Proc. Nat. Acad. Sci. USA Vol. 71, No. 10, pp. 3854-3857, October 1974

Relaxation Complexes of ColEl and ColE2: Unique Site of the Nick in the Open Circular DNA of the Relaxed Complexes (/supercoiled DNA/endonuclease/restriction /DNA replication) MICHAEL A. LOVETT, DONALD G. GUINEY, AND DONALD R. HELINSKI Department of Biology, University of California, San Diego, La Jolla, Calif. 92037 Communicated by Stanley L. Miller, June 26, 1974

ABSTRACT The product of the induced relaxation of volved in the production of a single-strand cleavage in the supercoiled DNA-protein relaxation complexes of colicino- initiation of replication and/or conjugal transfer of genic factors El (ColEl) and E2 (CoIE2) is an open circular DNA molecule with a strand-specific nick. Cleavage of the DNA. An involvement in such processes would most likely open circular DNA of each relaxed complex with the EcoRI require that the relaxation event take place at a unique site restriction endonuclease demonstrates that the single- in the DNA molecule. In this report, evidence will be pre- strand break is at a unique position. The site of the single- sented from studies using the EcoRI restriction endonuclease strand break in the relaxed ColEl complex is approximately the same distance from the EcoRI cleavage site as the that the strand-specific relaxation event takes place at a origin of ColEl DNA replication. unique site on both the ColEl and CoIE2 plasmid molecules. The location of this site, at least in the case of the ColEl Many of extrachromosomal circular DNA elements (plas- plasmid, is approximately the same distance from the single mids) of Escherichia coli, differing in molecular weight, num- EcoRI site as the origin of replication for this plasmid. ber of copies per cell, and in the properties they confer to their host, have been isolated as both supercoiled DNA- MATERIALS AND METIODS protein relaxation complexes and supercoiled DNA without Strains, Media, and Labeling Conditions. The E. coli K12 associated protein (1). Treatment of the plasmid DNA- strains JC411 thy-(ColEl) and JC411 thy-(ColE2) have protein relaxation complexes in vitro with agents capable of been described (6). M9 medium (7) contained 0.2% (w/v) altering protein structure induces the conversion of the super- glucose and 0.005% (w/v) of each of the required amino coiled DNA in the complex to the open circular (relaxed) acids. For [IH]thymine labeling, cultures contained 1 ug/ml DNA form. The relaxation complexes of the colicinogenic of nonradioactive thymine and 10-20 ,uCi/ml of [methyl-'H]- factors, ColEl (2) and ColE2 (3, 4), the sex factor F1 (5), thymine. For [(4C]thymine labeling, 10 pCi of ["4C]thymine and the R factor R6K (Kupersztoch, Lovett, and Helinski, containing 27 pg of thymine was added to each 15 ml of me- submitted for publication) have been studied in the greatest dium. The amount of plasmid DNA present as relaxation com- detail. Sodium dodecyl sulfate-induced relaxation of each of plex was maximized by the addition of cyclic adenosine mono- these complexes produces one strand-specific break. The phosphate (cAMP) to a final concentration of 2.5 mM (6). properties of the relaxation complexes suggest that they con- Under these conditions, about 80% of CoIEl DNA and 60% sist of supercoiled DNA and a latent strand-specific endo- of ColE2 DNA are isolated in the form of relaxation com- that is activated by treatment with certain protein- plex. denaturing agents. In each case the broken strand is the poly(U,G) binding or "heavy" strand. More recently, it Preparation of Complexed and Noncomplexed Plasmid DNA. has been shown that the supercoiled ColEl complex has Relaxation complexes of ColEl and ColE2 were prepared as three major protein components whose molecular weights are described by Clewell and Helinski (2), with the modification 60,000, 16,000, and 11,000 (Lovett and Helinski, manuscript that crude lysates were made by the addition of Triton X-100 in preparation). After the induction of relaxation with sodium to spheroplasts (6) followed by centrifugation at 46,000 X g dodecyl sulfate, the 60,000-molecular-weight protein alone for 25 min to pellet most of the chromosomal DNA. The remains in association with the broken strand even under a supernatant (cleared lysate) was centrifuged through 38-ml variety of conditions that normally dissociate protein from sucrose density gradients containing 50 mM Tris-HCl (pH DNA (Blair and Helinski, manuscript in preparation). The 8.0), 5 mM EDTA, 50 mM NaCl for 16 hr at 40 at 25,000 protein remains associated with the 5' end of the broken rpm in a Beckman SW 27 rotor. Plasmid-containing fractions strand (Guiney and Helinski, manuscript in preparation). were pooled and precipitated with ethanol. After resuspension It has been proposed that a covalent bond between the 5' with Tris-EDTA-NaCl buffer containing 0.5%7O sodium terminus of the DNA and the 60,000-dalton protein is formed dodecyl sulfate to induce relaxation, the DNA was centri- upon relaxation of the ColEl complex. The ColE2 relaxation fuged through 5-iml sucrose density gradients containing Tris- complex has been found to exhibit similar properties upon EDTA-NaCl buffer with 0.5 M NaCl for 140 min at 150 at treatment with sodium dodecyl sulfate. 50,000 rpm in a Beckman SW 50.1 rotor to separate 23S These novel properties of plasmid DNA protein complexes supercoiled plasmid DNA from 17S open circular DNA. have led to the suggestion that relaxation complexes are in- The open circular DNA was precipitated with ethanol and suspended in 0.1 M Tris HCl (pH 7.5), 50 mM NaCl, 5-10 Abbreviations: ColEl and ColE2, colicinogenic factors. mM MgCl2 for digestion with the EcoRI endonuclease. 3854 Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Unique Site of the Nick in Plasmid DNA 3855

For preparation of supercoiled ColEl and ColE2 DNA, cells were grown in M9 glucose medium without the addition of cAMP. To minimize the amount of Co1E2 DNA present as relaxation complex, cleared lysates of JC411 (CoIE2) were heated at 600 for 20 min to inactivate the relaxation com- plex (4); Sarkosyl was added to a final concentration of 0.05% (w/v) and the lysate was centrifuged to equilibrium in a cesium chloride-ethidium bromide density gradient (8). Supercoiled ColE1 DNA was also prepared by the dye-buoy- ant density procedure. After centrifugation, the supercoiled were extracted four to six times with CsCl-saturated isopropanol (9) and then precipitated with ethanol. After resuspension with Tris-EDTA-NaCl buffer, the plasmid DNA was centrifuged through 5-ml 5-20% sucrose density gra- Fraction Nunber dients as described above, and the 23S supercoiled DNA was FIG. 1. Cleavage of ColEl and ColE2 DNA with the EcoRI then precipitated with ethanol and suspended in RI buffer. restriction endonuclease. ['H] Thymine-labeled DNA of the ColEl relaxed complex, prepared as described in Materials and Agarose Gel Electrophoresis of DNA. Electrophoresis of Methods, was analyzed on neutral 5-20% sucrose density gra- DNA was performed in agarose gels set in cylindrical Plexi- dients before and after digestion with the EcoRI endonuclease. glas tubes of 6-mm internal diameter (15-cm lengths). Agarose [14C]Thymine-labeled DNA of the ColEl relaxed complex was of the indicated concentration was added to electrophoresis added as a sedimentation marker. Centrifugation was carried out buffer [Tris-phosphate-dodecyl sulfate buffer (Loening) (11) in a Spinco SW 50.1 rotor at 50,000 rpm for 140 min at 15°. mM 30 mM 1 mM Ten-drop fractions were collected from the bottom of the gra- containing 36 Tris, NaH2PO4, EDTA, dients directly into vials containing Triton scintillation fluid. and 0.05% dodecyl sulfate (pH 7.7)], dissolved by auto- [3H]Thymine-labeled DNA of the ColE2 relaxed complex was claving for 15 min, and poured into rubber-capped gel tubes. analyzed similarly before EcoRI digestion and after EcoRI After the gels hardened, the rubber caps were removed, the digestion with ColEl supercoiled and open circular [32P]DNA top 5 mm of the gels were sliced off to provide a flat surface, (prepared by Dr. P. Williams) added as a sedimentation marker. and the bottoms of the gel tubes were covered with dialysis ColEl DNA (A) before and (B) after digestion with the EcoRI tubing. DNA samples were denatured with 0.1 volume of endonuclease. ColE2 DNA (C) before and (D) after digestion 1 M NaOH, as described by Hayward and Smith (11), before with the EcoRI endonuclease. electrophoresis for analysis of single-stranded DNA. To assay radioactively labeled DNA, the gels were sliced into 1-mm discs single-stranded fragments of less than unit size derived from and their radioactivity was determined in 10 ml of a toluene the broken strand. solution containing 4 g/liter of Omnifluor and 5% Protosol The product of EcoRI digestion of 23S supercoiled or 17S (New England Nuclear Corp.) after they were shaken on a open circular ColEl DNA derived from the induced relaxa- rotary shaker for at least 2 hr at 37°. tion of the ColEl complex sediments as a linear 15S molecule EcoRr Endonuclease Digestion. The buffer for EcoRI di- in neutral sucrose density gradients (Fig. 1A and B). There gestion contained 0.1 M Tris- HCO (pH 7.5), 50 mM NaCl, are no detectable fragments of less than unit size (4.2 X 106 and 5-10 mM MgCl2. EcoRI endonuclease was the generous molecular weight), indicating that EcoRI cleavage produces gift of Herbert Boyer. Incubations were for 5-30 min at 37°. one break in the ColEl molecule. Analysis of ColEl DNA cleaved by EcoRI, by electron microscopy and by electro- Reagents. [methyl-3H]Thymine (40-60 Ci/mmol) and phoresis in a 1% agarose gel with the EcoRI cleavage prod- [2-14C]thymine (46 mCi/mmol) were obtained from New ucts of X DNA (13) as molecular weight markers have con- England Nuclear Corp., Boston, Mass. Agarose was pur- firmed that it is a linear molecule of unit size. chased from MCI Biomedical, Rockland, Md. cAMP was purchased from Sigma Chemicals, St. Louis, Mo. Sodium Cleavage of ColE2 DNA with EcoRI Endonuclease. EcoRI dodecyl sulfate was obtained from Gallard-Schlesinger, cleavage of supercoiled or open circular ColE2 DNA derived New York, N.Y. from the induced relaxation of ColE2 complex produces two fragments that sediment more slowly than intact ColE2 RESULTS DNA in neutral sucrose density gradients (Fig. 1C and D). Electrophoresis in a 2.3% agarose gel with the Hin restric- Cleavage of ColEl DNA with EcoRI Endonuclease. The tion endonuclease digestion products of simian virus 40 DNA EcoRI restriction endonuclease has been shown to cleave as molecular weight references (14) showed that the small double-stranded DNA at a unique sequence (12). fragment of ColE2 DNA cleaved by EcoRI has a molecular By use of EcoRI cleavage sites as reference points, it has weight of about 3.5 X 105. Electrophoresis in a 1% agarose been possible to test whether the single-strand breaks found gel with the EcoRI cleavage products of X DNA as markers in the relaxed DNA form of the plasmid relaxation complexes showed that the larger fragment has a molecular weight of are located at unique positions. If relaxation events break 3.85 X 106. one DNA strand at a unique site on the plasmid DNA, EcoRI cleavage of the relaxed DNA should produce a double- Site of the ColEl Relaxation Event. A portion of the EcoRI stranded fragment of the DNA containing the strand-specific digest of relaxed ColEl complex, shown in Fig. 1 to be a break which, when exposed to alkaline conditions, would linear, unit-sized molecule upon neutral sucrose density yield one single-stranded fragment of unit size and two gradient centrifugation, was analyzed in an alkaline sucrose Downloaded by guest on October 2, 2021 3856 Biochemistry: Lovett et al. Proc. Nat. Acad. Sci. USA 71 (1974)

3 - (A) COIEl-EcoRI CGIEZ-(coRI I lon 2 42 - 8 -1 so 6 - 12 so~~~~~~~~~~~~3o 0 I 6 to -I ®-I Co/El.EcoRI 0 A 8 CdE2.&coRI \-D./ A~~ 7 coK t6 4;7) 3 Doo l 5 - C -~~~~ (- 2 6 4 4 2 10 3 32 2 11 300 & C. 0 0 6I C6m, I0 20 30 40 50 60 70 80 4C ©> CoIEl .EcoRI D0L 4 F.) A CoIE2#EcoRf 160 24 100 A B 3 40 120 '00 2 80 I 300 C] 60 54 40 C ^ k J---k , -A-- D -, :L 20 10 20 30 40 50 60 70 _o-A . . n_fv - Il M - -W Fraction Number -l10 20 30 40" 80 0*0 ------Mo NO Fraction mbar FIG. 2. Site of the strand-specific break in the relaxed ColEl DNA-protein complex. [3H]Thymine-labeled DNA of the ColEl FIG. 3. Site of the strand-specific break in the relaxed ColE2 relaxed complex, prepared as described in Materials and Methods, DNA-protein complex. [3H]Thymine-labeled DNA of the was analyzed on alkaline (0.3 M NaOH) 5-20% sucrose density ColE2 relaxed complex and [14C]thymine-labeled supercoiled gradients (A) before and (B) after digestion with the EcoRI ColE2 DNA, prepared as described in Materia1s and Methods, endonuclease. [14C]Thymine-labeled DNA of the ColEl relaxed were mixed and analyzed on alkaline (0.3 M NaOH) 5-20% complex was added as a sedimentation marker. Centrifugation sucrose density gradients (A) before and (B) after cleavage with was carried out in a Spinco SW 50.1 rotor at 50,000 rpm for 230 the EcoRI endonuclease. Centrifugation was carried out in a min at 150. Four-drop fractions were collected from the bottom Spinco SW 50.1 rotor at 50,000 rpm for 240 min at 15°. Four- of the gradients onto filter paper squares. [3HIThymine-labeled drop fractions were collected from the bottom of the gradients DNA of the ColEl relaxed complex cleaved by EcoRI, denatured onto filter paper squares. ['HJThymine-labeled DNA of the with alkali, was analyzed (C) by electrophoresis in a 2.1% ColE2 relaxed complex cleaved by EcoRI, followed by denatura- agarose gel for 180 min at 90 V, as described in Materials and tion with alkali, was analyzed (C) by electrophoresis in a 2.5% Methods. agarose gel for 225 min at 90 V, as described in Materials and Methods. density gradient with an added marker of uncleaved, relaxed CoIEl complex (Fig. 2A and B). The products of EcoRI gradient (Fig. 3A and B). There are two fragments, A and D, cleavage of the relaxed ColEl complex are one fragment are derived from both ColE2 DNA and whose size is identical to linear single strands of ColEl DNA, that supercoiled -relaxed ColE2 complex. Fragments A and D exhibit sedimen- A, and two fragments of less than unit size, termed fragment tation coefficients for A and 6.5S for D) that correspond termed B and C. The size of fragments B and C has been de- (17S to molecular weights of 1.9 X 106 and 1.8 X 106, respectively. termined by alkaline sucrose density gradient centrifugation, are two additional fragments, B and C, whose sizes with the RI digestion products of X DNA and supercoiled There are 1.7 X 106 daltons (10.2S) and 2.5 X 105 daltons (7.68), ColE2 DNA as molecular weight markers. Correspondence which are derived only from the relaxed CoIE2 complex. The between size DNA fragments was determined and s2o, for the B C indicates that the relaxation by the equation of Studier (15) for denatured DNA in alkaline existence of fragments and event occurs at one unique site on the larger of the two sucrose containing 1 M NaCl. The size of fragment B (16.3S) double-stranded by RI cleavage of is 1.7 X 106 daltons and the size of fragment C (9.2S) is 4.0 fragments produced ColE2 DNA. Alkaline-denatured relaxed ColE2 complex, X 106 daltons, or about 19% of unit size. Similar results were was examined electro- obtained where the RI digestion products of relaxed ColEl cleaved by EcoRI, also by agarose gel phoresis with results similar to those found by alkaline sucrose complex were examined by agarose gel electTophoresis after denaturation with alkali (Fig. 2C). The generation of two gradient analysis (Fig. 3C). discrete fragments of less than unit size by EcoRI cleavage DISCUSSION of relaxed ColEl complex indicates that the strand-specific A feature of the plasmids studied in E. coli is their existence as break in the relaxed DNA takes place at one unique site on relaxation complexes of supercoiled DNA and protein. In the plasmid DNA. each case examined in detail the product of induced relaxa- Site of the ColE2 Relaxation Event. The products of RI tion of the plasmiid DNA is an open circular molecule with a cleavage of- relaxed ColE2 complex have been analyzed in a single strand-specific break. This report has shown that the similar manner as those of CoWE1. A portion of the RI digest strand-specific break is located at a unique site on the ColEl of a mixture of ColE2 relaxed complex and ColE2 supercoiled and ColE2 molecules with respect to an EcoRI endonuclease DNA (Fig. 1D) was analyzed in an alkaline sucrose density cleavage site. Recently, it has been shown also for the plas- Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Unique Site of the Nick in Plasmid DNA 3857

mids R6K (Kupersztoch, Lovett and Helinski, manuscript the M13 gene 2 and 4X174 gene A are the bacteriophage in preparation) and pSC101 (Lovett, unpublished observa- equivalents of plasmid relaxation complex proteins acting tions) that the site of the single-strand break in the DNA of at the origin of DNA replication. the relaxed complex is unique. While bacterial restriction We thank Dr. Herbert Boyer for his generous provision of endonucleases have been shown to break both strands of a EcoRI restriction endonuclease. This work was supported by DNA duplex at a unique nucleotide sequence (12, 16, 17), no U.S. Public Health Services Research Grant AI-07194 and other endonucleases have been described whose action is the National Science Foundation Grant GW329492. site-specific cleavage of one strand of a DNA duplex. 1. Helinski, D. R. (1973) Annu. Rev. Microbiol. 27, 437-470. 2. Clewell, D. B. & Helinski, D. R. (1969) Proc. Nat. Acad. Sci. By the approach of Fareed et al. (18) for the determination USA 62, 1159-1166. of the origin and direction of replication of DNA of simian 3. Clewell, D. B. & Helinski, D. R. (1970) Biochem. Biophys. virus 40, it has been demonstrated, by EcoRI cleavage of Res. Commun. 40, 608-613. replicating ColEl molecules isolated from intact E. coli cells, 4. Blair, D. B., Clewell, D. B., Sherratt, D. J. & Helinski, D. R. that ColEl DNA replication proceeds unidirectionally from (1971) Proc. Nat. Acad. Sci. USA 68, 210-214. 5. Kline, B. C. & Helinski, D. R. (1971) Biochemistry 10, an origin 18.3 i 0.9% of unit length from one end of the 4975- 4980. EcoRI-cleaved molecule (Lovett, Katz, and Helinski, sub- 6. Katz, L., Kingsbury, D. T. & Helinski, D. R. (1973) .J. mitted for publication). The site of the nick produced by in- Bacteriol. 114, 577-591. duced relaxation of the ColEl complex is approximately 19% 7. Roberts, R. B., Abelson, P. H., Cowie, D. B., Bolton, E. T. & Britten, R. J. (1963) Studies of Biosynthesis in Escherichia from one end of the EcoRi-cleaved molecule. Although these coli (Carnegie Institution of Washington Publication no. data do not distinguish between the' two ends of the EcoRI- 607). cleaved ColEl DNA molecule, it is most probable that the 8. Radloff, R., Bauer, W. & Vinograd, J. (1967) Proc. Nat. site of the nick in the DNA of the relaxed complex and the Acad. Sci. USA 57, 1514-1521. position of the origin/terminus are located approximately the 9. Sebring, E. D., Kelley, T. J., Jr., Thoren, M. M. & Salzman, N. P. (1971) J. Virol. 8, 478-490. same distance from the same end of the molecule. Location of 10. Loening, U. E. (1969) Biochem. J. 113, 131-138. the relaxation event at or near the origin/terminus of ColEl 11. Hayward, G. S. & Smith, M. G. (1972) J. Mol. Biol. 63, replication suggests an involvement of the protein compo- 383-396. nents of the relaxation complex in either the initiation or 12. Hedgpeth, J., Goodman, H. & Boyer, H. (1972) Proc. Nat. Acad. Sci. USA 69, 3448-3452. termination of replication, possibly satisfying a requirement 13. Allet, B., Jeppesen, P. G. N., Katagiri, K. J. & Delius, H. for a nicking event(s) at the origin/terminus. It is possible (1973) Nature 241, 120-123. that relaxation complexes are an integral feature of the 14. Danna, K. & Nathans, D. (1971) Proc. Nat. Acad. Sci. USA organization of E. coli plasmid DNA molecules as replicons; 68, 2913-2917. there be a for one 15. Studier, F. W. (1965) J. Mol. Biol. 11, 373-390. for every replicon may requirement relaxa- 16. Kelley, T. J. & Smith, H. 0. (1970) J. Mol. Biol. 51, 393-409. tion complex and one or more relaxation event(s) at the origin 17. Boyer, H. W., Chow, L. T., Dugaiczyk, A., Hedgpeth, J. or terminus of replication. Conversion of supercoiled M13 & Goodman, H. M. (1973) Nature New Biol. 244, 40-43. DNA (RF I) to the open circular form (RF II), required for 18. Fareed, G. C., Garon, C. F. & Salzman, N. P. (1972) J. both RF I and viral strand synthesis, involves the action of Virol. 10, 484-491. 19. Fidanidn, H. M. & Ray, D. S. (1972) J. Mol. Biol. 72, 51-63. the M13 gene 2 product (19-21). Nicking of OX174 DNA by 20. Lin, N. S. C. & Pratt, D. (1972) J. Mol. Biol. 72, 37-49. the gene A product has also been suggested as a requirement 21. Tseng, B. Y. & Marvin, D. A. (1972) J. Virol. 10, 384-391. for DNA replication (22). It is possible that the products of 22. Franke, B. & Ray, D. S. (1971) J. Mol. Biol. 61, 565-586. Downloaded by guest on October 2, 2021