Cell Biology

Proc. Nati. Acad. Sci. USA Vol. 73, No. 11, pp. 4125-4129, November 1976

Superstructure of linear duplex DNA

(electron microscopy/analytical ultracentrifugation/circular DNA/benzyldimethylalkylammonium chloride-spreading method/ ethidium bromide)

Montpellier, France; and tInstituto de Biologia Celular, C.S.I.C., Madrid, Spain

H. J. VOLLENWEIDER*, TH. KOLLER*, J. PARELLOt, AND J. M. SOGOt * Institut fur Zellbiologie, Eidgen6ssische Technische Hochschule, Zurich, Switzerland; tEquipe de recherche 140, C.N.R.S., Universit6 du Languedoc,

Communicated by A. Frey-Wyssling, September 7, 1976

ABSTRACT The superstructure of a covalently closed circular DNA (of bacteriophage PM 2) was compared by electron microscopy with that of a linear duplex DNA (of bacteriophage T7) when ionic strength and benzyldimethylalkylammonium chloride concentration were varied. In parallel studies the sedimentation behavior of these DNAs was studied by analytical ultracentrifugation, but for technical reasons these had to be without benzyldimethylalkylammonium chloride. By combining the information from the two methods one has to conclude that with increasing ionic strength the linear duplex T7 DNA spontaneously forms a structure similar to that of the superhelical structure of closed circular PM 2 DNA. The superstructure is destroyed under premelting conditions and in the presence of an excess of ethidium bromide.
The determination of the structure of DNA in solution has been the subject of numerous studies. From sedimentation analyses it is known that the structure of linear duplex DNA is dependent on ionic strength (1-4). Covalently closed circular duplex DNA with different degrees of supercoiling shows a very complex sedimentation behavior when the counterion and ionic strength are varied (5-8). Molecular dimensions have also been gauged by viscosimetry (4, 9) and light scattering (10, 11). Yet there is still considerable disagreement in the literature when it comes to determining just how extended a linear duplex DNA molecule is in solution and to what extent the ionic strength influences the coil dimensions. In order to learn more about the structure of linear duplex DNA molecules in solution, we compared, by electron microscopy and analytical ultracentrifugation, the linear DNA of bacteriophage T7 (12) with DNA of bacteriophage PM 2, which is the most tightly supercoiled DNA known (13). MATERIALS AND METHODS Materials. Benzyldimethylalkylammonium chloride (BAC) was a gift from Bayer, Leverkusen, Germany, and was used from a stock solution (14) in formamide. Ethidium bromide (EtdBr) was obtained from Sigma (St. Louis, Mo.). All other chemicals were purchased from Merck, Darmstadt, Germany. Phenol-extracted DNA from purified Escherichia coli bacteriophage T7 was a gift from Dr. R. Portmann. DNA from bacteriophage PM 2 (containing over 90% form I, covalently closed circular DNA) was given to us by Dr. W. Arber. Heavy metal ions were removed by the addition of EDTA to the dialysis buffer. DNA was dissolved in a buffer containing 30 mM triethanolamine-acetate at pH 7.9 and sodium acetate at concentrations between O-3 M and 2 M.
Abbreviations: superstructure, structure formed by folding of a duplex DNA molecule; BAC, benzyldimethylalkylammonium chloride (nalkyl mixture: C12H25 60% and CG4HN 40%); EtdBr, ethidium bromide; form I PM 2 DNA, the covalently closed circular form of DNA of bacteriophage PM 2; form II PM 2 DNA, circular form with one or
more

Electron Microscopy. DNA (0.2 Asg/ml) was adsorbed to carbon-coated grids by the BAC-droplet method (14). The BAC concentration was varied in different experiments. In control experiments the droplet method with 125 ,gg/ml of EtdBr (15) was used. After adsorption of the DNA strands, the specimens were floated on redistilled water for 10 min, dehydrated in 90% ethanol, and dried on filter paper. The grids were rotary shadowed at an angle of 70 with 1100 Hz carbon platinum from an electron gun (16). In order to eliminate the possibility that the DNA structure observed in the electron microscope might be due to the ethanol treatment of the grids (see ref. 17), we made several experiments with and without ethanol dehydration in parallel. Since no difference was found, we retained the ethanol step, since it gives a smoother background and thus a better contrast for the DNA molecules. Analytical Ultracentrifugation. An MSE ultracentrifuge (1968 model) was used with a four cell rotor, ultraviolet absorption optics, and a scanning device for direct recording of absorbance. Boundary sedimentation velocity experiments were performed at 35,000 rpm, 20 0C, and DNA concentrations of about 0.5 A o/ml, by a method similar to that described in ref. 18. Sedimentation coefficients s20 were measured by standard procedures and were corrected for viscosity effects. Form II PM 2 DNA (circular, with one or more single-strand chain scissions) never exceeded 40% of the total.

RESULTS
In order to investigate the influence of BAC on the appearance of the DNA molecules in the electron microscope, we varied the BAC concentration between 1 X 10-5 and 5 X 10-2% (wt/vol). Below 2 X 10-4% BAC, the number of DNA molecules bound to the support film decreased until at 1 X 10-5% no more molecules were adsorbed. This was true independent of the ionic strength tested. Between 2 X 10-4 and 1 X 10-2% BAC the amount of adsorbed DNA remained roughly constant, giving reproducible binding of molecules to the support film. High BAC concentrations were characterized by the presence of highly compacted forms (Fig. 1A and B), whereas at low BAC concentrations relaxed or folded forms for both T7 and PM 2 DNA were found. Qualitatively, T7 and PM 2 DNA behaved similarly. The results are summarized in Table 1. All further experiments were performed at 2 X 10-4% BAC. Fig. 1C-H shows representative forms of T7 and form I PM 2 DNA at 10 mM, 100 mM, and 1 M sodium acetate concentrations. Below 100 mM sodium acetate the strands of both DNAs were relaxed and unfolded. At 1 mM salt concentration the known superhelical structure of PM 2 DNA could not be observed, and at 10 mM it only appeared as a slight supertwist (Table 1; Fig. iD). Between 0.1 and 1 M both DNAs progressively adopted more folded forms. The T7 DNA assumed a form

single-strand chain scissions.

with frequent hairpin folds and thick filaments, whereas the

4125

4126

Cell

Biology:

Vollenweider

et

al.Prc

Proc. Natl. Acad. Sci. USA 73 (1976)

F
.I

I'\
I.

Jt
I.
/-

G
FIG. 1. T7 DNA: left column (A, C, E, and G). PM 2 DNA: right column (B, D, F, and H). Magnification X22,500. All experiments were performed at a DNA concentration of 0.2 j~g/ml in 30 mM triethanolamine-acetate at pH 7.9 using the droplet technique of Vollenweider et al. (14). (A and B) Solvent containing 0.01 M sodium acetate and 5 X 10-3% BAG. (C and D) Solvent containing 0.01 M sodium acetate and 2 X 10-4% BAC. (E and F) Solvent containing 0.1 M sodium acetate and 2 X 10-4% BAG. (G and H) Solvent containing 1 M sodium acetate and
2 X

10-4%

BAG. The

same

result

was

obtained when 3.5% formaldehyde

was

added.

typical
LE and

thin filaments of DNA

G). The forms of PM 2 DNA percoiled DNA (Fig. iF and H).

We

progressively disappeared (Fig. are compatible with a supremelting

conditions, which should lead

undetectable
were

to

local melted regions (5, 6)

by conventional
to 60

electron microscopy. The

75

samples
in 1 M

heated

0C

(T7)

or

'C (PM 2) for 15 min

compared the

forms of both DNAs under

sodium acetate and 3.5%

formaldehyde,

30 mM triethanol-

Cell Biology: Vollenweider et al.

Proc. Natl. Acad. Sci. USA 73 (1976)

4127

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B .S#
....

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FIG. 2. T7 DNA: left column (A and C). PM 2 DNA: right column (B and D). Magnification X25,000. The same DNA adsorption technique as described in the legend of Fig. 1 was used. Samples were treated in the following way before spreading: (A and B) T7 DNA was heated to 60 C and PM 2 DNA to 75 C in 1 M sodium acetate, 3.5% formaldehyde, and 30 mM triethanolamine-acetate, pH 7.9, for 15 min. After cooling, BAC was added to a concentration of 2 X 10-4%. (C and D) Same conditions as in Fig. 1G and H, except that BAC was replaced by 125 ,ug/ml of EtdBr. Note relaxed form II PM 2 DNA molecule on the left of micrograph D.

amine-acetate at pH 7.9. After cooling in ice, the DNA was prepared for electron microscopy with 2 X 1o-4% BAC (Fig. 2A and B). Unlike the results shown in Fig. IG and H, which were also obtained in the presence of formaldehyde but without heat treatment, both DNAs were relaxed (PM 2 DNA: about 3 Aim, T7 DNA: about 12 ,um contour length). The same was true when EtdBr at a concentration of 125 ,ug/ml was used (15) instead of BAC, implying that a partial denaturation must have taken place. The same experiment was repeated but without formaldehyde. After cooling, the DNA was adsorbed again in the presence of excess EtdBr instead of BAC (15). The form I PM 2 DNA appeared highly folded (Fig. 2D), proving the absence of nicking during the heat treatment. The T7 and form II PM 2 DNA strands, however, remained fully relaxed and extended (Fig. 2C and Fig. 2D, strand at left).
Table 1. Appearance of T7 and PM2 DNA by electron microscopy in the presence of BAC* BAC concentration (%, wt/vol) 1 mM
1 x 10-2 5 x 10-3 1 X 10-3

Fig. 3 shows the sedimentation behavior of T7 DNA compared with that of form I and form II of PM 2 DNA as a function of sodium acetate concentration. The experiments could not be done in the presence of BAC because of overlapping absorbance at 260 nm. Particularly interesting is the fact that both T7 and form I PM 2 DNA show transitions in the same salt concentration range, near 0.1 M, although the transitions go in opposite directions. The sedimentation coefficient of T7 DNA increases by about a factor of 2, in agreement, although not strictly comparable, with published data (1, 4). The sedimentation coefficient of form I PM 2 DNA decreases about 15%
S20

30-

T7
PM

2/1

Sodium acetate concentration

100

500

20 -

PM

2/l1

10 mM
c c r

mM
c sf r
f

mM
c

1M

c
c c
r

r/c
Sf f

f/c f f
f

0.001

0.01

0.1

1.0

M NaOAc

2 X 10-4

T7

PM2:sf r

* c = compacted (e.g., Fig. 1A and B); r = relaxed (e.g., Fig. 1C); sf = slightly folded (e.g., Fig. 1D); f = folded (e.g., Fig. 1G and H).

FIG. 3. Sedimentation coefficient S20 (corrected for viscosity) as a function of the molar concentration of sodium acetate (NaOAc). (-) form I PM 2 DNA; (0) form II PM 2 DNA; (A) T7 DNA. The value 0 on the abscissa corresponds to 30 mM triethanolamine-acetate, pH 7.9.

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Proc. Natl. Acad. Sci. USA 73 (1976)

towards that of form II PM 2, which remains nearly constant. Form I and form II behave as distinct species, even at high salt concentrations (up to 2 M). This behavior is very similar to that observed with PM 2 DNA in the presence of sodium perchlorate (5). At low salt concentrations T7 DNA has lower s20 values than form II PM 2 DNA, whereas at high salt concentrations the corresponding S20 values are higher than those of form I PM 2 DNA. DISCUSSION It is highly unlikely that the progressive folding of T7 DNA at increasing salt concentration is an artifact of specimen preparation. The superstructure was observed in the presence of formaldehyde, but disappeared when the samples were heated to 60 'C and 75 "C, respectively. It is unlikely that the folding of T7 DNA occurs by a change in the adsorption characteristics since the number of adsorbed DNA molecules was independent of salt and BAC concentration (within the ranges given in Table 1). Form I PM 2 DNA is characterized by a superhelical structure in solution (13); therefore, the obvious parallel in the behavior of both types of DNA in the electron microscope experiments (Table 1, Fig. 1) is an indication that the folding of the T7 DNA is also a real phenomenon. Furthermore, in similar experiments in which BAC was replaced by 1,12-diaminododecane or malouetine, a good correlation between analytical ultracentrifugation and electron microscopy performed under strictly comparable conditions could be established. This implies that under controlled preparation conditions (19) electron microscopy may reveal forms corresponding to those present in solution (Parello, Etienne, Raynaud, and Koller, in preparation). It has recently been reported that supertwisting in a circular DNA molecule must be a property of the DNA molecule and not dependent on circularization (20). It is known from analytical ultracentrifugation and viscosimetry that aliphatic diamines with 12 or more carbon atoms in the aliphatic part of the molecule (18) and steroid diamines (21, 22) induce an uncoiling of closed circular DNA. From the data presented (Table 1) it appears that BAC, which is a monocationic amphiphilic molecule, has a similar effect. The conditions in which this relaxation of form I PM 2 DNA was most pronounced were shifted towards higher BAC concentrations as the sodium acetate concentration was raised (Table 1). Above 10 mM sodium acetate a BAC concentration of 2 X lo-4% was too low to unfold form I PM 2 DNA completely. Because of the relaxing effect of BAC, we believe that the strands visualized in our electron micrographs of Fig. 1E-H are still less folded than they are in solution in the absence of BAG. In the BAC-salt system T7 DNA behaved like form I PM 2 DNA (Fig. 1G-H). This implies that similar mechanisms act on both types of DNA and that T7 DNA might also have a superstructure in solution. The fact that a transition can be detected by analytical ultracentrifugation for both T7 DNA and form I PM 2 DNA in the same salt concentration range at about 100 mM, suggests that the observed ionic strength dependence involves a common mechanism acting on the linear as well as on the circular DNA molecule. However, the transition does not take place in the same direction for both the molecules (Fig. 3). The reason for this is not understood, but it may be explained by their different topologies. The transition is not visible for the nicked circular PM 2 DNA since its sedimentation behavior depends very little upon increasing sodium acetate concentration (Fig. 3). Superhelical structures of covalently closed circular molecules are destroyed by mild denaturation (5, 6). Our experiments under premelting conditions using formaldehyde (24) show that

the folding of both DNAs observed at high sodium acetate concentrations and 2 X 10-4% BAC disappears. Both types of DNA appear as unfolded strands (Fig. 2A and B), which again suggests that a native linear DNA molecule possesses a superstructure very similar to that of a covalently closed circular DNA molecule. Intercalating drugs like EtdBr and those of the acridine group unwind DNA duplexes (25, 26). In the case of covalently closed circular DNA, an excess of intercalating drug leads to supertwisting in the reverse sense (27). Our findings are in agreement with this superhelical structure (Fig. 2D). The results of our experiments with excess of EtdBr show that for T7 (Fig. 2C) and form II PM 2 DNA (Fig. 2D, nicked strand to the left), once the relaxed form has been reached, no further recoiling takes place, probably because the ends of the strands are not fixed. This finding is in agreement with the results of other authors (26, 28), who showed that intercalating drugs reduce the sedimentation coefficient and markedly enhance viscosity of linear DNA. At high BAC concentration we observed compact DNA molecules (Fig. 1A and B). This form does not appear to be the same as the folding induced by sodium acetate. It occurred also under premelting conditions. We believe that the compact form occurs as the result of BAC micelle formation (14). For technical reasons the sedimentation analysis could not be performed in the presence of BAC and is therefore not strictly comparable to the electron microscope observations. Nevertheless, both methods are complementary. The progressive folding seen for T7 DNA as the salt concentration is increased to 1 M is in agreement with the s2o values, which also progressively increase up to 1 M. The crossing of the curves for T7 and form II PM 2 DNA is of interest. We assume that at low ionic strength T7 DNA sediments more slowly than form II PM 2 DNA since it is a longer and a linear molecule. However, when the strands are folded at high salt concentrations (around 1 M), the sedimentation velocity of these molecules is influenced more by the molecular weight than the form [molecular weight of 25.3 X 106 forT7 DNA (12) and 6 X 106(23) for PM 2 DNA]. The sedimentation data for form I PM 2 DNA, however, do not agree with the electron micrographs obtained at 2 X 10-4% BAC (Fig. ID, F, and H; Table 1). At very low salt concentrations the closed circular molecules are relaxed, and only at 100 mM salt and above are they twisted. This disagreement may be explained by the action of BAC unwinding the superhelix in low salt concentration (Table 1). Our combined data from electron microscopy and ultracentrifugation suggest that above 100 mM sodium acetate a linear DNA molecule possesses a structure similar to that of a covalently closed circular DNA molecule. It is likely that at very low salt concentrations T7 DNA loses its superstructure as a result of electrostatic repulsion (1, 4), whereas for form I PM 2 DNA, because the ends are fixed, electrostatic repulsion will not give rise to a relaxed structure (7). Thus, DNA molecules have the ability to assume a higher order of structural organization at ionic concentrations similar to those measured in the cell nucleus (29). This may have implications for understanding of the packing of DNA within chromosomes of higher organisms, viruses, and bacteriophages, as well as for recognition in the binding of proteins to nucleic acid molecules. Our results imply that some of the forms commonly considered as spreading artifacts may be present in solution and have biological significance.
We thank Mrs. H. Mayer-Rosa and Dr. G. Etienne for excellent technical assistance and Drs. Ken Downing, Barbara Dod, Charles Weissmann, and Jack Dunitz for critically reading the manuscript. This

Cell Biology: Vollenweider et al.

work was supported by Schweizerischer Nationalfonds zur F6rderung der wissenschaftlichen Forschung, Grant no. 3.1590.73.
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