Dehydrated circular DNA: Electron microscopy of ethanol-condensed molecules

J. Mol. Biol. (1976) 106, 97-107

Dehydrated Circular DNA: Electron Microscopy of Ethanol-condensed D. LANG, T. N. TAYLOR?,

Molecules

D. C. DOBYAN~ AND D. M. GRAY

Molecular Biology Program, The University of Texas at Dallas P.O. Box 688, Richardson, Tex. 75080, U.S.A. (Received 18 December1975, and in revisedform 14 May 1976) The ethanol-induced condensation of linear DNA from phage #29, and circular DNA from phage PM2 and plasmid R6K, on air-dried electron microscope specimen grids has been analyzed by measuring size and shape of the resulting particles. Upon such dehydration, single DNA molecules are condensed into tight particles of three different classes of superstructure, appearing shorter and thicker with increasing ethanol concentration, as described earlier with coliphage T7 DNA (Lang, 1973), except that circularity of DNA impairs the formation of condensates of second and third order to a degree that depends on the molecular weight. It is suggested that tight supercoilsare formed by induction of numerous kinks in the double helix between short, straight DNA segments. This would be consistent with DNA kinks proposed by Crick & Klug ( 1975).

1. Introduction The packing, or condensation, of DNA in eukaryotic chromosomeshas been the subject of many investigations. Based on X-ray results, the DNA in chromatin was thought. to be in a supercoiled conformation (Pardon & Wilkins, 1972; Pardon et al., 1974). New evidence (Van Holde et al., 1974; Burgoyne et al., 1974; Noll, 1974; Olins $ Olins, 1974) led to the view that DNA in chromatin is condensed together with two tetramers of four major histones into discrete, nearly spherical particles of about 70 to 100 A diameter, connected in tandem by more flexible stretches of chromatin (Kornberg, 1974). This proposal is supported through recent studies by Axe1 et al. (1974), Senior et al. (1975), Griffith (1975), Lohr & Van Holde (1975), Germond et al. (1975) and Thomas & Kornberg (1975). Most studies of DNA condensation have been done with chromatin from or in eukaryotic cells. All work using electron microscopy involved some sort of dehydration in the preparation of chromatin, raising the question of artifacts. Alternatively, one may start with purified, protein-free DNA and look for condensation after solvent substitution by alcohols (Geiduschek & Gray, 1956; Herskovits et al., 1961) or by simple environmental shifts such asapplied by Lerman (1974) and Jordan et al. (1972) for solutions of water, salt, and neutral polymers in which the DNA assumesa folded t Part of the work described in this paper has been carried out by T. N. T. in partial fulfillment of the requirements for the degree of Master of Science in Molecular Biology at the University of Texas at Dallas. $ Present address: Department of Pathology, Universit,y of Maryland at Baltimore, Medical Sohool, Baltimore, Md 21201, U.S.A. 7

97

OH

I ) . 1, A s u ) ‘1’.

s.

‘l’;\YI~OK,

I).

(‘.

I)OHF;\,I‘

ANI)

I).

II.

c:lt.-\\

conformation by volume exclusion. Xnot hrr type of DSA condensation OWUI’S itt aqueous salt solution at pH values of about 2 (Dore ut ccl.. 1972, 1973). The finding in this laboratory that the collapse of a DNA molecule, upon sub st’itution of its aqueous salt solvent by ethanol, results in a particle having a MUrandom tertiary conformation (Lang, 1969) initiated a more detailed study of t,he dehydration of purified bact,eriophage DNA (Lang. 1973). By subjecting linear DKA to various concentrations of ethanol followed by air-drying on electron microscope specimen grids, a condensation of individual DKA molecules into three markedly different size classes of space-filling. asymmetric, linear particles was found. A dimensional analysis of these particles, taking advanhage of the uniform molecular weight of the DNA, suggested a hierarchy of tightly-wound supercoils. This effect of ethanol clearly involves competition with DNA f or water, a component essential for maintaining the structure of the double helix. It also indicates that DNAwithout any protein-is sufficiently versatile to assume superstructures of high order, and that dehydration by ethanol may be used as a simple analogue of more complex dehydration processes in vivo. This paper compares ethanol-induced condensation of linear DNA with that of either covalently-closed (supertwisted) or nicked (relaxed) circular DNA.

2. Materials (a)

and Methods DNA

Bacteriophage PM2 and its host, Pseudomonas BAL31 (Espejo & Cafielo, 1968) wert’ gifts from Dr H. B. Gray (University of Houston) who also supervised and helped in preparing the phage by one of us (T. N. T.) in his laboratory. Initial preparations of phage PM2 DNA were donated by Drs J. C. Wang and W. A. Baase (University of California at Berkeley). Bacillus subtilis phage 429 was a gift from Dr D. L. Anderson (University of Minnesota) and bacterial plasmid DNA (RBK), extracted from detergent-lysed cells and purified by isopycnic centrifugation in the presence of ethidium bromide (Kontomichalou et al., 1970), was contributed by Dr R. C. Clowes and Mrs P. Holmans (University of Texas at Dallas). Phages $29 and PM2 were disrupted with Sarkosyl and the DNA was extracted by phenol and stored at 4°C in 0.01 M-phosphate buffer (pH 7.8) containing 0.001 M-Na,EDTA, 0.018 M-Na+ (429 DNA) or at, -60°C in 0.1 M-NaCl, 0.02 M-Tris, 0.001 nr-Na,EDTA (pH 7.5) (PM2 DNA). The composition of PM2 DNA with regard to forms I (circular supertwisted), II (circular relaxed) and III (linear) was frequently monitored by electron microscopy by counting between 200 and 1700 molecules for each determination on non-overlapping fields on the fluorescent screen at a magnification of 20,000. Circular molecules with more than 3 strand crossings were counted as form I; fragments were counted (as form III) only when they appeared longer than half-size. (Supertwists are here defined as interwound sl;psrhelical turns, and supercoils as toroidal superhelical turns; see Bloomfield e2 al., 1974.) DNA samples predominantly in form II were obtained by diluting form I DNA into 2 M-ammonium acetate and heating in a 90°C water bath for 8 min and then dialyzing against 0.2 M-ammonium acetate, 0.001 M-Na,EDTA. (b)

Electron

microscopy

(i) Samples without ethanol were prepared by adsorption to cytochrome c monolayers from droplets (Lang & Mitani, 1970) containing 0.20 M-NH&~ (pH 5) in place of ammonium acetate. (ii) Ethanol treatment (at room temperature), staining, and sampling of DNA proceeded according to Lang (1973). Note that no cytochrome c is involved in the ethanol experiments. For the experiments listed in Table 1, the salt concentrations in the initial

ELECTRON

MICROSCOPY

OF

DEHYDRATE

L) DNA

99

DNA solutions before the first ethanol step were 0.2 M-NH,acetate and 0.001 M-Na,EDTA (pH 6) ; but some solutions contained instead 0.5 M-NH,acetate (the first of each PMZ(1) experiment at a given ethanol concentration), 0.48 M-NaCl (R6K, 10% ethanol), 0.2 W-NH&Y, pH 5 (R6K, 95% ethanol), or 0.04 M-phosphate buffer, pH 7 (R6K, 30% ethanol). Earlier experiments showed no significant change of lengths at these salt concentrations. No preferential adsorption of supertwisted or relaxed DNA to specimen grids was noticed. Some samples were shadowed with platinum after the first ethanol step instead of staining with phosphotungstic acid. (iii) Micrographs of DNA were optically projected and traced on paper for contour length measurements of the DNA with a map measurer. Magnifications were calibrated by a replica of a cross-lined opt’ical grating (2160 lines/mm).

3. Results lt will on linear

be shown here that the previous conclusions regarding the ethanol effect T7 DNA as seen by electron microscopy (Lang, 1973) are equally valid for another linear DNA, from phage 429, that is only half the size of T7 DNA. Then the ethanol effect on circular DNA from phage PM2 and from plasmid R6K will be presented and compared with that’ on linear DNA. (a) Analysis

of condensed

DNA

The analysis of ethanol-treated, condensed DNA molecules is based on the following previous observations by electron microscopy (Lang, 1973). Linear DNA of uniform size, at concentrations of the order of 3 pg/ml or less and processed through ethanol, yielded predominantly single DNA molecules t’hat were condensed into particles. These particles remained linear but their size and shape changed in that they became shorter and thicker with increasing ethanol concentration. This change was not a gradual one but such that, at ethanol concentrations of lo,30 and 950/b, condensations of successive orders 1, 2 and 3 could be seen. In each order, the particles had characteristic lengths and diameters. The particles appeared compact and the failure to detect any internal structure by conventional staining methods indicated that this condensation was approximately space-filling. The high asymmetry of the particles (ratio of length to diameter) strongly suggested a non-random tertiary conformation. The only feasible alternatives are folding and supercoiling. Folding was considered to be unlikely because three sets of approximately equidistant bending sites along the DNA are required to explain a folding which produces the observed, quite uniform particle size and diameter within each order of condensation. There was no evidence or compelling argument for such sites. Consequently, an entire DNA molecule was thought) to be coiled about its imaginary long axis, then coiled again (coiled coil), and so on. This model and the experimental results were described by the equation log L, = log I,, -

n log c;

n -= 0, I, 2. .

(1)

relating the length L, of a DNA particle to n, the order of supercoiling (Lang, 1973). L, is the contour length before coiling (DNA B-conformation). Combining equation (1) with the demand that the volume V of the (dry) DNA molecule is conserved during condensation, i.e. V = qn L, = constant, it follows that qn = CP*- 1. The value of the parameter c is the factor by which nth coil. qnz increases over the area of the cross-section

(2) the area of the cross-section of the of the (n - I)th coil, qn- 1.

100

D.

(i) Molecular

LANG,

T.

N.

TAYLOR,

I).

C’. DOBYAN

AND

I).

M.

GRAY

weights

At zero ethanol concentration, the DNA has been prepared for electron microscopy by a protein monolayer technique according to Materials and Methods, section (b) (i). Under these standard conditions, duplex DNA is extended and has a molar linear density of M’ = (2.07 f 0.04) x lOlo dalton/cm as determined previously (Lang, 1970). This is close to the B-conformation which has a calculated value of 1.958 x lOlo dalton/cm assuming a rise per base-pair of 3.38 A (Arnott & Hukins, 1972) and 661.87 dalton per average disodium base-pair (a GC pair differs from an A.T pair by only 2 dalton). The contour length the DNA would have in the B-conformation L,, can thus be obtained by multiplying the measured contour lengths as listed in Table 1 at zero ethanol concentration by (2.07 Y 101°)/(1.958 x lOlo) = 1.057. 1

TABLE

Length measurements DNA from

429 ~-.

PM2 (1)

PM2 (11)

R6K

Ethanol concn (% v/v)

No. of molecules

DNA (1)

form 11 (%)

IT1

L

Length SSDt km)

SDMS

100 100 100 100

5.92 1.68 0.30 0.074

0.16 0.17 0.03 0.019

0.09 0.02 0.003 0.001

0 10 30 95

85 60 122 170

10 10

88 185

s 80

17 16

1 6

0.78 0.08 0.88 0.12 0.83 (Av.)

0.01 0.01

30 30

71 160

82 80

17 16

1 6

0.36 0.29 0.32 (Av.)

0.07 0.05

0.01 0.004

95 9.5 95

278 504 604

82 80 59

17 16 36

I 5 6

0.039 0.042 0.038

0.002 0.002 0.002

3.07 3.06 3.06 3.06 (Bv.)

0.13 0.16 0.11

0.02 0.02 0.01

100 100 100

0.196 0.166 0.171 0.177

(Av.)

0 0 0

49 64 133

10

17

5

83

12

0.70

0.07

0.02

30 30

49 59

13 5

78 83

9 12

0.32 0.10 0.36 0.08 0.34 (Av.)

0.01 0.01

96

278

15

73

12

0.133

0.036

0.002

0 10 30 95

56 31 69 98

87 87 87

100 12 12 12

1 1 1

11.66 2.91 9.740 0.199

0.40 0.26 0.065 0.038

0.05 0.06 0.008 0.004

t SSD, sample standard deviation. $ SDM, standwd deviation of the mean. Av., average length for a given ethanol concentration.

-

ELECTRON

MICROSCOPY

OF TABLE

Contour

DEHYDRATED

101

DNA

2

lengths, L (measured), L, (in B conformation), a& molecular weights, DNA from $29 PM2 R6K

Length, From

M

L

literature

This

work

(rm) 5.78*0.15t 3.02+0.11: 12.8 &0.3§

592*0.15 3.06&0.01 11.56+0.40

t Anderson t Moshanafa (1968). $ Espejo et al. (1969). § Kontomichalou et al. (1970). ~/ The indicated error of M is the square

root

6.26 3.24 12.22

of the sum of the squares

12.3hO.4 6.350.1 23.9kO.9

of the errors

in M’ and

L.

The molecular weight, M, of duplex DNA is given by M = M’L

= (2.07 + 0.04)

x IO’O L.

(3)

where L is the contour length in cm measured under standard conditions. Table 2 lists L, L, and M. Our length measurements are close to values based on DNA preparations from 0.15 al-ammonium acetate and reported earlier by others (Table 2). (ii) Linear

DNA

The morphology of condensed 429 DNA was found to be identical with that of T7 DNA except for particle length. Again, three fairly uniform size classes(about f 10 to + 26% standard deviation within a class)of linear particles were observed. Dimers and higher aggregatesappeared also, depending on the DNA concentration. At 95% ethanol, circular particles with joined ends were found as before with T7 DNA. Figure I(a) showsa plot according to equation (1) for 429 DNA. The point at n = 0 refers to the length in the B-conformation (from Table 2), the other values were taken from Table 1; the values I,2 and 3 of n correspond to IO,30 and 95% (v/v) ethanol, respectively. The straight line through the pivot point L, at n = 0 has been calculated by the method of least squares. From the slope, one calculates c = 4.408 in good agreement with c = 4.308 (broken line) found previously for T7 DNA (Lang, 1973). It follows that c is essentially independent of n and L, or molecular weight of the two DNAs tested and that the data are consistent with the supercoil model. (iii) Circular DNA The particles of condensed,circular DNA from phage PM2 and plasmid R6K are shown in Figure 2. They are representative of both supertwisted (form I) and relaxed (form II) DNA, since no significant difference in condensation has been detected between them. This remarkable fact will be interpreted later in the Discussion. The staining is generally very light and, at 10% ethanol, the contrast is low. Therefore, a shadowed preparation (R6K) has been included in Figure 2. All micrographs are shown at a magnification of 5.4 x lo4 illustrating the large size changes between transitions. Not shown are moleculesat zero ethanol concentration (n = 0); at this magnification they would have contour lengths of 16.6 cm (PM2 DNA, form II) and 69.4 cm (R6K, form II). The particles at 10% and 30% ethanol are obviously circular, the former being mostly aligned during drying on the grid and showing few if any

(bl

FIG. 1. (a) The average lengths, L,, of condensed $29 DNA are plotted according to equation (1) as a function of the order of condensation, n; n = 0, 1, 2, 3 correspond to zeroth (B conformation), lst, 2nd and 3rd order of condensation, respectively. The slope of the solid line gives c ~ 4.408 in good agreement with c = 4.308 (broken line) found previously for T7 DNA. (b) Similar plot as in (a) for circular PM2 DNA, form I (0) and form II ( n ). The measured particle lengths et 95% ethanol (Table 1) have been doubled for reasons given in the text and are plotted at n = 3. Significent deviations from the behavior of a linear DNA that would have the same molecular weight as PM2 DNA (broken line; c = 4.308) occur at n = 2 and n :: 3. The deviations between forms I and II are insignificant. (c) Similar plot as in (a) for circular R6K DNA, form I. The deviations from a linear D?JA (broken line; c = 4.308) are less than those found with PM2 DNA.

crossovers, and the latter appearing less flexible and DNA was almost entirely in either form I or II (see dominantly linear particles at 95 ‘$6 ethanol, as listed by 2. The torus shown in Figure 2 (PM2, 95% ethanol) joined ends, as is also found occasionally with particles

more open. Since the original Table l), the length of the prein Table 1, must be multiplied is presumably a particle with derived from linear DNA.

R6K

PM2

104

1).

LANG,

T.

N.

TAYLOR,

I).

c’.

UOBYAN

.i,sl)

1).

AI.

GR.A\

The existence of circular particles at 10% and 30% ethanol shows that this COIIdensation proceeds by shortening along the circular contour, clearly excluding the circle about its midpoint. This possibility of a repeated folding of a “squeezed” suggests that circular DNA condenses by supercoiling. A two- or three-dimensional zig-zag folding over very short lengths, equal to the particle thickness, would be a special case of supercoiling. The analyses of circular PM2 DNA and R6K DNA are shown in Figure l(b) and (c). The condensation of both DNAs from n = 0 to )I = 1 follows that of linear DNA (broken line; c = 4.308) whereas it deviates at 12 = 2 and 3. The differences between circular forms I and II of PM2 DNA shown in Figure l(b) are not significant. The conclusion is that condensation of orders 2 and 3 is incomplete owing to a constraint imposed by the circularity of these DNAs. This topological constraint has a greater effect on the condensation of PM2 DNA than on R6K DNA (see Fig. l(b) versus (c)), the effect decreasing with increasing DNA molecular weight since R6K DNA is 3.8 times larger than PM2 DPI’A. (b) Additional

observations

(i) Ethanol precipitation of form 1 PM2 DNA in O-2 M-salt, drying of the precipitated DNA in warm air, redissolving in water, and electron microscopy, did not change the fraction (80%) of form I. Therefore, drying as described does not introduce singlestrand nicks in DNA. (ii) DNA from coliphage T4, in which cytosine is replaced by glucosylated hydroxymethyl cytosine, forms third-order condensates as easily as does T7 and $29 DNA. (iii) Third-order condensation is not observed if the initial aqueous DNA solution in O-2 M-N&acetate also contains 3 x 10m3 M-ethidium bromide which elongates (Lang, 1971) and stiffens (Freifelder, 1971) the DNA molecule.

4. Discussion Condensation of double-stranded DNA from aqueous salt solution by ethanol and drying on electron microscope specimen grids proceeds, as we wish to argue, by generation of supercoils of multiple order. This transition is independent of molecular weight (6 x lo6 and 25 x 106) and of the DNA source and appears to be a general response of the purified (protein-free) double helix. It must be emphasized that the the DNA while in aqueous ethanol micrographs are those of dried preparations; solution may be in a much less condensed conformation. The topological constraint imposed by circular DNA is too weak to restrict firstorder condensation but inhibits second- and third-order condensation to an extent inversely related to the DNA size. The observation that second-order condensates of circular DNA are open rings offers a new argument in favor of the supercoil model as opposed to lengthwise folding. Rather unexpected was the failure to distinguish between condensation of covalently-closed (supertwisted) and nicked (relaxed) PM2 DNA. An interpretation may be that the work available to generate a tight supercoil is considerably greater than the work required to overcome a few natural supertwists in a given molecule. This

ELECTRON

MICROSCOPY

OF

DEHYDRATED

10.5

DNA

is supported by an estimate of the number of supercoil windings, N,, for each order. n, on the basis of the supercoil model, if we make additional assumptionst. The result is N, = 669, N, = 78, and N, = 8. The number N, may be compared with the number of supertwists in covalently-closed circular PM2 DNA, which is believed to be about 100 (in 3 ~-&cl; Wang, 1974) and thus only 15% of N, = 669. If generating one supertwist and one supercoil winding involve equal energy changes, it follows that the conformational effect of dehydration on circular DNA is about the same irrespective of whether the DNA is relaxed or supertwisted. The absence of significant additional supertwisting in DNA after condensation by loo/;, ethanol could be interpreted in several ways. (i) The dehydration produces one (or more) single-strand nicks somewhere in the backbone of the circular DNA. thereby introducing a swivel point for relaxation of conformational strains imposed by supercoiling. Such nicking has been experimentally excluded (see Results, section (b)(i)). (ii) The primary effect of dehydration on the covalently-closed circular double helix is a uniform increase (or decrease) of the winding angle between adjacent base-pairs and is compensated by left-handed (or right-handed) supercoiling result,ing in a circular particle with no additional supertwists. If PM2 DNA (of about 9600 base-pairs) is assumed to have 669 first-order supercoil windings, the change in winding angle would have to be about 25 degrees per base pair. Although there is evidence that the winding angle of DNA in solution slightly increases in the presence of salt (Wang, 1969) and ethanol (Gray, unpublished data), an increase in winding angle as large as 25” has never been reported and seems to be unlikely. (iii) Dehydration leaves the winding angle essentially unchanged but produces a supercoil. Its coiling sense may, if necessary. alternate from right- to left’-handed around the circular particle to minimize topological constraints. Since the coils of space-filling first-order supercoils would have a radius of curvat,ure of roughly 10 A, implying large conformational energy changes, it may be energetically favourable that the DNA in the supercoil is not uniformly bent but sharply kinked with straight segments between kinks. Such a supercoil structure would be consistent with model-building by Crick & Klug (1975) who showed that 90” t.o 98” kinks in DNA are feasible and that kinks may be involved in the known types of DNA condensation. A feature of such a model is that left- or right-handed supercoils could exist simultaneously in a circular DNA depending on the number of base-pairs between kinks. The energy required to generate large numbers of kinks could he supplied as water is removed from the DNA. t \Ve reasonably expect that the cross-section, qn, of a supercoil of order n is circular and that t.he cross-section of order n - 1 (and lower) in this particle is deformed during the transition from I? --- 1 to n in order to generate a space-filling particle, but that the arecl of the cross-section Q” _ I is conserved in this transition. The winding number N, is then N, = V/V,, where 8, is the volume of one winding and equal to the volume of a toroidal body generated by rotating the area of qn _ 1 by 2a around the long axis of the coil. This volume is V, = 2aRq, _ 1 where R is the distance of the ocnter of mass of the area from the axis of rotation. Assuming that R = d,/4, where d, is the diameter of qn, we obtain with equations (1) and (2) X”

= V/[2n(d”/4)q,-

1] = 2q,L,/nd,q,..

The best experimental vaIue of d, is d, = 6.4 x IO-? cnez, one obtains rl, = &c(“-~)‘~. Thus. S” For r = 4.308

and

L, = 3.24x

L’I,,c2-3”‘2/ml*:

10m4 cm (PM2

DNA),

, z 2L,c’

em (Lang, ,I :

-“/nd,.

1973).

Since

qin/qz = (d,/d,)z

I, 2. .,.

X1 = ti69, S,

(-I) = 78, and S,

= 8

lOti

I).

LANG,

‘I’.

K.

T.~TLOR,

I).

(‘.

I,ORP;1S

;\NI)

I).

11. (:R.-\1-

While this work deals with dehydration by ethanol and subsequent air-drying. studies of DNA remaining in aqueou s ethanol do not support the notion of tight supercoiling at and below 414:) (v/v) ethanol although the Kuhn st~at’istical element is reduced by a factor of about 3 (J. E. Hearst. personal communicat’ion). However. it may be possible that ethanol, even below 41’:!; (v/v), introduces kinks which would shorten the st’atistical clement and t,hen permit t,ight, supercoiling. if airdrying follows. If the idea is adopted that interaction of DNA with histones involves not onl,v electrostatic shielding but also local displacement of water. one may expect thrb DNA in chromatin to be condensed, irrespective of the details of the int’eraction.

d. t’o the and A.

We thank D. L. Anderson, W. A. Baase, R. C. Clowes, H. B. Gray, P. Holmans and C. Wang for generous gifts of purified virus or DNA ; J . E. Hearst for his permission cite unpublished results; B. Bruton, L. Lewis, Jr, for assistance in the laboratory ; U.S. Public Health Service for National Institutes of Health grants GM34964, GM2085 1 GM 19060; the National Science Foundation for grant GB31158XI ; and the Robert, Welch Foundation for grant AT-503. REFERENCES

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MICROS(I’OPY

OF

Thomas, J. 0. & Kornberg, K. II. (1975). Proc. Van Holde, K. E.. Sahasrabuddhr. C.‘. G. & Shnw, 1586. \Vang, ,J. C’. (1969). .I. Mol. Biol. 43, 25-39. \Vang. .I. (‘. (1974). ,J. Mol. Biol. 89, 78%801.

DEHYDRATEI)

K’at. I?. Ii.

DNA

107

Acad. Sci., II’.S.A. 72, 2626.-2630. (I 974). XwI. Acids Res. 1, 1579