- Email: [email protected]
Electron microscopy of superhelical circular λ DNA
J. Mol. Biol. (1968) 32, 673-679
Electron Microscopy of Superhelical Circular h DNA VERNON C. BODE Department of Biochemistry, University of Maryland Medical School Baltimore, Maryland, U.S.A. AND
L. A. MACHATTIE~ Department of Biophysics, Johns Hopkins University Baltimore, Maryland, U.S.A. (Received 12 September 1967) Electron microscopy conf%rns that circular h DNA molecules contain supertwists. The average number of primary supertwists at low ionic strength (0.06) is 117, or 3.8 per million molecular weight. This number is not constant but decreases to an average of 12 when the ionic strength is increased to 2.0. The sedimentation rate of the superhelical molecules also decreaseswhen the ionic strength is increased through this range. The change in number of twists as revealed by both sedimentation and electron microscopy indicates that the pitch of the primary DNA helix varies with ionic strength.
1. Introduction A h DNA molecule whose ends have been covalently joined in vivo exhibits sedimentation properties that suggest it is superhelical, i.e. the primary helix is twisted about itself (Bode & Kaiser, 1965). It seems likely that when formed within the cells such circular molecules are not superhelical, but only become so when placed in an environment that differs in some significant aspect from the intracellular one. The significant factors are those that exert an influence on the natural pitch (base-pairs per turn) of the primary helix. A change in the concentration of such factors could effect a change from open circle to superhelix in any circular duplex DNA molecule both of whose component strands are intact circles. This view is consistent with all of our current evidence on superhelical DNA molecules. For example, double-stranded circular molecules that are closed in vivo contain twists when examined in the buffer solutions used for DNA isolation (Vinograd $ Lebowitz, 1966), but molecules whose ends are covalently sealed in vitro do not supertwist in the same buffers since the solutions provide an environment very similar to that in which the molecules were closed (Gellert, 1967; Gefter, Becker & Hurwitz, 1967). A more direct line of evidence is the observation that superhelices untwist when ethidium bromide intercalates between base pairs of the primary helix (Crawford & Waring, 1967). This result, as do those of Vinograd, Lebowitz, Radloff, Watson & Liapis (1965) with partial melting, indicates that in superhelical polyoma t Present address: Department Mass. 02115, U.S.A.
of Biologicrtl Chemistry, Harvard 673
Medical School, Enston.
674
V. C. BODE
AND
L. A. MAOHATTIE
DNA the primary helix is underwound, i.e. in linear duplex molecules the two singlestrands normally wrap around each other a fewer number of times in the in viva environment than they do in the in vitro environment. Rhoades & Thomas (personal communication) in a study of phage P22 DNA superhelices show, by partial-melting unwinding and by visualizing in electron micrographs the sense of the superhelical twist (right-handed), that the primary helix of this twisted circular DNA also is underwound. Crawford & Waring (1967) estimate the degree of supertwisting (underwinding) in polyoma DNA as 12 turns per molecule of 3-2 million molecular weight or 3-S primary super-twists per million molecular weight. This paper (i) confirms by electron microscopy of circular h DNA molecules the supertwisting deduced from their sedimentation properties (Bode &I Kaiser, 1965) and (ii) presents both electron microscopic and sedimentation data that indicate that the degree of supertwisting (and therefore the pitch of the DNA helix) varies with NaCl concentration.
2. Materials and Methods (a) Preparation of DNA from mature phuge A DNA was prepared by phenol extraction as described by Kaiser & Hogness (1960). The extracted DNA was dialyzed and stored in Tris-EDTA (lOma a6-Tris-HCl, IO-3 MEDTA, pH 7.1). A solution of purified h DNA with an Azao = I.0 contains about 60 pg of DNA/ml. The purity of intracellular forms was estimated by comparing the ratio of tritium disintegrations/min to optical density at 260 rnp for the extract of infected cells with the ratio obtained for DNA isolated from a sample of the mature phage used for superinfection. (b) Putification
of tuk2.d circular
X DNA
Lysogenic bacteria were infected with 3H-labeled h phage (I.5 x IO5 cts/min/60 pg of I\ DNA) and the nucleic acids were extracted from the infected bacteria as previously reported (Bode & Kaiser, 1965). Where necessary, extracts were concentrated by dialysis against saturated sucrose. All solutions were prepared in Tris-EDTA and maintained below 6’C. A crude infected-cell extract (3 or 4 ml. of a solution with Azeo = 10 was layered on a linear 6 to 26% sucrose gradient (62 ml. in a Spinco SW25,2 tube) and centrifuged for 6 hr at 25,000 rev./min or 16 hr at 18,000 rev./mm in a Spinco model L2 centrifuge. After collection through a hole in the bottom of the tube, fractions containing the intracellular forms of h DNA were identified by the distribution of tritium. Fractions rich in twisted circular molecules were pooled, dialyzed, concentrated by dialysis against saturated sucrose, stirred to shear the long molecules of high molecular weight host DNA, and then re-sedimented through a sucrose gradient. Judging by the ratio of tritium disintegrations to &mot the fractions selected from the first sedimentation contained tritiated superhelioal DNA of about 10% purity and the second sedimentation improved this to about 60% purity ( 100.fold purified over the crude extract). The pH was maintained near 7 during all steps of the isolation. (Although purification by melting the DNA in alkali followed by selective renaturation of circular duplex molecules might be more efficient, we wished to avoid melting and renaturation in the initial characterization of the superhelical species. Gellert (1967), using X DNA circles whose ends were joined in vitro, reports that purification by sedimentation in alkaline gradients altered the subsequent sedimentation rate of those circular molecules at neutral PH.) (c) Preparatti
of aampl8.8 for electron microscopy
Samples of purified superhelical ,I DNA were prepared for electron microscopy by the protein-film technique (Kleinschmidt $ Zahn, 1969; Kleinschmidt, Lang, Jacherts & Zahn, 1962), as modified by MacHattie & Thomas (1964). The purified DNA solution in Tr&EDTA was diluted 1 :I with 0.04% cytochrome c dissolved in either (i) 0.075 M-NaCI,
ELECTRON
MICROSCOPY
OF
SUPERHELICAL
h DNA
6;s
M-sodium citrate or (ii) 4.0 M-N&~. Of this mixture, 0.06 ml. WM spread on a level flame-cleaned stainless steel blade, then gently lowered into contact with a clean water surface at room temperature (20 to 23°C). This allowed a DNA-containing cytochrome c monolayer to spread out from the blade to cover the 60 cma water surface. Samples of the monolayer 2 to 4 mm from the blade were picked up by touching it with carbon f%.nbearing electron microscope grids. The grids were touched to 95% ethanol to remove water and rotary shadowed at a low angle with platinum-palladium.
0.0075
3. Results (a) CharacteGution
of the circular
X DNA
preparation
by sedimentations
The preparation of superhelical h DNA used for electron microscopy contained about 4 pg of 3H-labeled A DNA per ml. The optical density at 260 rnp exceeded that. expected from this amount of labeled DNA by O-12 unit, indicating the presence of contaminating host DNA at a concentration not greater than 6 pg/ml. As judged by sedimentation in a sucrose gradient (Fig. 1) about 80% of the tritiated X DNA
Dtstance
FIG. 1. Sedimentation analysis of the tritiated A sample (0.2 ml.) containing 0.8 pg of purified to 26% sucrose gradient (5 ml.) in Tris-EDTA,
from
meniscus
(cm)
h DNA preparation used in electron microscopy. intracellular species I A DNA was layered on a 5 pH 7.1. After sedimentation for 3 hr at 37,500
rev./min, 5’C, in the Spinco SW39 rotor, fractions were collected from the bottom of the tube. The distribution of the sH-labeled A DNA was determined by drying and counting B O-l-ml. portion from each fraction. Linear duplex h DNA (0.3 pg) isolated from the mature phage particles was sedimented in a duplicate tube. The distribution of this DNA is plotted as a reference (dashed line).
molecules were circular. Considering only circular molecules, 87% sedimented at a rate (3.2 cm in three hours) charaoteristio of species I, i.e. twisted circular molecules, while 13% sedimented at a rate (2.1 cm in three hours) characteristic of species II, i.e. untwisted circular molecules. After a period of one month during which grids were prepared for electron microscopy, re-analysis by sedimentation indicated that the fraction of twisted circular molecules, species I, had decreased to 78% and the fraction of species II had increased to 22%. (b) Electron microscopy Random samples of the DNA molecule population present on the electron microscope grids were obtained by photographing large fields unselected except for
676
V. C. BODE
AND
L.
TABLE
A. MAcHATTIE 1
Configuration of h DNA molecules in the puri$ed prepar&m DNA floated in High salt Low salt
Number of molecules 149 193
Number of circles 116 148
Percentage Less than 40 crossings 89 6
of circles
showing: More then 90 crossings 3 77
distinctness of DNA at low magnifications (2100 or 3700). Each micrograph showed an average of nine isolated DNA molecules of about h size, which constituted from 10 to 60% of the total DNA present in different areas of the grids. Of these isolated X-sized DNA molecules, 264 out of 342, or 77%, were circular (Table 1). In the grids for which the protein monolayer was spread from high-salt solution, the majority of the circular X DNA molecules were relaxed-looking open circles, while in the low-salt case the majority had a distinctly twisted appearance. Examples of the two forms are shown in Plate I. The length distributions of small samples from the two preparations are shown in Fig. 2.
High salt
“%-Length (+)
FIG. 2. moleoules (av. 113) crossings
Length distribution for circular molecules of h DNA. High 8&t: average length of 24 = 17.6 p. Lozo salt: length distribution of 22 molecules with greater than ninety crossings is shown by the dashed line. Length distribution of 38 molecules with less than 90 (av. 48) is shown by the dotted line.
(c) EstimaGm of superhelical twisting A quantitative comparison of the two preparations with respect to twisting was made by counting the number of times the duplex helix crossed over itself in each circular molecule. The results, shown in Fig. 2 and Table 1, clearly differentiate the two populations of molecules. In the number of crossings distribution of Fig. 3, the lowsalt preparation shows a major peak, comprising 77% of the total number of circular molecules (almost exactly the expected proportion of species I) in which the mean is 117 crossings f 11 8.~. In the high-salt preparation 89% of the circular molecules showed less than 40 crossings, with an average of 12 orossings per molecule in this major component. This low number may or may not represent a coherent superhelical twistedness, since the sense of the twists could not be reliably distinguished in these micrographs.
ELECTRON
MICROSCOPY
OF
SUPERHELICAL
h DNA
677
Number of crossings
FIQ. 3. Distribution of the number of times the primary DNA helix crossed itself in electron micrographs of circular h DNA molecules spread from solutions with low (0.06) and high (2.0) ionia strength at 20 to 23%. A statistically random sample of circular molecules wms examined in each case. The visually estimated upper limit of the major peak for crossing frequency in high salt w&s 40 crossings, end the lower limit of the major peak in low ealt was 90 crossings.
(d) The effect of ionic strength on the sedimentation of circular X DNA The sedimentation coefficient for linear X DNA molecules changes very little in the ionic strength range O-01 to 1.0 (Studier, 1966). In contrast to linear molecules, and also circular molecules with single-strand breaks (species II), the sediment&ion rate of A circular superhelices (species I) decreases with increasing salt concentration (Fig. 4).
001
0IO Ionic
I0
strength
FIG. 4. The effect of ionic strength on the sediment&ion rate of twisted circular h DNA (specie8 I) and untwisted circles (species II). Sucrose gradients (6 to 20%) were prepared in Trie-EDTA (or diluted Tris-EDTA for the lowest ionic strength) with N&l added to yield the indicated ionic strengths. A partially purified preparation of species I (9 pg/ml. of DNA of which 5% was tritieted h DNA) w&e mixed with 32P-labeled linear X DNA and N&l wee added to bring the ionic strength of the sample to a value near that of the gradient through which it would be centrifuged. The molecules in the sample were sedimented for 3 to 4 hr (37,000 rev./min, Spinco SW39 rotor, 5’C). The distance sedimented by twisted circular molecules (speoies I) relative to that traveled by the linear marker is plotted es e function of ionic strength (-O-O--). The relative sedimentation rate of species II (circuler molecules with single-strand breaks formed from species I ee e result of tritium decays) is also plotted for aomparieon (-O-O-). 48
678
V. C. BODE
AND
L.
A. MAcHATTIE
4. Discussion These results support the conclusion that h DNA species I is a circular molecule which in low-salt media is superhelically twisted, but in higher ionic strengths untwists to approach the open circular configuration. This would suggest that some dimensions of the duplex DNA molecular structure must vary slightly with ionic strength, in such a way as to change the pitch of the helix (number of base-pairs per 360” turn of helix). Though the molecular configurations seen in the electron microscope grids are well supported by the sedimentation data, some additional comment is called for regarding the relation of the molecular configurations seen on the grids to those present in solutions. The preparative technique for electron microscopy as was used here in the high-salt samples has been shown in a number of past determinations on DNA from a variety of phages to yield molecular lengths which, when compared with independent molecular weight determinations, are consistent with the B form of the Watson-Crick helix (MacHattie & Thomas, 1964, Thomas, 1966). However, Lang, Bujard, Wolff & Russell (1967) have shown thatunbrokenviralDNAmolecules incorporated into a protein monolayer by diffusion through solutions ofionic strength less than 0.14 are more thanusually heterogeneous in length, and are on the average longer than would be expected for the B form of the helix. Inman (1967) reports a similar finding. In the present experiments, this length effect is seen in the sample floated from a solution of ionic strength O-06, but not in that floated from ionic strength 2.0. This lends credence to the idea that in our technique the DNA-containing protein monolayer forms predominantly over the salt solution lying on the horizontal blade, and that the configuration and length of DNA molecules once embedded in the monolayer remain essentially unchanged as the monolayer expands out over the water hypophase for at least 2 to 4 mm. Thus the configurations of the X DNA molecules reported here may be believed to represent the conditions in the respective salt solutions in which they were layered on the blade, rather than the low ionic strength found in the hypophase onto which the monolayer was allowed to spread. The limits of accuracy of this statement are indicated by the occasional highly twisted molecules (3% showed more than 90 crossings) found in the high-salt sample. These most probably represent a small minority which through mixing and diffusion became exposed to low ionic strengths before incorporation into the monolayer. The relatively constant length of the highly twisted molecules in the low-salt sample (see Fig. 2) is of interest, in that it suggests that the twisting stress exerted on the DNA helix by the constraint of superhelical configuration may prevent it from undergoing the type of change that is responsible for the length heterogeneity in low ionic strengths reported by Lang et al. (1967) and Inman (1967) and seen in the less twisted members of the same population. These less twisted molecules most probably contained single-chain breaks, corresponding to sedimenting species II, and therefore were not subject to the superhelical constraint. The shortness in apparent length of the highly twisted molecules may not be significant, since it could represent a consistent error in measurement of moleoules with many crossings. The sedimentation rate of species I varies inversely with ionic strength below values near 1-O (Fig. 4). This behavior can be rationalized if, as is indicated in the electron micrographs, this circular form is less twisted in solutions of high ionic strength. At
ELECTRON
MICROSCOPY
OF
SUPERHELICAL
h DNA
670
sodium chloride concentrations above 1 M, the rate at which species I sediments appears to be independent of ionic strength but is significantly faster than that for species II. This suggests that it is still twisted and that the low number of crossings seen in the electron micrographs of molecules from the solution with high salt represents coherent superhelical twists rather than just random crossings. Presumably the molecules would untwist completely if placed in a solution which mimicked the intracellular environment. Salts other than sodium chloride might be more effective in this respect. Only one has been examined. Magnesium sulfate at 0.001, 0.01 and 0.1 &I did not significantly reduce the sedimentation rate of h superhelices below the value predicted from Fig. 4 on the basis of ionic strength alone. The number of twists in X superhelices seen on the low-salt electron-microscope grids agrees remarkably well with Crawford & Waring’s (1967) and with Vinograd’s (personal communication, 1967) estimate of the twistedness of polyoma DNA molecules in low salt. It will be realized that if all the crossings of a circular molecule over itself represent twists of the same sense (and this is likely to be true for highly twisted molecules), each crossing is equivalent to a half-twist of duplex about duplex, or to one full 360” twist imposed on the primary DNA helix. This is what Crawford & Waring term one supercoiling twist. The frequency of superhelical twists that we observe in circular A DNA prepared for electron microscopy from solutions of ionic strength 0.06 at 20 to 23°C is then 117 primary supertwists per 31 million molecular weight, or 3.8 per million. This is exactly what Crawford & Waring (1967) estimated by a completely different method for polyoma DNA in solutions of similar (0.05) ionic strength and temperature (ZO’C). If this agreement is not taken to be simply coincidental, it suggests that whatever factors in the intracellular environment are responsible for superhelix formation, their effect is constant per unit length of DNA, and of the same magnitude in cells as different as mammalian and bacterial. The present work indicates that high ionic strength mimics, at least partially, the effect of the intracellular environment on the pitch of the DNA double helix. This work was supported by grants from the National Science Foundation (GB-2793) and the Public Health Service (AI-06493). One of us (L. A. M.) was a Career Development Awardee (5-K3-GM-25,342) of the Public Health Service. REFERENCES Bode, V. C. & Kaiser, A. D. (1965). J. Mol. Biol. 14, 399. Crawford, L. V. & Waring, M. J. (1967). J. Mol. Biol. 25, 23. Gefter, M., Becker, A. & Hurwitz, J. (1967). PTOC. Nat. Acd Sci., Wash. 58, 240. Gellert, M. (1967). Proc. Nat. Acad. Sci., Wash. 57, 148. Inman, R. B. (1967). J. MOE. Biol. 25, 209. Kaiser, A. D. & Hogness, D. S. (1960). J. Mol. Biol. 2, 392. Kleinschmidt, A. K., Lang, D., Jacherts & Zahn, R. K. (1962). Biochim. biophys. Actu, 61, 857. Kleinschmidt, A. K. & Zahn, R. K. (1959). 2. Natur-. 146, 770. Lang, D., Bujard, H., Wolff, B. & Russell, D. (1967). J. Mol. Biol. 23, 163. MacHattie, L. A. & Thomas, C. A. (1964). Science, 144, 1142. Studier, F. W. (1965). J. Mol. Biol. 11, 373. Thomas, C. A. (1966). J. Gen. Phyeiol. 49, part 2, 143. Vinograd, J. & Lebowitz, J. (1966). J. Gen. Phyaiol. 49, part 2, 103. Vinograd, J., Lebowitz, J., Radloff, R. Watson, R. & Laipis, P. (1965). Proc. Nut. Acud. Sci., Wash. 53, 1104.
Electron Microscopy of Superhelical Circular h DNA VERNON C. BODE Department of Biochemistry, University of Maryland Medical School Baltimore, Maryland, U.S.A. AND
L. A. MACHATTIE~ Department of Biophysics, Johns Hopkins University Baltimore, Maryland, U.S.A. (Received 12 September 1967) Electron microscopy conf%rns that circular h DNA molecules contain supertwists. The average number of primary supertwists at low ionic strength (0.06) is 117, or 3.8 per million molecular weight. This number is not constant but decreases to an average of 12 when the ionic strength is increased to 2.0. The sedimentation rate of the superhelical molecules also decreaseswhen the ionic strength is increased through this range. The change in number of twists as revealed by both sedimentation and electron microscopy indicates that the pitch of the primary DNA helix varies with ionic strength.
1. Introduction A h DNA molecule whose ends have been covalently joined in vivo exhibits sedimentation properties that suggest it is superhelical, i.e. the primary helix is twisted about itself (Bode & Kaiser, 1965). It seems likely that when formed within the cells such circular molecules are not superhelical, but only become so when placed in an environment that differs in some significant aspect from the intracellular one. The significant factors are those that exert an influence on the natural pitch (base-pairs per turn) of the primary helix. A change in the concentration of such factors could effect a change from open circle to superhelix in any circular duplex DNA molecule both of whose component strands are intact circles. This view is consistent with all of our current evidence on superhelical DNA molecules. For example, double-stranded circular molecules that are closed in vivo contain twists when examined in the buffer solutions used for DNA isolation (Vinograd $ Lebowitz, 1966), but molecules whose ends are covalently sealed in vitro do not supertwist in the same buffers since the solutions provide an environment very similar to that in which the molecules were closed (Gellert, 1967; Gefter, Becker & Hurwitz, 1967). A more direct line of evidence is the observation that superhelices untwist when ethidium bromide intercalates between base pairs of the primary helix (Crawford & Waring, 1967). This result, as do those of Vinograd, Lebowitz, Radloff, Watson & Liapis (1965) with partial melting, indicates that in superhelical polyoma t Present address: Department Mass. 02115, U.S.A.
of Biologicrtl Chemistry, Harvard 673
Medical School, Enston.
674
V. C. BODE
AND
L. A. MAOHATTIE
DNA the primary helix is underwound, i.e. in linear duplex molecules the two singlestrands normally wrap around each other a fewer number of times in the in viva environment than they do in the in vitro environment. Rhoades & Thomas (personal communication) in a study of phage P22 DNA superhelices show, by partial-melting unwinding and by visualizing in electron micrographs the sense of the superhelical twist (right-handed), that the primary helix of this twisted circular DNA also is underwound. Crawford & Waring (1967) estimate the degree of supertwisting (underwinding) in polyoma DNA as 12 turns per molecule of 3-2 million molecular weight or 3-S primary super-twists per million molecular weight. This paper (i) confirms by electron microscopy of circular h DNA molecules the supertwisting deduced from their sedimentation properties (Bode &I Kaiser, 1965) and (ii) presents both electron microscopic and sedimentation data that indicate that the degree of supertwisting (and therefore the pitch of the DNA helix) varies with NaCl concentration.
2. Materials and Methods (a) Preparation of DNA from mature phuge A DNA was prepared by phenol extraction as described by Kaiser & Hogness (1960). The extracted DNA was dialyzed and stored in Tris-EDTA (lOma a6-Tris-HCl, IO-3 MEDTA, pH 7.1). A solution of purified h DNA with an Azao = I.0 contains about 60 pg of DNA/ml. The purity of intracellular forms was estimated by comparing the ratio of tritium disintegrations/min to optical density at 260 rnp for the extract of infected cells with the ratio obtained for DNA isolated from a sample of the mature phage used for superinfection. (b) Putification
of tuk2.d circular
X DNA
Lysogenic bacteria were infected with 3H-labeled h phage (I.5 x IO5 cts/min/60 pg of I\ DNA) and the nucleic acids were extracted from the infected bacteria as previously reported (Bode & Kaiser, 1965). Where necessary, extracts were concentrated by dialysis against saturated sucrose. All solutions were prepared in Tris-EDTA and maintained below 6’C. A crude infected-cell extract (3 or 4 ml. of a solution with Azeo = 10 was layered on a linear 6 to 26% sucrose gradient (62 ml. in a Spinco SW25,2 tube) and centrifuged for 6 hr at 25,000 rev./min or 16 hr at 18,000 rev./mm in a Spinco model L2 centrifuge. After collection through a hole in the bottom of the tube, fractions containing the intracellular forms of h DNA were identified by the distribution of tritium. Fractions rich in twisted circular molecules were pooled, dialyzed, concentrated by dialysis against saturated sucrose, stirred to shear the long molecules of high molecular weight host DNA, and then re-sedimented through a sucrose gradient. Judging by the ratio of tritium disintegrations to &mot the fractions selected from the first sedimentation contained tritiated superhelioal DNA of about 10% purity and the second sedimentation improved this to about 60% purity ( 100.fold purified over the crude extract). The pH was maintained near 7 during all steps of the isolation. (Although purification by melting the DNA in alkali followed by selective renaturation of circular duplex molecules might be more efficient, we wished to avoid melting and renaturation in the initial characterization of the superhelical species. Gellert (1967), using X DNA circles whose ends were joined in vitro, reports that purification by sedimentation in alkaline gradients altered the subsequent sedimentation rate of those circular molecules at neutral PH.) (c) Preparatti
of aampl8.8 for electron microscopy
Samples of purified superhelical ,I DNA were prepared for electron microscopy by the protein-film technique (Kleinschmidt $ Zahn, 1969; Kleinschmidt, Lang, Jacherts & Zahn, 1962), as modified by MacHattie & Thomas (1964). The purified DNA solution in Tr&EDTA was diluted 1 :I with 0.04% cytochrome c dissolved in either (i) 0.075 M-NaCI,
ELECTRON
MICROSCOPY
OF
SUPERHELICAL
h DNA
6;s
M-sodium citrate or (ii) 4.0 M-N&~. Of this mixture, 0.06 ml. WM spread on a level flame-cleaned stainless steel blade, then gently lowered into contact with a clean water surface at room temperature (20 to 23°C). This allowed a DNA-containing cytochrome c monolayer to spread out from the blade to cover the 60 cma water surface. Samples of the monolayer 2 to 4 mm from the blade were picked up by touching it with carbon f%.nbearing electron microscope grids. The grids were touched to 95% ethanol to remove water and rotary shadowed at a low angle with platinum-palladium.
0.0075
3. Results (a) CharacteGution
of the circular
X DNA
preparation
by sedimentations
The preparation of superhelical h DNA used for electron microscopy contained about 4 pg of 3H-labeled A DNA per ml. The optical density at 260 rnp exceeded that. expected from this amount of labeled DNA by O-12 unit, indicating the presence of contaminating host DNA at a concentration not greater than 6 pg/ml. As judged by sedimentation in a sucrose gradient (Fig. 1) about 80% of the tritiated X DNA
Dtstance
FIG. 1. Sedimentation analysis of the tritiated A sample (0.2 ml.) containing 0.8 pg of purified to 26% sucrose gradient (5 ml.) in Tris-EDTA,
from
meniscus
(cm)
h DNA preparation used in electron microscopy. intracellular species I A DNA was layered on a 5 pH 7.1. After sedimentation for 3 hr at 37,500
rev./min, 5’C, in the Spinco SW39 rotor, fractions were collected from the bottom of the tube. The distribution of the sH-labeled A DNA was determined by drying and counting B O-l-ml. portion from each fraction. Linear duplex h DNA (0.3 pg) isolated from the mature phage particles was sedimented in a duplicate tube. The distribution of this DNA is plotted as a reference (dashed line).
molecules were circular. Considering only circular molecules, 87% sedimented at a rate (3.2 cm in three hours) charaoteristio of species I, i.e. twisted circular molecules, while 13% sedimented at a rate (2.1 cm in three hours) characteristic of species II, i.e. untwisted circular molecules. After a period of one month during which grids were prepared for electron microscopy, re-analysis by sedimentation indicated that the fraction of twisted circular molecules, species I, had decreased to 78% and the fraction of species II had increased to 22%. (b) Electron microscopy Random samples of the DNA molecule population present on the electron microscope grids were obtained by photographing large fields unselected except for
676
V. C. BODE
AND
L.
TABLE
A. MAcHATTIE 1
Configuration of h DNA molecules in the puri$ed prepar&m DNA floated in High salt Low salt
Number of molecules 149 193
Number of circles 116 148
Percentage Less than 40 crossings 89 6
of circles
showing: More then 90 crossings 3 77
distinctness of DNA at low magnifications (2100 or 3700). Each micrograph showed an average of nine isolated DNA molecules of about h size, which constituted from 10 to 60% of the total DNA present in different areas of the grids. Of these isolated X-sized DNA molecules, 264 out of 342, or 77%, were circular (Table 1). In the grids for which the protein monolayer was spread from high-salt solution, the majority of the circular X DNA molecules were relaxed-looking open circles, while in the low-salt case the majority had a distinctly twisted appearance. Examples of the two forms are shown in Plate I. The length distributions of small samples from the two preparations are shown in Fig. 2.
High salt
“%-Length (+)
FIG. 2. moleoules (av. 113) crossings
Length distribution for circular molecules of h DNA. High 8&t: average length of 24 = 17.6 p. Lozo salt: length distribution of 22 molecules with greater than ninety crossings is shown by the dashed line. Length distribution of 38 molecules with less than 90 (av. 48) is shown by the dotted line.
(c) EstimaGm of superhelical twisting A quantitative comparison of the two preparations with respect to twisting was made by counting the number of times the duplex helix crossed over itself in each circular molecule. The results, shown in Fig. 2 and Table 1, clearly differentiate the two populations of molecules. In the number of crossings distribution of Fig. 3, the lowsalt preparation shows a major peak, comprising 77% of the total number of circular molecules (almost exactly the expected proportion of species I) in which the mean is 117 crossings f 11 8.~. In the high-salt preparation 89% of the circular molecules showed less than 40 crossings, with an average of 12 orossings per molecule in this major component. This low number may or may not represent a coherent superhelical twistedness, since the sense of the twists could not be reliably distinguished in these micrographs.
ELECTRON
MICROSCOPY
OF
SUPERHELICAL
h DNA
677
Number of crossings
FIQ. 3. Distribution of the number of times the primary DNA helix crossed itself in electron micrographs of circular h DNA molecules spread from solutions with low (0.06) and high (2.0) ionia strength at 20 to 23%. A statistically random sample of circular molecules wms examined in each case. The visually estimated upper limit of the major peak for crossing frequency in high salt w&s 40 crossings, end the lower limit of the major peak in low ealt was 90 crossings.
(d) The effect of ionic strength on the sedimentation of circular X DNA The sedimentation coefficient for linear X DNA molecules changes very little in the ionic strength range O-01 to 1.0 (Studier, 1966). In contrast to linear molecules, and also circular molecules with single-strand breaks (species II), the sediment&ion rate of A circular superhelices (species I) decreases with increasing salt concentration (Fig. 4).
001
0IO Ionic
I0
strength
FIG. 4. The effect of ionic strength on the sediment&ion rate of twisted circular h DNA (specie8 I) and untwisted circles (species II). Sucrose gradients (6 to 20%) were prepared in Trie-EDTA (or diluted Tris-EDTA for the lowest ionic strength) with N&l added to yield the indicated ionic strengths. A partially purified preparation of species I (9 pg/ml. of DNA of which 5% was tritieted h DNA) w&e mixed with 32P-labeled linear X DNA and N&l wee added to bring the ionic strength of the sample to a value near that of the gradient through which it would be centrifuged. The molecules in the sample were sedimented for 3 to 4 hr (37,000 rev./min, Spinco SW39 rotor, 5’C). The distance sedimented by twisted circular molecules (speoies I) relative to that traveled by the linear marker is plotted es e function of ionic strength (-O-O--). The relative sedimentation rate of species II (circuler molecules with single-strand breaks formed from species I ee e result of tritium decays) is also plotted for aomparieon (-O-O-). 48
678
V. C. BODE
AND
L.
A. MAcHATTIE
4. Discussion These results support the conclusion that h DNA species I is a circular molecule which in low-salt media is superhelically twisted, but in higher ionic strengths untwists to approach the open circular configuration. This would suggest that some dimensions of the duplex DNA molecular structure must vary slightly with ionic strength, in such a way as to change the pitch of the helix (number of base-pairs per 360” turn of helix). Though the molecular configurations seen in the electron microscope grids are well supported by the sedimentation data, some additional comment is called for regarding the relation of the molecular configurations seen on the grids to those present in solutions. The preparative technique for electron microscopy as was used here in the high-salt samples has been shown in a number of past determinations on DNA from a variety of phages to yield molecular lengths which, when compared with independent molecular weight determinations, are consistent with the B form of the Watson-Crick helix (MacHattie & Thomas, 1964, Thomas, 1966). However, Lang, Bujard, Wolff & Russell (1967) have shown thatunbrokenviralDNAmolecules incorporated into a protein monolayer by diffusion through solutions ofionic strength less than 0.14 are more thanusually heterogeneous in length, and are on the average longer than would be expected for the B form of the helix. Inman (1967) reports a similar finding. In the present experiments, this length effect is seen in the sample floated from a solution of ionic strength O-06, but not in that floated from ionic strength 2.0. This lends credence to the idea that in our technique the DNA-containing protein monolayer forms predominantly over the salt solution lying on the horizontal blade, and that the configuration and length of DNA molecules once embedded in the monolayer remain essentially unchanged as the monolayer expands out over the water hypophase for at least 2 to 4 mm. Thus the configurations of the X DNA molecules reported here may be believed to represent the conditions in the respective salt solutions in which they were layered on the blade, rather than the low ionic strength found in the hypophase onto which the monolayer was allowed to spread. The limits of accuracy of this statement are indicated by the occasional highly twisted molecules (3% showed more than 90 crossings) found in the high-salt sample. These most probably represent a small minority which through mixing and diffusion became exposed to low ionic strengths before incorporation into the monolayer. The relatively constant length of the highly twisted molecules in the low-salt sample (see Fig. 2) is of interest, in that it suggests that the twisting stress exerted on the DNA helix by the constraint of superhelical configuration may prevent it from undergoing the type of change that is responsible for the length heterogeneity in low ionic strengths reported by Lang et al. (1967) and Inman (1967) and seen in the less twisted members of the same population. These less twisted molecules most probably contained single-chain breaks, corresponding to sedimenting species II, and therefore were not subject to the superhelical constraint. The shortness in apparent length of the highly twisted molecules may not be significant, since it could represent a consistent error in measurement of moleoules with many crossings. The sedimentation rate of species I varies inversely with ionic strength below values near 1-O (Fig. 4). This behavior can be rationalized if, as is indicated in the electron micrographs, this circular form is less twisted in solutions of high ionic strength. At
ELECTRON
MICROSCOPY
OF
SUPERHELICAL
h DNA
670
sodium chloride concentrations above 1 M, the rate at which species I sediments appears to be independent of ionic strength but is significantly faster than that for species II. This suggests that it is still twisted and that the low number of crossings seen in the electron micrographs of molecules from the solution with high salt represents coherent superhelical twists rather than just random crossings. Presumably the molecules would untwist completely if placed in a solution which mimicked the intracellular environment. Salts other than sodium chloride might be more effective in this respect. Only one has been examined. Magnesium sulfate at 0.001, 0.01 and 0.1 &I did not significantly reduce the sedimentation rate of h superhelices below the value predicted from Fig. 4 on the basis of ionic strength alone. The number of twists in X superhelices seen on the low-salt electron-microscope grids agrees remarkably well with Crawford & Waring’s (1967) and with Vinograd’s (personal communication, 1967) estimate of the twistedness of polyoma DNA molecules in low salt. It will be realized that if all the crossings of a circular molecule over itself represent twists of the same sense (and this is likely to be true for highly twisted molecules), each crossing is equivalent to a half-twist of duplex about duplex, or to one full 360” twist imposed on the primary DNA helix. This is what Crawford & Waring term one supercoiling twist. The frequency of superhelical twists that we observe in circular A DNA prepared for electron microscopy from solutions of ionic strength 0.06 at 20 to 23°C is then 117 primary supertwists per 31 million molecular weight, or 3.8 per million. This is exactly what Crawford & Waring (1967) estimated by a completely different method for polyoma DNA in solutions of similar (0.05) ionic strength and temperature (ZO’C). If this agreement is not taken to be simply coincidental, it suggests that whatever factors in the intracellular environment are responsible for superhelix formation, their effect is constant per unit length of DNA, and of the same magnitude in cells as different as mammalian and bacterial. The present work indicates that high ionic strength mimics, at least partially, the effect of the intracellular environment on the pitch of the DNA double helix. This work was supported by grants from the National Science Foundation (GB-2793) and the Public Health Service (AI-06493). One of us (L. A. M.) was a Career Development Awardee (5-K3-GM-25,342) of the Public Health Service. REFERENCES Bode, V. C. & Kaiser, A. D. (1965). J. Mol. Biol. 14, 399. Crawford, L. V. & Waring, M. J. (1967). J. Mol. Biol. 25, 23. Gefter, M., Becker, A. & Hurwitz, J. (1967). PTOC. Nat. Acd Sci., Wash. 58, 240. Gellert, M. (1967). Proc. Nat. Acad. Sci., Wash. 57, 148. Inman, R. B. (1967). J. MOE. Biol. 25, 209. Kaiser, A. D. & Hogness, D. S. (1960). J. Mol. Biol. 2, 392. Kleinschmidt, A. K., Lang, D., Jacherts & Zahn, R. K. (1962). Biochim. biophys. Actu, 61, 857. Kleinschmidt, A. K. & Zahn, R. K. (1959). 2. Natur-. 146, 770. Lang, D., Bujard, H., Wolff, B. & Russell, D. (1967). J. Mol. Biol. 23, 163. MacHattie, L. A. & Thomas, C. A. (1964). Science, 144, 1142. Studier, F. W. (1965). J. Mol. Biol. 11, 373. Thomas, C. A. (1966). J. Gen. Phyeiol. 49, part 2, 143. Vinograd, J. & Lebowitz, J. (1966). J. Gen. Phyaiol. 49, part 2, 103. Vinograd, J., Lebowitz, J., Radloff, R. Watson, R. & Laipis, P. (1965). Proc. Nut. Acud. Sci., Wash. 53, 1104.