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Isolation of closed-circular duplex DNA by chromatography on hydroxyapatite in the presence of ethidium bromide
ANALYTICAL
BIOCHEMISTRY
Isolation Chromatography
67. 372-383 (1975)
of Closed-Circular
Duplex
on Hydroxyapatite of Ethidium
DNA
by
in the Presence
Bromide
WERNER PAKROPPA, WERNER GOEBEL, AND WERNER MUELLER Gesellschaft fidfirr- Molekltlarbiologisclle Forschun!: mbH and Lehrstuhlj~ir Biochemie der Technischen Unirsersitiit Braunschweig BRD-3300 Braunschweig / Stikkhrim, German)
Received December 18. 1974: accepted February 6. 1975 A method is described for the preparative purification of supercoiled DNA of bacterial cell extracts without an ultracentrifugal step. DNA of crude cell lysates, containing supercoiled plasmid DNA and chromosomal DNA fragments is freed of protein by chromatography on Sepharose B4. The separation of closed-circular supercoiled DNA from linear and open-circular DNA is performed by chromatography on hydroxyapatite in the presence of ethidium bromide by taking advantage of the different binding ability of the dye for linear and closed-circular DNA. The best separations are obtained when the linear or nicked DNA in the sample is complexed at a frequency of one dye molecule per three-five base-pairs. The extent of separation seems to depend only on the intrinsic superhelical density of the supercoiled DNA molecules and not on their sizes.
The isolation of supercoiled circular duplex DNA is performed in general by density gradient centrifugation in the presence of the intercalating dye ethidium bromide (1). The separation from linear duplex DNA or nicked-circular DNA by this technique is based on the fact that the supercoiled DNA has a restricted capacity of binding the dye when more than one dye molecule per about ten base-pairs are bound, due to the formation of supercoils with opposite directions of winding. The limited uptake of dye molecules leads to a smaller change in buoyant density of the supercoiled than of the linear DNA. The two DNA species can thus be separated in a density gradient. Recently we described a method for separating mixtures of DNA of varying G + C content on hydroxyapatite in the presence of a GCspecific intercalating DNA ligand (2). We concluded that the separation is based on the structural change produced in the DNA backbone by the intercalator. This led us to the assumption that supercoiled circular duplex DNA can be separated from linear duplex or nicked-circular DNA by the same technique by taking advantage of the different binding capacities of the two species. The results communicated in this paper 372 Copyright @ 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
CHROMATOGRAPHIC
DNA
373
FRACTIONATION
demonstrate that this assumption is basically correct. The techniques described allow the separation of supercoiled DNA of various sizes from bacterial lysates without any centrifugation steps. The high binding-capacity of the hydroxyapatite for DNA allows the preparative isolation of large amounts of circular DNA within a relatively short time. METHODS Bacterial strains and groM,th conditions. The two strains JC411 (ColEI) and E. co/i 15 THU have described (3). E. coli W945(Rldrdl9) was obtained from The bacterial strains were grown in phosphate-buffered (4). Preparation
Escherichia
coli
been previously Dr. E. Meynell. minimal medium
and purijcation of cleared lysates. DNA of the bacteria was labeled with [3H]thymidine (5 &i/ml: sp act, 23 Ci/mmol). Cells were lysed with lysozyme and Brij 58 according to the procedure described by Clewell and Helinski (5). “Cleared lysates” were obtained by centrifugation of the crude lysates at 8,OOOg and 2°C. The DNA was separated from protein and low molecular weight material by passing the cleared lysate (2.0 ml) through a Sepharose 4B column (6) (50 X 1 cm) which was equilibrated with TES buffer (Tris-HCl, 0.1 M, NaCI, 0.015 M, EDTA, 0.0025 M, pH 8.0). Fractions containing the labeled DNA were used directly for hydroxyapatite-ethidium bromide chromatography. Hydroxyapatite-ethidiwn bromide chromatography. Hydroxyapatite type B mixed with Celite, as previously described (2), was used in all experiments. Chromatographic columns, Pharmacia Type K16, were filled with the hydroxyapatite-Celite mixture suspended in 0. I M sodium phosphate buffer, pH 7.1, to give about 12 cm of packed length. The columns were maintained at 25°C by means of a thermostatically controlled water jacket. DNA mixture to be fractioned was’adsorbed from 0. I M sodium phosphate, pH 7.1, onto 3 ml of sedimented hydroxyapatiteCelite suspended in the same medium. Sufficient ethidium bromide was added to this suspension to allow complex formation with linear DNA in the desired dye-base-pair ratio. This mixture was then layered on the column and washed with 0.1 M sodium phosphate, pH 7.1, containing an amount of ethidium bromide sufficient to maintain the desired DNAdye-binding ratio. For calculations of the required dye concentrations. the binding data presented in Fig. 8 were used. When the column effluent had reached a constant absorbance level at 254 nm, a linear eluting gradient consisting of 150 ml of 0.1 M and 150 ml of 0.5 M sodium phosphate. pH 7.1, was applied. Both solutions contained the same ethidium bromide concentration as the buffer used for equilibration of the column.
374
PAKROPPA,
GOEBEL
AND
MijLLER
A constant flow rate of about 17 ml/hr was maintained by means of a peristaltic pump. Fractions of about 4 ml were collected. The removal of ethidium bromide from fractions used for further analysis was achieved by extraction with isobutanol and chloroform as described (2). Centrifugation conditions. Cleared lysates were centrifuged to equilibrium in cesium chloride-ethidium bromide gradients as previously described (5). Sucrose gradient centrifugation was performed in an SW 27 rotor. Portions of DNA samples, 1.O ml in dilute SSC (0.015 M NaCl, 0.0015 M sodium citrate, pH 7.0), were layered onto 30 ml of 5-20% linear sucrose gradients containing 0.1 M NaCl. 0.001 M EDTA, and 0.01 M Tris (pH 8.0). Samples were centrifuged at 25,000 rpm. for appropriate times at 20°C. Fractions (60 drops) were collected from the bottom of the tube in small vials. Counting of radioisotopes. Samples of the gradient fractions were spotted on filter disks (1 X 1 cm). The disks were treated with 10% trichloroacetic acid and then washed twice with alcohol and finally with ether. The dried filter papers were placed in scintillation vials containing 10 ml of a solution of PPO-POPOP [2,5-diphenyloxazole/1.4-&s-2-(5phenyloxazolyl)benzene] in toluene and counted in a liquid scintillation counter (SL 30, Intertechnique, France). Samples of the fractions of the hydroxyapatite-ethidium bromide column were directly placed into scintillation vials containing 5 ml of Bray’s solution (7) and counted in a scintillation counter. Binding of ethidium bromide to E. co&-DNA in 0.2 M sodium phosphate. The binding of ethidium bromide in the region of r = 0.09 bound dye molecules per DNA nucleotide-pair up to r = 0.42 was studied by equilibrium dialysis in 0.2 M sodium phosphate, pH 7.1, at 25°C. The concentrations of bound and free dye for each equilibrium were determined spectrophotometrically at 5 10 nm in a Zeiss DMR 2 1 spectrophotometer after addition of sodium dodecyl sulfate to yield a final concentration of 2.5% (w/v). Under these conditions the DNA-dye complex is split completely. For evaluation, the molar extinction coefficient of 5600 M-’ cm -’ was used. The binding data are presented according to Scatchard (8) in Fig. 8. Sources of reagents: [Methyl-3H]thymidine (sp act, 23 Ci/mmole) was purchased from the Radiochemical Centre, Amersham, Great Britain. Cesium chloride was obtained from Merck and ethidium bromide from Calbiochem. Sepharose 4B was purchased from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Hydroxyapatite (type B) was prepared as described previously (2). Celite, Type 545 (20-45 -pm particle size), was purchased from Serva, Heidelberg, Germany. All other chemicals used were of analytical grade from E. Merck, Darmstadt.
CHROMATOGRAPHIC
DNA
FRACTIONATION
375
RESULTS Separation of Purijed Supercoiled ColEl DNA from Linear Chromosomal DNA of E. coli by Chromatography on a Hydroxyapatite-Ethidium Bromide Column
Bourgaux and Bourgaux-Ramoisy (9) reported that circular duplex DNA from polyoma virus is eluted from hydroxyapatite at slightly lower phosphate concentration than linear double-stranded DNA. We have confirmed this result. The difference in the phosphate concentration used for eluting supercoiled circular ColEl-DNA (10) and linear chromosomal DNA of E. coli prepared by the procedure of Marmur (11) was found to be 0.013 M in sodium phosphate. However, this difference is insufficient for a clearcut separation of both DNA species. The addition of ethidium bromide to the eluting phosphate gradient as weli as to the DNA mixture reversed the elution order. In Fig. 1 the elution profile of the same mixture in the presence of the dye at a ration (r) of 0.3 bound dye molecules per nucleotide pair is shown. This r-value corresponds to the dye-binding capacity of the linear DNA. Under these conditions the separation of the two species is complete. Note that in the present paper the dye-binding ratio r is defined as bound dye molecules per nucleotide pair instead of the more commonly used definition of dye per nucleotide. We think that this definition is more appropriate to describe the binding of intercalating compounds to double-helical polymers. A260 0.5
30
LO FRACTION
50 NUMBER
FIG. 1. Chromatography of purified closed-circular ColEl-DNA on hydroxyapatite in the presence of ethidium bromide. The dye/DNA ratio r is 0.3 (bound dye molecules per nucleotide pair) based on the binding of the linear E. co/i DNA. The concentration of free dye in the eluting gradient is I X 10M5 M. Supercoiled ColEl-DNA purified by dyebuoyant density centrifugation as described in Methods was mixed with 1 mg of nonradioactive chromosomal DNA of E. coli and subjected to a hydroxyapatite-ethidium bromide column (1.6 X 12 cm; flow rate, 17 ml/hr). Open circles represent Ale, in a 0.3-cm cuvette; filled circles indicate [RH] radioactivity; dashed line shows the sodium phosphate gradient.
376
PAKROPPA.
GOEBEL
01
AND
0.2
MijLLER
0.3
r
0.4
FIG. 2. (A), Variation of the eluting sodium phosphate concentration with increasing r. The conditions are described in Methods. Open circles represent linear E. co/i DNA: filled circles show closed-circular ColEI-DNA. For definition of Y see legend to Fig. 1. (B), Variation of the difference between the eluting sodium phosphate concentration for linear E. c&i-DNA and closed circular ColE I-DNA with increasing Y as calculated from Fig. 2A. For definition of I’ see legend to Fig. I.
A set of further experiments with different v-values revealed a rather broad optimum for the separation of the two DNA species at v-values between 0.09 and 0.43; the decrease in the eluting phosphate concentration to lower values becomes substantial for both DNA’s at r-values above 0.1. The results of these experiments are shown in Figs. 2A and B. Sepurution of the DNA DNA by chromatography Bromide Column
of u Cleared Lysute Containing on u Hydroxyuputite-ethidium
Supercoiled
Cells of JC411 (ColEl) labeled with [3H]thymidine were lysed according to the lysozyme-Brij 58 procedure (12). The crude lysate was cleared by low speed centrifugation. This step removes the bulk of the chromosomal DNA. Passing of the supernatant (cleared lysate) which contains most of the plasmid DNA (> 80%) and the residual chromosomal DNA through a Sepharose B4 column (6) separates the total DNA from protein, most of the RNA and low molecular weight material
CHROMATOGRAPHIC
DNA FRACTIONATION
A A260
0'
3H Cpm
OL - ‘lOOdd 03--75
?
0 i
377
0-o
\,
0
/
AZM) OL 03
0 i 30
FRACTION
NUMBER
3H Cpm LO.103
t j i.
30
LO FRACTION NUMBER
50
FIG. 3. (A), Chromatography of a cleared lysate of JC41 l(ColE1) on a Sepharose B4 column. A cleared lysate (2.0 ml) was passed through a Sepharose B4 column as described in Methods. Open circles represent A 260in a l-cm cuvette. Aliquots (0.1 ml) of each fraction were taken for the determination of the 3H radioactivity (filled circles). (B), Chromatography of the radioactive DNA fraction from the Sepharose column (A). Fractions 4 and 5 from the Sepharose column were pooled and mixed with I mg of nonradioactive chromosomal DNA of E. coli and subjected to chromatography on a hydroxyapatiteethidium bromide column as described in the Methods. The dye/DNA ratio r was 0.3. Open circles represent Az6,, in a 0.3-cm cuvette. Filled circles indicate the sH radioactivity.
(Fig. 3A). The fractions containing the radioactively labeled DNA (about 20 pg) were mixed with a large excess of nonradioactive chromosomal DNA of E. cofi (1 mg) in order to recognize linear DNA by its optical density. The DNA mixture was applied to a hydroxyapatiteethidium bromide column and eluted as before with a linear sodium phosphate gradient. Figure 3B demonstrates that the radioactively labeled DNA is separated into two bands. The first one roughly overlaps with the band of nonradioactive chromosomal DNA, whereas the second one elutes at the same salt concentration as the supercoiled ColEl DNA shown in Fig. 1. Sucrose gradient analyses of the radioactive DNA in the two bands (Fig. 4) reveal that band I contains residual chrosomal DNA and open-circular ColEl-DNA (17 s), whereas band II contains pure supercoiled ColEI-DNA without detectable contamination by chromosomal DNA. The shoulder, which sediments in front of the main DNA peak, consists of supercoiled dimeric ColE 1-DNA (3 1
378
PAKROPPA,
GOEBEL
AND
MiiLLER
3H Cpm 750
250
5000
3HCPm 3750 32P Cm 1000
2500
FRACTION
NUMBER
4. Sucrose gradient analyses of the DNA’s separated on the hydroxyapatiteethidium bromide column (Fig. 3B). A portion (0.2 ml) of the peak fractions (fractions 37 and 43, Fig. 3B) of the two separated DNA bands was mixed together with 32P-labeled supercoiled ColEl-DNA and layered on neutral 5-20% sucrose gradients and centrifuged for 75 min (at 20°C; 45,000 rpm, Spinco SW50.1). Fractions (10 drops) were collected from the bottom of the tube directly on filter squares which were assayed for radioactivity. (A), DNA of fraction 37 (-C-C): 32P-labeled ColEI-DNA (......a....), (B), DNA of fraction 43 (-C-U); 32P-labeled ColEI-DNA (..a...@....). FIG.
-H Cpr
A260 0.L 0.3 02 01
L
30
35
LO FRACTION
L5
50
NUMBER
FIG. 5. Chromatography of a protein-free cleared lysate of W945(Rldrd19) on hydroxyapatite-ethidium bromide (r = 0.3). A cleared lysate of W945(Rldrdl9) was freed of protein by passage through a Sepharose B4 column as described in Fig. 3A. The DNA-containing fractions were mixed with 0.5 mg of nonradioactive chromosomal DNA of E. coli and subjected to a hydroxyapatite-ethidium bromide column. Open circles represent A,,, in a 0.3~cm cuvette. Filled circles indicate the 3H radioactivity.
CHROMATOGRAPHIC
DNA
FRACTIONATION
379
s). Similar results were obtained when the cleared lysate of the E. coli strain W945 (Rldrdl9) which harbors the large antibiotic-resistance factor Rldrd19 (M,, 65 X 106), was chromographed together with an excess of nonradioactive chromosomal DNA of E. co/i (Fig. 5). Chromatography of a Mixture of Supercoiled DNA’s of Different Sizes on a Hydroxyapatite-Ethidium Bromide Column To test whether the ethidium bromide hydroxyapatite columns also has the potential of separating two supercoiled DNA’s of different sizes, supercoiled Rldrdl9-DNA (M,, 6.5 X 106) and minicircular DNA of E. coli 15 (M,, 1.5 X 106) (13), each corresponding to 10,000 cpm, were mixed with nonradioactive chromosomal DNA of E. cob. The mixture was chromatographed on a hydroxyapatite-ethidium bromide column as before (Fig. 6). Most of the radioactively labeled DNA elutes in a rather broad band at a phosphate concentration of 0.19 M. This band contains a mixture of unseparated supercoiled Rldrdl9-DNA and supercoiled minicircular DNA, as demonstrated by sucrose gradient analysis. The sharp peak at fraction 32 contains almost exclusively open-circular Rldrdl9DNA. It is interesting to note that this DNA does not overlap with the peak of the chromosomal duplex DNA but elutes from the column at a higher salt concentration than the latter one. Similar results were obtained with a mixture of purified ColEl-DN A (M,, 4.3 x 106) (10) and Rldrdl9-DNA. A260
FRACTION
NUMBER
FIG. 6. Chromatography of a mixture of purified minicircular DNA of E. coli 15 and Rldrdl9-DNA on a hydroxyapatite-ethidium bromide column (r = 0.3). 3H-labeled minicircular DNA of E. coli IS and Rldrdl9-DNA, purified by dye-buoyant density gradient centrifugation as described in Methods. were mixed with 0.44 mg of nonradioactive chromosomal DNA of E. coli. The mixture was subjected to a hydroxyapatite-ethidium bromide column (0.9 X 12 cm; flow rate, 6 ml/hr) and eluted with a gradient consisting of 70 ml of 0.1 M and 70 ml of 0.5 M sodium phosphate, pH 7.1. Fractions of 1.5 ml were collected. Open circles represent A,,, in a l.O-cm cuvette. Filled circles indicate 3H radioactivity.
380
PAKROPPA,
GOEBEL
AND
FRACTION
NUMBER
MtiLLER
7. Chromatographic separation of covalently closed-circular PMZ-DNA from small amounts of its nicked or linear form on hydroxyapatite in the presence of ethidium bromide (r= 0.28). PM?-DNA, 0.27 mg, was subjected to a hydroxyapatite column of 0.9 x 10.5 cm and eluted by a linear gradient consisting of 55 ml of 0.1 M and 55 ml of 0.5 M sodium phosphate, pH 7. I. Flow rate was 6 ml/hr. Both solutions contained ethidium bromide at a concentration of 8 X 10mfiM. Fractions of 1.2 ml were collected. FIG.
Separation of Closed-circular of Its Nicked-Circular Form
PM2-DNA
from Small Amounts
In most of the preceding experiments DNA mixtures were separated in which the amount of linear or nicked-circular DNA exceeded the amount of closed-circular DNA by at least one order of magnitude. In order to test if the chromatographic procedure can also be used to separate small amounts of nicked material from a large excess of the corresponding closed-circular species, a mixture of 0.25 mg of PM2-DNA containing 10.5% of the nicked-circular (or linear) form was chromatographed on a hydroxyapatite column (0.9 X 10.5 cm) in the presence of ethidium bromide at a binding ratio of r = 0.28. The amount of nicked DNA was determined previously by analytical ultracentrifugation in a CsCl gradient in the presence of the dye. The elution profile shown in Fig. 7 reveals that a complete separation of the two DNA species is not obtained by the chromatographic technique described here. The material eluted earlier than the main peak (fractions 26-29) consisted exclusively of nicked or linear DNA as confirmed by analytical CsCl density centrifugation in the presence of ethidium bromide (36 pg/ml). These four fractions contained 9.1% of the total DNA and about nine-tenths of the nicked material originally present in the sample. It should be noted that the eluting phosphate concentrations for the nicked and closed-circular forms differ only by 0.018 M instead of 0.025-0.03 M as observed in the runs with ColEI-DNA.
CHROMATOGRAPHIC
DNA
FRACTIONATION
381
DISCUSSION
Recently we were able to show that the affinity of DNA for hydroxyapatite is reduced if intercalating ligands are bound to this doublehelical polymer (2). The sigmoid-shaped decrease in the eluting phosphate concentration with increasing amounts of bound ligand as shown in Fig. 2A for ethidium bromide confirms this result. This effect is possibly caused by the distortion of the DNA helix due to the intercalator leading to irregularities in the phosphate distances. This in turn might result in a reduced binding to the crystal surface of the hydroxyapatite matrix, in agreement with the idea of Bernardi (14). Otherwise, the intercalation might also turn the phosphate groups at the binding sites slightly to the inside of the helix if hydrogen bonds are formed with the ligand (15). This could also reduce the binding affinity to hydroxyapatite, as proposed by Martinson (16). The behavior of the supertwisted circular DNA in the experiments reported in this paper may best be explained by taking into account both effects. At low ratios of ethidium bromide to nucleotide pair (Y < 0.09) where the super-twisted circular DNA binds more dye than the linear DNA (17), we find an increase in the binding of this DNA to the adsorbant, while the binding of the linear DNA to the absorbant is barely changed. This effect is most probably a consequence of the increased availability of the phosphate groups for the adsorbant in the opencircular form. At higher r-values (Y > 0.09), the uptake of dye by the closed-circular DNA is limited (17). One would therefore expect a continuous increase in the difference between the binding affinities of the two dye-DNA complexes for the adsorbant with increasing I’. The fact that this is not observed may be explained by the reformation of twists in the opposite sense in the circular DNA. The accessibility of the phosphate groups for the adsorbant would therefore again be reduced. At a ratio of about 0.42, which is substantially above the highest possible charging of circular DNA with ethidium bromide and near the saturation of linear DNA, as shown in Fig. 8. both forms are eluted at roughly the same phosphate concentration (Fig. 2). The obvious implication is that the effect of the higher r-value obtained for the linear DNA is compensated by the formation of positive twists in the closed-circular DNA. More quantitative evaluations of our results require an exact knowledge of the binding isotherm for the closed-circular DNA under the conditions of our experiments i.e., in about 0.2 M sodium phosphate buffer. The evaluations require in addition that the amount of dye bound to both DNA species is not appreciably altered when the DNA is fixed on hydroxyapatite. No assumptions can be made about the second point; we only know that the fixed DNA’s bind enough dye to produce
382
PAKROPPA,
0.1
GOEBEL
0.2
AND
0.3
MijLLER
OL
r
8. Isotherm for the binding of ethidium bromide to linear DNA from E. coli in 0.2 M sodium phosphate, pH 7.1, at 25°C as determined by equilibrium dialysis (solid line). The broken line corresponds to the isotherm for a closed-circular duplex DNA with an intrinsic superhelical twist u = -0.026 per ten nucleotide-pairs as calculated from Bauer and Vinograd’s data (17) for SV40-DNA. FIG.
strongly colored pink bands on the hydroxyapatite. Concerning the first point, one could construct a binding isotherm for the closed circular DNA on the basis of the data reported by Bauer and Vinograd (17) for closed-circular and nicked SV40-DNA in 5.8 M C&l. This implies however that the different uptake of dye by the two forms is caused mainly by the different mechanical properties of the two molecular species and is not influenced by the ionic strength of the medium. Assuming that these preconditions are fulfilled, we constructed the binding isotherm for the ColEl-DNA (Fig. 7, broken line) from the isotherm for linear E. coli-DNA and the data of Bauer and Vinograd (17). A comparison of the two isotherms with the curve in Fig. 2B reveals that the best separation of the two DNA species in our system occurs in an r-range in which the difference between their v-values at a given free-dye concentration shows a maximum. This range is around r = 0.25 for the linear DNA which corresponds to r = 0.165 for the closed-circular DNA. From this finding it might be concluded that the affinities of the two dye-DNA complexes for the hydroxyapatite depend more on the amount of structural change introduced by the intercalated dye than on the number of induced positive twists. At very high Yvalues, however, the effect of the twists seems to dominate again. On the basis of the arguments presented it is not surprising that a separation of large and small supercoiled circles by chromatography on hydroxyapatite-ethidium bromide failed. If both DNA’s have the same intrinsic superhelical density their dye-binding properties and consequently their structural changes should be the same. For this reason
CHROMATOGRAPHIC
DNA
FRACTIONATION
383
different binding affinities to hydroxyapatite cannot be expected. It remains unclear, however, why nicked-circular DNA (as clearly observed with the large nicked Rldudl9-DNA) is eluted somewhat later than the linear chromosomal DNA from E. co/i as the dye binding of this DNA should be the same as of linear DNA. The situation is altered however when the intrinsic superhelical density changes. In the last experiment reported PM2-DNA was used instead of ColEl-DNA. The superhelical density of the closed circular form of this DNA was reported to be - 0.053 (18) which is nearly twice as high as the corresponding value for SV40- or ColEl -DNA (17,19). Since this higher degree of negative coiling allows substantially higher dye-binding and thus reduces the difference in the total uptake of dye for the closed-circular and the nicked or linear form it is not surprising that the separation of these forms is not as good in the case of PM2-DNA as in the cases reported before. We think that the described procedure provides a valuable new method for a rather rapid isolation of covalently closed-circular DNA’s on an analytical as well as on a preparative scale. ACKNOWLEDGMENTS We are very grateful to Miss U. Nielander for skillful technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft. We thank Dr. G. Bezanson for careful reading of the manuscript.
REFERENCES 1. Radloff, R., Bauer, W. R., and Vinograd. J. (1967) Proc. Nut. Acad. Sci. USA 57, 1514-1.521. 2. Pakroppa, W., and Miller, W. (1974) Proc. Nut. Acad. Sci. USA 71, 699-703. 3. Goebel, W., and Schrempf, H. (1972) J. Bncteriol. 111, 696-704. 4. Dewitt, W.. and Helinski, D. R. (1965) J. Mol. Biol. 13, 692-703. 5. Clewell, D. B., and Helinski, D. R. (1969) Proc. Nut. Acad. Sci. USA 62, 1159-l 166. 6. Satava, J., Zadraizil, S., and Sormova, Z., ( 1973) Collect. Czech. Chem. Commun. 38, 2167-2173. 7. Bray, G. A. (1960) Anal. Biochem. 1, 279-285. 8. Scatchard, G. (1949) Arm. N.Y. Acad. Sci. 51, 660-672. 9. Bourgaux, P., and Bourgaux-Ramoisy, D. (1967) .I. Gen. Virol. 1, 323-332. IO. Bazaral, M., and Helinski. D. R. (1970) Biochemistry 9, 399-406. 1 I Marmur, J. (I 961) J. Mol. Biol. 3, 208-2 18. 12. Goebel, W. ( 1970) Eur. J. Biochem. 15, 3 1 l-320. 13. Cozzarelli, N. R., Kelley. R. B.. and Kornberg. A. (1968) Proc. Nat. Acud. Sci. USA 60, 992-999. 14. Bemardi, G. (1971) in Methods in Enzymology. (Colowick, S. P., and Kaplan, N. 0.. eds.), Vol. 21, pp. 95-l 39. Academic Press, New York and London. 15. Fuller, W., and Waring, M. (1964) Ber. Bunsenges. 68, 805-808. 16. Martinson, H. G. (I 973) Biochemistry 12, 2737-2746. 17. Batter, W., and Vinograd, J. ( 1968) J. Mol. Biol. 33, 14 I - I7 I. 18. Upholt, W. B., Grayjr, H. B., and Vinograd. J. ( 197 1) J. MO/. Biol. 61, 2 l-38. 19. Bazaral, M., and Helinski. D. R. (1970) Biochemistry 9, 399-406.
BIOCHEMISTRY
Isolation Chromatography
67. 372-383 (1975)
of Closed-Circular
Duplex
on Hydroxyapatite of Ethidium
DNA
by
in the Presence
Bromide
WERNER PAKROPPA, WERNER GOEBEL, AND WERNER MUELLER Gesellschaft fidfirr- Molekltlarbiologisclle Forschun!: mbH and Lehrstuhlj~ir Biochemie der Technischen Unirsersitiit Braunschweig BRD-3300 Braunschweig / Stikkhrim, German)
Received December 18. 1974: accepted February 6. 1975 A method is described for the preparative purification of supercoiled DNA of bacterial cell extracts without an ultracentrifugal step. DNA of crude cell lysates, containing supercoiled plasmid DNA and chromosomal DNA fragments is freed of protein by chromatography on Sepharose B4. The separation of closed-circular supercoiled DNA from linear and open-circular DNA is performed by chromatography on hydroxyapatite in the presence of ethidium bromide by taking advantage of the different binding ability of the dye for linear and closed-circular DNA. The best separations are obtained when the linear or nicked DNA in the sample is complexed at a frequency of one dye molecule per three-five base-pairs. The extent of separation seems to depend only on the intrinsic superhelical density of the supercoiled DNA molecules and not on their sizes.
The isolation of supercoiled circular duplex DNA is performed in general by density gradient centrifugation in the presence of the intercalating dye ethidium bromide (1). The separation from linear duplex DNA or nicked-circular DNA by this technique is based on the fact that the supercoiled DNA has a restricted capacity of binding the dye when more than one dye molecule per about ten base-pairs are bound, due to the formation of supercoils with opposite directions of winding. The limited uptake of dye molecules leads to a smaller change in buoyant density of the supercoiled than of the linear DNA. The two DNA species can thus be separated in a density gradient. Recently we described a method for separating mixtures of DNA of varying G + C content on hydroxyapatite in the presence of a GCspecific intercalating DNA ligand (2). We concluded that the separation is based on the structural change produced in the DNA backbone by the intercalator. This led us to the assumption that supercoiled circular duplex DNA can be separated from linear duplex or nicked-circular DNA by the same technique by taking advantage of the different binding capacities of the two species. The results communicated in this paper 372 Copyright @ 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
CHROMATOGRAPHIC
DNA
373
FRACTIONATION
demonstrate that this assumption is basically correct. The techniques described allow the separation of supercoiled DNA of various sizes from bacterial lysates without any centrifugation steps. The high binding-capacity of the hydroxyapatite for DNA allows the preparative isolation of large amounts of circular DNA within a relatively short time. METHODS Bacterial strains and groM,th conditions. The two strains JC411 (ColEI) and E. co/i 15 THU have described (3). E. coli W945(Rldrdl9) was obtained from The bacterial strains were grown in phosphate-buffered (4). Preparation
Escherichia
coli
been previously Dr. E. Meynell. minimal medium
and purijcation of cleared lysates. DNA of the bacteria was labeled with [3H]thymidine (5 &i/ml: sp act, 23 Ci/mmol). Cells were lysed with lysozyme and Brij 58 according to the procedure described by Clewell and Helinski (5). “Cleared lysates” were obtained by centrifugation of the crude lysates at 8,OOOg and 2°C. The DNA was separated from protein and low molecular weight material by passing the cleared lysate (2.0 ml) through a Sepharose 4B column (6) (50 X 1 cm) which was equilibrated with TES buffer (Tris-HCl, 0.1 M, NaCI, 0.015 M, EDTA, 0.0025 M, pH 8.0). Fractions containing the labeled DNA were used directly for hydroxyapatite-ethidium bromide chromatography. Hydroxyapatite-ethidiwn bromide chromatography. Hydroxyapatite type B mixed with Celite, as previously described (2), was used in all experiments. Chromatographic columns, Pharmacia Type K16, were filled with the hydroxyapatite-Celite mixture suspended in 0. I M sodium phosphate buffer, pH 7.1, to give about 12 cm of packed length. The columns were maintained at 25°C by means of a thermostatically controlled water jacket. DNA mixture to be fractioned was’adsorbed from 0. I M sodium phosphate, pH 7.1, onto 3 ml of sedimented hydroxyapatiteCelite suspended in the same medium. Sufficient ethidium bromide was added to this suspension to allow complex formation with linear DNA in the desired dye-base-pair ratio. This mixture was then layered on the column and washed with 0.1 M sodium phosphate, pH 7.1, containing an amount of ethidium bromide sufficient to maintain the desired DNAdye-binding ratio. For calculations of the required dye concentrations. the binding data presented in Fig. 8 were used. When the column effluent had reached a constant absorbance level at 254 nm, a linear eluting gradient consisting of 150 ml of 0.1 M and 150 ml of 0.5 M sodium phosphate. pH 7.1, was applied. Both solutions contained the same ethidium bromide concentration as the buffer used for equilibration of the column.
374
PAKROPPA,
GOEBEL
AND
MijLLER
A constant flow rate of about 17 ml/hr was maintained by means of a peristaltic pump. Fractions of about 4 ml were collected. The removal of ethidium bromide from fractions used for further analysis was achieved by extraction with isobutanol and chloroform as described (2). Centrifugation conditions. Cleared lysates were centrifuged to equilibrium in cesium chloride-ethidium bromide gradients as previously described (5). Sucrose gradient centrifugation was performed in an SW 27 rotor. Portions of DNA samples, 1.O ml in dilute SSC (0.015 M NaCl, 0.0015 M sodium citrate, pH 7.0), were layered onto 30 ml of 5-20% linear sucrose gradients containing 0.1 M NaCl. 0.001 M EDTA, and 0.01 M Tris (pH 8.0). Samples were centrifuged at 25,000 rpm. for appropriate times at 20°C. Fractions (60 drops) were collected from the bottom of the tube in small vials. Counting of radioisotopes. Samples of the gradient fractions were spotted on filter disks (1 X 1 cm). The disks were treated with 10% trichloroacetic acid and then washed twice with alcohol and finally with ether. The dried filter papers were placed in scintillation vials containing 10 ml of a solution of PPO-POPOP [2,5-diphenyloxazole/1.4-&s-2-(5phenyloxazolyl)benzene] in toluene and counted in a liquid scintillation counter (SL 30, Intertechnique, France). Samples of the fractions of the hydroxyapatite-ethidium bromide column were directly placed into scintillation vials containing 5 ml of Bray’s solution (7) and counted in a scintillation counter. Binding of ethidium bromide to E. co&-DNA in 0.2 M sodium phosphate. The binding of ethidium bromide in the region of r = 0.09 bound dye molecules per DNA nucleotide-pair up to r = 0.42 was studied by equilibrium dialysis in 0.2 M sodium phosphate, pH 7.1, at 25°C. The concentrations of bound and free dye for each equilibrium were determined spectrophotometrically at 5 10 nm in a Zeiss DMR 2 1 spectrophotometer after addition of sodium dodecyl sulfate to yield a final concentration of 2.5% (w/v). Under these conditions the DNA-dye complex is split completely. For evaluation, the molar extinction coefficient of 5600 M-’ cm -’ was used. The binding data are presented according to Scatchard (8) in Fig. 8. Sources of reagents: [Methyl-3H]thymidine (sp act, 23 Ci/mmole) was purchased from the Radiochemical Centre, Amersham, Great Britain. Cesium chloride was obtained from Merck and ethidium bromide from Calbiochem. Sepharose 4B was purchased from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Hydroxyapatite (type B) was prepared as described previously (2). Celite, Type 545 (20-45 -pm particle size), was purchased from Serva, Heidelberg, Germany. All other chemicals used were of analytical grade from E. Merck, Darmstadt.
CHROMATOGRAPHIC
DNA
FRACTIONATION
375
RESULTS Separation of Purijed Supercoiled ColEl DNA from Linear Chromosomal DNA of E. coli by Chromatography on a Hydroxyapatite-Ethidium Bromide Column
Bourgaux and Bourgaux-Ramoisy (9) reported that circular duplex DNA from polyoma virus is eluted from hydroxyapatite at slightly lower phosphate concentration than linear double-stranded DNA. We have confirmed this result. The difference in the phosphate concentration used for eluting supercoiled circular ColEl-DNA (10) and linear chromosomal DNA of E. coli prepared by the procedure of Marmur (11) was found to be 0.013 M in sodium phosphate. However, this difference is insufficient for a clearcut separation of both DNA species. The addition of ethidium bromide to the eluting phosphate gradient as weli as to the DNA mixture reversed the elution order. In Fig. 1 the elution profile of the same mixture in the presence of the dye at a ration (r) of 0.3 bound dye molecules per nucleotide pair is shown. This r-value corresponds to the dye-binding capacity of the linear DNA. Under these conditions the separation of the two species is complete. Note that in the present paper the dye-binding ratio r is defined as bound dye molecules per nucleotide pair instead of the more commonly used definition of dye per nucleotide. We think that this definition is more appropriate to describe the binding of intercalating compounds to double-helical polymers. A260 0.5
30
LO FRACTION
50 NUMBER
FIG. 1. Chromatography of purified closed-circular ColEl-DNA on hydroxyapatite in the presence of ethidium bromide. The dye/DNA ratio r is 0.3 (bound dye molecules per nucleotide pair) based on the binding of the linear E. co/i DNA. The concentration of free dye in the eluting gradient is I X 10M5 M. Supercoiled ColEl-DNA purified by dyebuoyant density centrifugation as described in Methods was mixed with 1 mg of nonradioactive chromosomal DNA of E. coli and subjected to a hydroxyapatite-ethidium bromide column (1.6 X 12 cm; flow rate, 17 ml/hr). Open circles represent Ale, in a 0.3-cm cuvette; filled circles indicate [RH] radioactivity; dashed line shows the sodium phosphate gradient.
376
PAKROPPA.
GOEBEL
01
AND
0.2
MijLLER
0.3
r
0.4
FIG. 2. (A), Variation of the eluting sodium phosphate concentration with increasing r. The conditions are described in Methods. Open circles represent linear E. co/i DNA: filled circles show closed-circular ColEI-DNA. For definition of Y see legend to Fig. 1. (B), Variation of the difference between the eluting sodium phosphate concentration for linear E. c&i-DNA and closed circular ColE I-DNA with increasing Y as calculated from Fig. 2A. For definition of I’ see legend to Fig. I.
A set of further experiments with different v-values revealed a rather broad optimum for the separation of the two DNA species at v-values between 0.09 and 0.43; the decrease in the eluting phosphate concentration to lower values becomes substantial for both DNA’s at r-values above 0.1. The results of these experiments are shown in Figs. 2A and B. Sepurution of the DNA DNA by chromatography Bromide Column
of u Cleared Lysute Containing on u Hydroxyuputite-ethidium
Supercoiled
Cells of JC411 (ColEl) labeled with [3H]thymidine were lysed according to the lysozyme-Brij 58 procedure (12). The crude lysate was cleared by low speed centrifugation. This step removes the bulk of the chromosomal DNA. Passing of the supernatant (cleared lysate) which contains most of the plasmid DNA (> 80%) and the residual chromosomal DNA through a Sepharose B4 column (6) separates the total DNA from protein, most of the RNA and low molecular weight material
CHROMATOGRAPHIC
DNA FRACTIONATION
A A260
0'
3H Cpm
OL - ‘lOOdd 03--75
?
0 i
377
0-o
\,
0
/
AZM) OL 03
0 i 30
FRACTION
NUMBER
3H Cpm LO.103
t j i.
30
LO FRACTION NUMBER
50
FIG. 3. (A), Chromatography of a cleared lysate of JC41 l(ColE1) on a Sepharose B4 column. A cleared lysate (2.0 ml) was passed through a Sepharose B4 column as described in Methods. Open circles represent A 260in a l-cm cuvette. Aliquots (0.1 ml) of each fraction were taken for the determination of the 3H radioactivity (filled circles). (B), Chromatography of the radioactive DNA fraction from the Sepharose column (A). Fractions 4 and 5 from the Sepharose column were pooled and mixed with I mg of nonradioactive chromosomal DNA of E. coli and subjected to chromatography on a hydroxyapatiteethidium bromide column as described in the Methods. The dye/DNA ratio r was 0.3. Open circles represent Az6,, in a 0.3-cm cuvette. Filled circles indicate the sH radioactivity.
(Fig. 3A). The fractions containing the radioactively labeled DNA (about 20 pg) were mixed with a large excess of nonradioactive chromosomal DNA of E. cofi (1 mg) in order to recognize linear DNA by its optical density. The DNA mixture was applied to a hydroxyapatiteethidium bromide column and eluted as before with a linear sodium phosphate gradient. Figure 3B demonstrates that the radioactively labeled DNA is separated into two bands. The first one roughly overlaps with the band of nonradioactive chromosomal DNA, whereas the second one elutes at the same salt concentration as the supercoiled ColEl DNA shown in Fig. 1. Sucrose gradient analyses of the radioactive DNA in the two bands (Fig. 4) reveal that band I contains residual chrosomal DNA and open-circular ColEl-DNA (17 s), whereas band II contains pure supercoiled ColEI-DNA without detectable contamination by chromosomal DNA. The shoulder, which sediments in front of the main DNA peak, consists of supercoiled dimeric ColE 1-DNA (3 1
378
PAKROPPA,
GOEBEL
AND
MiiLLER
3H Cpm 750
250
5000
3HCPm 3750 32P Cm 1000
2500
FRACTION
NUMBER
4. Sucrose gradient analyses of the DNA’s separated on the hydroxyapatiteethidium bromide column (Fig. 3B). A portion (0.2 ml) of the peak fractions (fractions 37 and 43, Fig. 3B) of the two separated DNA bands was mixed together with 32P-labeled supercoiled ColEl-DNA and layered on neutral 5-20% sucrose gradients and centrifuged for 75 min (at 20°C; 45,000 rpm, Spinco SW50.1). Fractions (10 drops) were collected from the bottom of the tube directly on filter squares which were assayed for radioactivity. (A), DNA of fraction 37 (-C-C): 32P-labeled ColEI-DNA (......a....), (B), DNA of fraction 43 (-C-U); 32P-labeled ColEI-DNA (..a...@....). FIG.
-H Cpr
A260 0.L 0.3 02 01
L
30
35
LO FRACTION
L5
50
NUMBER
FIG. 5. Chromatography of a protein-free cleared lysate of W945(Rldrd19) on hydroxyapatite-ethidium bromide (r = 0.3). A cleared lysate of W945(Rldrdl9) was freed of protein by passage through a Sepharose B4 column as described in Fig. 3A. The DNA-containing fractions were mixed with 0.5 mg of nonradioactive chromosomal DNA of E. coli and subjected to a hydroxyapatite-ethidium bromide column. Open circles represent A,,, in a 0.3~cm cuvette. Filled circles indicate the 3H radioactivity.
CHROMATOGRAPHIC
DNA
FRACTIONATION
379
s). Similar results were obtained when the cleared lysate of the E. coli strain W945 (Rldrdl9) which harbors the large antibiotic-resistance factor Rldrd19 (M,, 65 X 106), was chromographed together with an excess of nonradioactive chromosomal DNA of E. co/i (Fig. 5). Chromatography of a Mixture of Supercoiled DNA’s of Different Sizes on a Hydroxyapatite-Ethidium Bromide Column To test whether the ethidium bromide hydroxyapatite columns also has the potential of separating two supercoiled DNA’s of different sizes, supercoiled Rldrdl9-DNA (M,, 6.5 X 106) and minicircular DNA of E. coli 15 (M,, 1.5 X 106) (13), each corresponding to 10,000 cpm, were mixed with nonradioactive chromosomal DNA of E. cob. The mixture was chromatographed on a hydroxyapatite-ethidium bromide column as before (Fig. 6). Most of the radioactively labeled DNA elutes in a rather broad band at a phosphate concentration of 0.19 M. This band contains a mixture of unseparated supercoiled Rldrdl9-DNA and supercoiled minicircular DNA, as demonstrated by sucrose gradient analysis. The sharp peak at fraction 32 contains almost exclusively open-circular Rldrdl9DNA. It is interesting to note that this DNA does not overlap with the peak of the chromosomal duplex DNA but elutes from the column at a higher salt concentration than the latter one. Similar results were obtained with a mixture of purified ColEl-DN A (M,, 4.3 x 106) (10) and Rldrdl9-DNA. A260
FRACTION
NUMBER
FIG. 6. Chromatography of a mixture of purified minicircular DNA of E. coli 15 and Rldrdl9-DNA on a hydroxyapatite-ethidium bromide column (r = 0.3). 3H-labeled minicircular DNA of E. coli IS and Rldrdl9-DNA, purified by dye-buoyant density gradient centrifugation as described in Methods. were mixed with 0.44 mg of nonradioactive chromosomal DNA of E. coli. The mixture was subjected to a hydroxyapatite-ethidium bromide column (0.9 X 12 cm; flow rate, 6 ml/hr) and eluted with a gradient consisting of 70 ml of 0.1 M and 70 ml of 0.5 M sodium phosphate, pH 7.1. Fractions of 1.5 ml were collected. Open circles represent A,,, in a l.O-cm cuvette. Filled circles indicate 3H radioactivity.
380
PAKROPPA,
GOEBEL
AND
FRACTION
NUMBER
MtiLLER
7. Chromatographic separation of covalently closed-circular PMZ-DNA from small amounts of its nicked or linear form on hydroxyapatite in the presence of ethidium bromide (r= 0.28). PM?-DNA, 0.27 mg, was subjected to a hydroxyapatite column of 0.9 x 10.5 cm and eluted by a linear gradient consisting of 55 ml of 0.1 M and 55 ml of 0.5 M sodium phosphate, pH 7. I. Flow rate was 6 ml/hr. Both solutions contained ethidium bromide at a concentration of 8 X 10mfiM. Fractions of 1.2 ml were collected. FIG.
Separation of Closed-circular of Its Nicked-Circular Form
PM2-DNA
from Small Amounts
In most of the preceding experiments DNA mixtures were separated in which the amount of linear or nicked-circular DNA exceeded the amount of closed-circular DNA by at least one order of magnitude. In order to test if the chromatographic procedure can also be used to separate small amounts of nicked material from a large excess of the corresponding closed-circular species, a mixture of 0.25 mg of PM2-DNA containing 10.5% of the nicked-circular (or linear) form was chromatographed on a hydroxyapatite column (0.9 X 10.5 cm) in the presence of ethidium bromide at a binding ratio of r = 0.28. The amount of nicked DNA was determined previously by analytical ultracentrifugation in a CsCl gradient in the presence of the dye. The elution profile shown in Fig. 7 reveals that a complete separation of the two DNA species is not obtained by the chromatographic technique described here. The material eluted earlier than the main peak (fractions 26-29) consisted exclusively of nicked or linear DNA as confirmed by analytical CsCl density centrifugation in the presence of ethidium bromide (36 pg/ml). These four fractions contained 9.1% of the total DNA and about nine-tenths of the nicked material originally present in the sample. It should be noted that the eluting phosphate concentrations for the nicked and closed-circular forms differ only by 0.018 M instead of 0.025-0.03 M as observed in the runs with ColEI-DNA.
CHROMATOGRAPHIC
DNA
FRACTIONATION
381
DISCUSSION
Recently we were able to show that the affinity of DNA for hydroxyapatite is reduced if intercalating ligands are bound to this doublehelical polymer (2). The sigmoid-shaped decrease in the eluting phosphate concentration with increasing amounts of bound ligand as shown in Fig. 2A for ethidium bromide confirms this result. This effect is possibly caused by the distortion of the DNA helix due to the intercalator leading to irregularities in the phosphate distances. This in turn might result in a reduced binding to the crystal surface of the hydroxyapatite matrix, in agreement with the idea of Bernardi (14). Otherwise, the intercalation might also turn the phosphate groups at the binding sites slightly to the inside of the helix if hydrogen bonds are formed with the ligand (15). This could also reduce the binding affinity to hydroxyapatite, as proposed by Martinson (16). The behavior of the supertwisted circular DNA in the experiments reported in this paper may best be explained by taking into account both effects. At low ratios of ethidium bromide to nucleotide pair (Y < 0.09) where the super-twisted circular DNA binds more dye than the linear DNA (17), we find an increase in the binding of this DNA to the adsorbant, while the binding of the linear DNA to the absorbant is barely changed. This effect is most probably a consequence of the increased availability of the phosphate groups for the adsorbant in the opencircular form. At higher r-values (Y > 0.09), the uptake of dye by the closed-circular DNA is limited (17). One would therefore expect a continuous increase in the difference between the binding affinities of the two dye-DNA complexes for the adsorbant with increasing I’. The fact that this is not observed may be explained by the reformation of twists in the opposite sense in the circular DNA. The accessibility of the phosphate groups for the adsorbant would therefore again be reduced. At a ratio of about 0.42, which is substantially above the highest possible charging of circular DNA with ethidium bromide and near the saturation of linear DNA, as shown in Fig. 8. both forms are eluted at roughly the same phosphate concentration (Fig. 2). The obvious implication is that the effect of the higher r-value obtained for the linear DNA is compensated by the formation of positive twists in the closed-circular DNA. More quantitative evaluations of our results require an exact knowledge of the binding isotherm for the closed-circular DNA under the conditions of our experiments i.e., in about 0.2 M sodium phosphate buffer. The evaluations require in addition that the amount of dye bound to both DNA species is not appreciably altered when the DNA is fixed on hydroxyapatite. No assumptions can be made about the second point; we only know that the fixed DNA’s bind enough dye to produce
382
PAKROPPA,
0.1
GOEBEL
0.2
AND
0.3
MijLLER
OL
r
8. Isotherm for the binding of ethidium bromide to linear DNA from E. coli in 0.2 M sodium phosphate, pH 7.1, at 25°C as determined by equilibrium dialysis (solid line). The broken line corresponds to the isotherm for a closed-circular duplex DNA with an intrinsic superhelical twist u = -0.026 per ten nucleotide-pairs as calculated from Bauer and Vinograd’s data (17) for SV40-DNA. FIG.
strongly colored pink bands on the hydroxyapatite. Concerning the first point, one could construct a binding isotherm for the closed circular DNA on the basis of the data reported by Bauer and Vinograd (17) for closed-circular and nicked SV40-DNA in 5.8 M C&l. This implies however that the different uptake of dye by the two forms is caused mainly by the different mechanical properties of the two molecular species and is not influenced by the ionic strength of the medium. Assuming that these preconditions are fulfilled, we constructed the binding isotherm for the ColEl-DNA (Fig. 7, broken line) from the isotherm for linear E. coli-DNA and the data of Bauer and Vinograd (17). A comparison of the two isotherms with the curve in Fig. 2B reveals that the best separation of the two DNA species in our system occurs in an r-range in which the difference between their v-values at a given free-dye concentration shows a maximum. This range is around r = 0.25 for the linear DNA which corresponds to r = 0.165 for the closed-circular DNA. From this finding it might be concluded that the affinities of the two dye-DNA complexes for the hydroxyapatite depend more on the amount of structural change introduced by the intercalated dye than on the number of induced positive twists. At very high Yvalues, however, the effect of the twists seems to dominate again. On the basis of the arguments presented it is not surprising that a separation of large and small supercoiled circles by chromatography on hydroxyapatite-ethidium bromide failed. If both DNA’s have the same intrinsic superhelical density their dye-binding properties and consequently their structural changes should be the same. For this reason
CHROMATOGRAPHIC
DNA
FRACTIONATION
383
different binding affinities to hydroxyapatite cannot be expected. It remains unclear, however, why nicked-circular DNA (as clearly observed with the large nicked Rldudl9-DNA) is eluted somewhat later than the linear chromosomal DNA from E. co/i as the dye binding of this DNA should be the same as of linear DNA. The situation is altered however when the intrinsic superhelical density changes. In the last experiment reported PM2-DNA was used instead of ColEl-DNA. The superhelical density of the closed circular form of this DNA was reported to be - 0.053 (18) which is nearly twice as high as the corresponding value for SV40- or ColEl -DNA (17,19). Since this higher degree of negative coiling allows substantially higher dye-binding and thus reduces the difference in the total uptake of dye for the closed-circular and the nicked or linear form it is not surprising that the separation of these forms is not as good in the case of PM2-DNA as in the cases reported before. We think that the described procedure provides a valuable new method for a rather rapid isolation of covalently closed-circular DNA’s on an analytical as well as on a preparative scale. ACKNOWLEDGMENTS We are very grateful to Miss U. Nielander for skillful technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft. We thank Dr. G. Bezanson for careful reading of the manuscript.
REFERENCES 1. Radloff, R., Bauer, W. R., and Vinograd. J. (1967) Proc. Nut. Acad. Sci. USA 57, 1514-1.521. 2. Pakroppa, W., and Miller, W. (1974) Proc. Nut. Acad. Sci. USA 71, 699-703. 3. Goebel, W., and Schrempf, H. (1972) J. Bncteriol. 111, 696-704. 4. Dewitt, W.. and Helinski, D. R. (1965) J. Mol. Biol. 13, 692-703. 5. Clewell, D. B., and Helinski, D. R. (1969) Proc. Nut. Acad. Sci. USA 62, 1159-l 166. 6. Satava, J., Zadraizil, S., and Sormova, Z., ( 1973) Collect. Czech. Chem. Commun. 38, 2167-2173. 7. Bray, G. A. (1960) Anal. Biochem. 1, 279-285. 8. Scatchard, G. (1949) Arm. N.Y. Acad. Sci. 51, 660-672. 9. Bourgaux, P., and Bourgaux-Ramoisy, D. (1967) .I. Gen. Virol. 1, 323-332. IO. Bazaral, M., and Helinski. D. R. (1970) Biochemistry 9, 399-406. 1 I Marmur, J. (I 961) J. Mol. Biol. 3, 208-2 18. 12. Goebel, W. ( 1970) Eur. J. Biochem. 15, 3 1 l-320. 13. Cozzarelli, N. R., Kelley. R. B.. and Kornberg. A. (1968) Proc. Nat. Acud. Sci. USA 60, 992-999. 14. Bemardi, G. (1971) in Methods in Enzymology. (Colowick, S. P., and Kaplan, N. 0.. eds.), Vol. 21, pp. 95-l 39. Academic Press, New York and London. 15. Fuller, W., and Waring, M. (1964) Ber. Bunsenges. 68, 805-808. 16. Martinson, H. G. (I 973) Biochemistry 12, 2737-2746. 17. Batter, W., and Vinograd, J. ( 1968) J. Mol. Biol. 33, 14 I - I7 I. 18. Upholt, W. B., Grayjr, H. B., and Vinograd. J. ( 197 1) J. MO/. Biol. 61, 2 l-38. 19. Bazaral, M., and Helinski. D. R. (1970) Biochemistry 9, 399-406.