided by Dr. Gordon

E6

)ICci

FRACTION NUMBERFIG. 1. Alkaline CsCl equilibrium centrifugation of a mixture

of CM and non-CM ColE1 DNA. Covalently-closed ColE1 DNAwas purified from logarithmically growing JC411 (ColEi) beforeand after incubation of the cells in the presence of chloramphen-icol (150 Mg/ml) for 18 hr. Conditions of alkaline CsCl centrifuga-tion, fractionation, and counting were as described in Methods.(0-0) 3H-labeled non-CM ColE1 DNA. (0- - -0) 32P-labeledCM ColE1 DNA.

Gill. Rifampicin (rifampin) was a gift of the CIBA Pharma-ceutical Company, Summit, N.J.

RESULTSAlkaline sensitivity of covalently-closed ColE1 DNA

The differential behavior in E. coli strains JC411 (ColE1) orCR34 (ColEj) of supercoiled ColE1 DNA synthesized in thepresence and absence of CM was first noted when purifiedmixtures (see Methods) of the two types of DNA were centri-fuged to equilibrium in alkaline CsCl (pH 12.8). As shown inFig. 1, a large fraction of the supercoiled DNA from CM-treated JC411 (ColE1) cells (CM ColEj) is converted from thecovalently-closed form, banding in the dense position, to aform banding in the lower density (single-stranded) positionby the introduction of one or more breaks in the closed DNAhelix. ColE1 DNA isolated from cells growing logarithmicallyin the absence of chloramphenicol (non-CM ColE1) is lesssensitive to conditions of alkaline centrifugation, since a muchsmaller fraction bands in the single-stranded position. Thishigh pH-induced conversion was examined in more detail inthe experiment shown in Fig. 2. Mixtures of CM and non-CM ColE1 supercoils were incubated for various times at pH13 in phosphate buffer, after which the pH was reduced to 8and the mixtures were analyzed by sucrose gradient velocitysedimentation to determine the fraction of covalently-closedDNA remaining. As shown in Fig. 2, CM ColE1 DNA loses itscovalently-closed structure during incubation at high pH,while non-CM ColE1 is only slightly affected by the alkalineconditions during the experiment. Denaturation of CM-ColE1DNA at pH 13 at 40 followed immediately by neutralizationdoes not result in a significant loss of its covalently-closedstructure. The alkaline-sensitive supercoiled CM ColE1 DNAwas indistinguishable from supercoiled non-CM ColE1 DNAby other criteria. Both exhibit identical sedimentationbehaviorin neutral sucrose density gradients and the same bouyantbehavior during neutral CsCl and CsCl-ethidium bromideequilibrium centrifugation, and appear the same when ex-amined in the electron microscope.

Sensitivity ofCM ColE1 DNA to ribonucleasesThe observation by Clewell et al. (5) that ColE1 DNA syn-thesis in the presence of chloramphenicol could be inhibited

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100 Lnormal supercoiled Cal El

60 - CM supercoiled

40 \

20 CM-supercoiled Col El

0 Jo0 6 12 18 24 30 40

MINUTES OF INCUBATION

e

C=

300 E.X-

FRACTION NUMBER70 100

FIG. 2. Stability of covalently-closed ColE1 DNA duringtreatment at pH 13 in 0.18 M P04-3* Samples of ColE1 DNA,similar to that described in Fig. 1 (prepared by Dr. P. Williams),were incubated at the temperatures and for the times indicated,then neutralized and analyzed on 5-20% sucrose gradients con-taining 1.05 M NaCl. Supercoiled DNA is expressed as a per-centage of total ColE1 DNA recovered from the gradients. Re-coveries of 70% or more of the counts layered were obtained forboth labels in all cases but one, the 1-min incubation at 370,where the 3H recovery was 64%, compared to a 32p recovery of95%. (- * and 0---O) incubations at 370; (A--A andA- - -A) incubations at 600.

by the inhibitor of RNA synthesis, rifampicin (12), implicatedRNA involvement in the ColE1 replication process. Since thepresence of RNA in supercoiled CM ColE1 DNA could accountfor its alkaline sensitivity, it was decided to test the sensitivityof this DNA to various ribonucleases. The results of incubat-ing a mixture of differentially labeled CM and non-CM ColE1supercoiled DNA in the presence of pancreatic ribonuclease Aare shown in Fig. 3. The sucrose gradient profiles (a and b)show that a lO-min treatment with 1 mg/ml of RNase A con-verts over 40% of the CM ColE1 DNA from a form sedimentingas a covalently-closed molecule to one sedimenting at the ratecharacteristic of an open circular molecule. The differentiallylabeled non-CM ColE1 DNA, present as an internal control,is not affected by this treatment. No further conversion of thesupercoils could be induced by the subsequent addition offresh ribonuclease. The RNase A used in all incubations hadbeen treated by heating for 5 min at 1000 to inactivate anydeoxyribonucleases that might be present. Prior phenol treat-ment of CM ColE1 DNA does not alter its sensitivity to RNaseA. The fraction of CM-supercoils that were resistant to pan-creatic RNase were no more sensitive to alkali treatment [40hr, alkaline CsCl (pH 12.8)] than non-CM supercoiled ColE1DNA. The incubations in this experiment were done with anexcess of RNase over that needed to induce a detectable con-version, in that 25 ,g/ml of RNase A was sufficient to producedetectible nicking in 60 min at 370 under these conditions.For determination of the nature of the products of the RN-

ase A treatment, the CM CoLE1 DNA, after incubation withRNase A, was banded in a dye-CsCl gradient and the lower(covalently-closed) and upper (open) bands, as well as un-treated starting material, were examined in the electron micro-scope. Essentially 100% of the untreated CM ColE1 DNA waspresent in the supercoiled form. The heavy peak isolated fromthe RNase-treated sample was also predominately super-coiled DNA, while the light peak was essentially all open cir-cular material, with C->

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RNase-Sensitive ColE1 DNA 2521

from one such experiment is shown in Fig. 4a. The ratio ofcircular to linear strands indicates that at least 80% of thestarting open circular DNA duplexes contained only onenicked strand. In other experiments this fraction has ap-proached 100%. The absence of a significant amount of trail-ing from the slower sedimenting peak of linear material indi-cates that most of the nicked DNA strands contained onlyone RNase-sensitive site.The separated linear and circular pools were then analyzed

by poly(UG)-CsCl centrifugation by the method of Szybalski(14) as described (9). The profiles for the two pools (Fig. 4band c) indicate that both the circular and linear strands con-sisted of equal numbers of each of the complementary ColE1DNA strands. The RNase-sensitive site thus appears to bedistributed randomly with respect to the two strands in CMColE1 DNA.

Effect of rifampicin on the formation of RNaseA-sensitive ColE, supercoilsAs has been shown, supercoiled ColEj DNA made in the ab-sence of chloramphenicol is not sensitive to RNase or alkali.

to

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18 _

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(a)

(b)

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heay

I(c)

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FRACTION NUMBER

FIG. 4. Analysis of the strand specificity of the nick(s) inducedby RNase A in CM ColE, supercoiled DNA. Covalently-closedColE, DNA was isolated by equilibrium centrifugation fromJC411 (ColE,) cells incubated in the presence of 150 Ag/mlchloramphenicol for 7 hr. The [3H]thymine label was added tothe culture after 2 hr in chloramphenicol. The supercoiled ColE,DNA was further purified on a 5-20% sucrose gradient. Thismaterial was treated for 60 min at 370 (see Methods) and rebandedin a dye-CsCl gradient. The upper band (open circular DNA)was then pooled and the ethidium bromide and CsCl were re-moved. A 0.2-ml portion of this material was then layered on a5 ml, 5-20% alkaline sucrose density gradient and centrifugedfor 225 min at 55,000 rpm, 15°, in an SW65 rotor; 4-drop frac-tions were collected, and 1O-ul aliquots of each fraction werespotted and counted. The peak fractions were then pooled,neutralized, and analyzed on CsCl gradients containing poly(UG)as has been described (9). (a) Alkaline sucrose velocity sedi-mentation; (b) poly(UG)-CsCl equilibrium centrifugation of thematerial from the single-stranded linear pool of (a); (c) poly-(UG)-CsCl equilibrium centrifugation of the material from thesingle-stranded circular pool of (a).

RNase sensitivity can not be detected until 1-2 hr after theaddition of CM to a logarithmically growing culture of JC411(ColEj). The relative percentage of sensitive ColE, supercoilsthen continues to increase at roughly a linear rate. WhenDNA synthesis, as detected by the incorporation of radio-active label into DNA, ceases in the culture, the increase inthe fraction of sensitive ColE, supercoils stops. When the cul-ture is incubated, with aeration, at 370 for 10-20 hr afterDNA synthesis has ceased, there is no change in the level ofRNase- or alkali-sensitive ColE1 DNA. These results are con-sistent with a requirement for active ColE, DNA synthesis forthe generation of the sensitive form of CoLE, DNA.

In support of this hypothesis, rifampicin prevents both thesynthesis of ColE, DNA and the generation of the RNase- oralkali-sensitive form of ColE, in the presence of chlorampheni-col. When chloramphenicol-treated cultures that are activelysynthesizing ColE, DNA (a fraction of which is RNase- andalkaline-sensitive) are treated with 3 Ag/ml of rifampicin,DNA synthesis ceases and no further increase in the percent-age of sensitive ColE, supercoils is detected over a 4-hr period.Control cultures that are not treated with rifampicin continueto synthesize ColE, DNA and to generate sensitive ColE,supercoils.

DISCUSSION

The direct dependence of DNA replication on RNA synthesishas been proposed on the basis of scattered observations fromseveral systems (15, 16). Recently, the number of cases wheresuch A relationship appears to exist has increased rapidly withthe finding that DNA synthesis in several systems is sensitiveto inhibition of RNA synthesis by inhibitors of RNA poly-merase such as rifampicin and streptolydigin (5-8). Thissensitivity does not appear to be a universal property, how-ever, since DNA synthesis of the replicative form of phage,X174 is resistant to rifampicin (17).

Covalently-closed

Insertion of DNA(W) RNA Primer Replication -

Completionof

Replication

Removal g ( )Repar or

/~Z blocked ord, I+('(f\~ reduced by\ /CM addition)

FIG. 5. Primer model for the formation of RNA-containingColE, supercoiled DNA. We propose that the initiation of DNAsynthesis involves the rifampicin-sensitive synthesis of RNA thatis complementary to a single site on either DNA strand. DNAsynthesis involves the addition of deoxyribonucleotides to thisprimer RNA. Either a single RNA primer, complementary toeither strand, is involved in the duplication of any one circularDNA molecule and synthesis proceeds unidirectionally, or syn-thesis proceeds bidirectionally from two RNA primer moleculespresent in a single replicating DNA molecule. Normally, theRNA primer is removed upon completion of replication of thecircular DNA, however, in the presence of chloramphenicol theRNA-containing, supercoiled DNA molecules accumulate.

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Where it appears to be required, RNA synthesis has beenpostulated to function either directly or indirectly in theinitiation of DNA synthesis. For conversion of M13 single-stranded phage DNA to the double-stranded RFII form, ithas been demonstrated that, at least in vitro, RNA acts as aprimer and is covalently joined to the newly synthesizedDNA strand (18). The idea that RNA might serve as a primerfor DNA synthesis is attractive, since it offers a way of initiat-ing DNA synthesis using existing DNA polymerizing enzymes,none of which are capable of initiating de novo synthesis invitro in their purified forms (19, 20).The evidence we have presented here suggests that when

protein synthesis is inhibited by chloramphenicol, covalently-closed ColE1 DNA containing one or more ribonucleotides, ineither one strand or the other, accumulates in colicinogenicE. coli cells during the period that these cells are activelysynthesizing ColE, DNA. Such structures, containing a shortsegment of DNA-RNA hybrid, would clearly be expected tobe sensitive to alkaline and RNase H hydrolysis, and evidencesupports the notion that these molecules would also be sensi-tive to RNase A (21, 22). The lack of sensitivity to RNase T,may imply the absence of ribosylguanine in the molecules orperhaps some unknown structural limitations to T, action inthe case of a hybrid substrate. The possibility that an unusualdeoxyribonucleotide might be responsible for these unusualproperties of the ColE1 DNA cannot be rigorously excluded,but it seems unlikely that such a base would be sensitive toalkali and to two different ribonucleases.The existence of a covalently-closed DNA duplex containing

RNA raises the question of its origin and function in DNAmetabolism. One possibility is that it arises as an artifact ofthe long-term exposure of the colicinogenic cells to chlor-amphenicol, which perhaps induces infrequently the erroneousinsertion of a ribonucleotide during DNA polymerization.Polymerase I will make such errors in the presence of Mn++(22), and ColE1 requires polymerase I for normal plasmidmaintenance (23). This model cannot be ruled out at this time,but in view of the rifampicin sensitivity of CotE1 synthesis, anessential role of RNA in the process is suggested. Evidence hasbeen obtained for a role for RNA as a primer in the initiationof DNA synthesis in the conversion of M13 viral single strandsto the double-stranded replicative form (6, 18). Fig. 5 illus-trates a possible model for the formation of RNA-containingCotE1 supercoils as a result of such an initiation step. Theobservation that the RNase-sensitive segment of ColE1 DNAis not strand-specific requires, according to this model, thateither initiation is unidirectional and random with respect tothe DNA strand, or synthesis is bidirectional and initiated

with short RNA segments from both strands of the replicatingmolecule. One might expect, however, that the site of initia-tion on a particular strand is specific.We thank Mr. Bernard Ashcraft for his excellent technical

assistance and Dr. Gordon Gill for his generous provision ofthe enzyme RNase H. This work was supported by U.S. PublicHealth Service Research Grant AI-07194 and National ScienceFoundation research grant GB-29492. D. G. B. was supportedby a U.S. Public Health Service Predoctoral Traineeship (2-T01-6M-1045). D. R. H. is a U.S. Public Health Service ResearchCareer Development Awardee (K04-6M07821).

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2522 Biochemistry: Blair et al.

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