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Formation of a Giant Toroid from Long Duplex DNA† Yuko Yoshikawa,‡ Kenichi Yoshikawa,*,§ and Toshio Kanbe| Department of Food and Nutrition, Nagoya Bunri College, Nagoya 451-0077, Japan, Department of Physics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan, and Laboratory of Medical Mycology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466-0064, Japan Received September 3, 1998. In Final Form: December 31, 1998 It is well-known that polyamines, such as spermidine, have the effect of condensing DNA molecules into a toroidal morphology with an outer diameter of 80-100 nm. We have investigated the collapse of long duplex T4 DNA induced by spermidine with use of fluorescence and electron microscopy. We found that giant toroids with an outer diameter of ∼200 nm are generated from single T4 DNA under a high concentration of spermidine in the buffer solutions with rather high salt.
Introduction A number of in vitro studies have shown that the binding of polyamines such as spermidine to DNA causes the condensation of DNA into compact particles.1-4 This phenomenon is of focal interest in biology, since, in living cells, DNA is often found in a tightly packaged state and requires polyamines to achieve and stabilize the compact structure.5,6 To make clear the mechanism of the condensation of DNA chains, in vitro experimental systems have been developed, indicating that, besides the polyamines, various chemical species are effective to induce the condensation, such as positively charged peptides,7,8 proteins,9 neutral polymers,10-12 cationic surfactants,13 alcohols,14,15 and inorganic cations.16-18 Because of these in vitro studies, it has been established that a toroidalshaped product is the typical morphology in the condensed DNA chains. It has been reported that a typical toroid is around 80 nm in diameter, independent of the length of the DNAs.19,20 The only condition required for condensation * To whom all correspondence should be addressed: e-mail,
[email protected]; tel, +81-75-753-3812; fax, +8175-753-3779. † Presented at Polyelectrolytes ‘98, Inuyama, Japan, May 31June 3, 1998. ‡ Nagoya Bunri College. § Kyoto University. | Nagoya University School of Medicine. (1) Gosule, L. C.; Schellman, J. A. Nature 1976, 259, 333. (2) Chattoraj, D. K.; Gosule, L. C.; Schellman, J. A. J. Mol. Biol. 1978, 121, 327. (3) Plum, G. E.; Arscott, P. G.; Bloomfield, V. A. Biopolymers 1990, 30, 631. (4) Sikorav, J.-L.; Pelta, J.; Livolant, F. Biophys. J. 1994, 67, 1387. (5) Flink, I.; Pettijohn, D. E. Nature 1975, 253, 62. (6) Bloomfield, V. A. Curr. Opin. Struct. Biol. 1996, 6, 334. (7) Laemmli, U. K. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 4288. (8) Emi, N.; Kidoaki, S.; Yoshikawa, K.; Saito, H. Biochem. Biophys. Res. Commun. 1977, 231, 421. (9) Hsiang, W. M.; Cole, R. D. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 4852. (10) Lerman, L. S. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 1886. (11) Evdokimov, Yu. M.; Platonov, A. L.; Tikhonenko, A. S.; Varshavskii, Ya. M. FEBS Lett. 1972, 23, 180. (12) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Khokhlov, A. R.; Doi, M. Biopolymers 1994, 34, 555. (13) Melnikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 2401. (14) Lang, D. J. Mol. Biol. 1973, 78, 247. (15) Ueda, M.; Yoshikawa, K. Phys. Rev. Lett. 1996, 77, 2133. (16) Widom, J.; Baldwin, R. L. J. Mol. Biol. 1980, 144, 431. (17) Ma, C.; Bloomfield, V. A. Biophys. J. 1994, 67, 1678. (18) Yamazaki, Y.; Yoshikawa, K. J. Am. Chem. Soc. 1997, 119, 10573.
is that DNAs have a chain length several times larger than its persistence length; the persistence length of DNA is ∼50 nm (150 bp) in usual aqueous environments.20 It is also to be mentioned that, in most of the current studies on the toroidal DNA, the toroid is found to be the product of a plural number of chains. In the present study, we have performed the study of a toroid induced from a single long duplex DNA chain. As the DNA sample, we have adapted T4 phage DNA (166 kbp) with the full length of 57 µm. The manner of collapse of single DNAs induced by spermidine has been monitored using fluorescence microscopy. On the basis of the result of the DNA collapse in aqueous environment, the experimental condition for the formation of somewhat swelled collapsed DNAs has been established. Then, we have carried out the electron microscopic measurement on such a swelled collapsed DNA and found the generation of a giant toroidal structure with a diameter of 200 nm. Experimental Section Materials. T4 phage DNA, 166 kbp with a contour length of 57 µm, was purchased from Nippon Gene (Toyama, Japan). A fluorescent dye, 4′,6-diamidino-2-phenylindole (DAPI), was purchased from Wako Pure Chemical Industries (Osaka, Japan). Spermidine‚3HCl was obtained from Nacalai Tesque Inc. (Kyoto, Japan). Fluorescence Microscopic Measurements. T4DNA was dissolved in 10 mM Tris/HCl buffer solution with 50 mM NaCl, 10 mM MgCl2, and 1 mM dithiothreitol (DTT) at pH 7.5. For fluorescence microscopic measurements, 1 µM DAPI was added to the DNA solution. The final DNA concentration was 1 µM in nucleotide unit (about 0.1 µg/300 µL). Compaction was induced by the addition of spermidine to the DNA solution. The concentration of spermidine was varied from 1 mM up to 9 mM. Fluorescence DNA images were obtained using an Axiovert 135 TV microscope (Carl Zeiss, Germany) equipped with a 100× oilimmersion objective lens and a high-sensitivity Hamamatsu SIT TV camera, that allowed recording of images on videotapes. The video image was analyzed with an Argus 50 image processor (Hamamatsu Photonics, Hamamatsu, Japan). Observations were performed at 20 °C. Electron Microscopic Measurements. Samples used for electron microscopy were prepared by the addition of spermidine to 1 µM DNA solutions. They were mounted on carbon-coated copper grids (200 mesh), negative-stained with 1% uranyl acetate, and observed with a JEOL 1200EX transmission electron (19) Arscott, P. G.; Li, A.-Z.; Bloomfield, V. A. Biopolymers 1990, 30, 619. (20) Bloomfield, V. A. Biopolymers 1991, 31, 1471.
10.1021/la981159g CCC: $18.00 © 1999 American Chemical Society Published on Web 03/11/1999
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Figure 1. (left) Fluorescence microscopic images of T4 DNA stained with DAPI: (a) elongated coil state in aqueous solution; (b) collapsed globule state in the presence of 6 mM spermidine. (right) Corresponding quasi-three-dimensional representations, where the vertical axis indicates the fluorescence intensity. microscope (Tokyo, Japan) at 100 kV. To obtain the DNA image with higher resolution, some samples were observed with a JEOL 2010 transmission electron microscope (Tokyo, Japan) at 200 kV.
Results Figure 1 exemplifies the fluorescence images of T4DNA molecules together with the corresponding quasi-threedimensional representation of the fluorescence intensity distribution. The DNA molecules exhibit extended conformation, i.e., a coil state, in the buffer solution (Figure 1a). While, the DNA molecules take shrunken, globular conformation with 6 mM spermidine (Figure 1b). To characterize the morphology of spermidine-collapsed DNA particles, we observed the DNA structure using transmission electron microscopy. Figure 2a shows a typical structure of the compacted DNA induced by spermidine, i.e., a doughnut-shaped toroidal structure with concentrations of 0.3 µM DNA and 600 µM spermidine in the buffer solution of 10 mM Tris/HCl and 60 mM NaCl at pH 7.2.21 The diameter of the DNA particle given in Figure 2a is about 90 nm, which is the same order as have been reported on many other studies with different sized
DNA chains. On the other hand, under the condition with 1 µM DNA and 6 mM spermidine in the buffer of 10 mM Tris/HCl with 50 mM NaCl and 10 mM MgCl2 (pH 7.5), giant toroids with diameters of 200-250 nm were observed as is shown in Figure 2b. Through repeated experiments on these conditions, we have confirmed that the formation of the giant toroids is an event with rather high reproducibility. Essentially the same ordered giant toroidal structure was also obtained under the condition without Mg2+ (data are not shown). It can be interpreted to mean that high concentrations of both spermidine and DNA under high ionic condition were required for the formation of giant toroids. The addition of a much higher concentration of spermidine (9 mM) does not make the particles any more compact but leads to a somewhat swollen particle (Figure 2c). Discussion In the present study, we have found that a giant toroid with outer diameter of ∼200 nm is generated most (21) Yoshikawa, Y.; Yoshikawa, K.; Kanbe, T. Biophys. Chem. 1996, 61, 93.
Toroid Formation from DNA
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Figure 2. Transmission electron microscopic photographs of spermidine-collapsed T4 DNA observed at 100 kV (detailed experimental conditions are given in the text: (a) Tightly packed toroid with 0.6 mM spermidine; (b) giant toroid with 6 mM spermidine; (c) swollen and partially unfolded object with 9 mM spermidine. Scale bar is 100 nm.
probably from a single T4 DNA molecule in the presence of a considerable amount of spermidine (6 mM) under high ionic conditions. Previous reports showed that the outer diameter of toroids averages 80-100 nm and does not depend on the molecular weight of DNAs from 400 bp to 40 kbp.19,20 In contrast, our result obtained from T4 DNA (166 kbp) shows that the condensate has an outer diameter ∼200 nm. To achieve such a giant toroidal structure, the ionic condition must be high, where 6 mM of spermidine was added to the solution of 1 µM DNA
containing 10 mM Tris/HCl, 50 mM NaCl, and 10 mM MgCl2. Thus, we have established the experimental conditions to obtain the giant toroids, although the possibility19 of the formation of giant toroids with plural numbers of T4 DNAs has not been excluded in a definite manner yet. Then, let us estimate the degree of packing of chain in the giant toroid. To obtain the electron microscopic image on the giant toroid with better quality, we have performed the measurement at 200 kV. The result in Figure 3 was
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Figure 3. Transmission electron microscopic image on a giant toroid observed with higher acceleration of the electron at 200 kV. Scale bar is 100 nm.
obtained with such a measurement, where the toroid was formed in the presence of 6 mM spermidine. The outer (D) and inner (d) diameters in this toroid are D ) 200 nm and d ) 70 nm, respectively. The effective volume of the toroid, V, is calculated as V ) 2π2r2R, where R ) (D + d)/4 and r ) (D - d)/4. Thus, the volume of the giant toroid is estimated as V ) 1.4 × 106 nm3. The contour length, or the full length, of T4 DNA is L ) 57 µm. With the assumption of the hexagonal packing of the DNA chains in the cross section of the toroidal arm, the average radius per single duplex DNA segment is calculated to be F ) 2.7 nm. As the diameter of DNA itself is known to be 2.0 nm, the average distance between the surface of the double stranded DNA segments is (2‚2.7 - 2.0) ) 3.4 nm. A similar calculation is carried out for the standard size DNA (Figure 2a) with D ) 90 nm and d ) 24 nm. As the average radius per single duplex DNA, F ) 0.9 nm. This means that the DNA is packed in the closest manner. Compared to such a high packing density, the giant toroid swells very much,
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suggesting that the attractive interaction between the DNA segments is rather weak (Figure 3). The decrease of the attractive interaction in the presence of excess spermidine may induce the experimental trend that the toroid becomes partially unfolded with the further increase of the spermidine concentration. From recent theoretical consideration on the stability of a toroid, it has been suggested that the toroid swells considerably in the case of a small attractive interaction between the polymer segments, for the collapsed DNA with rather large contour length, or molecular weight.22 The result in the present study, thus, fits well to such a theoretical prediction. The most significant contribution on the attractive interaction induced by multivalent cation has been attributed to the gain in the net translational entropy of small ions, owing to the ion-exchange effect.23 When an excess amount of multivalent cation is added to the system, the entropy gain is expected to decrease markedly, which results in the decrease of the attractive interaction. We have observed a giant toroidal form of DNA under the condition of 6 mM spermidine and a partially collapsed structure at 9 mM spermidine. Polyamines have a role in control of cell proliferation and growth; the concentration of polyamine is low in resting cells and increases rapidly when cells are stimulated to divide.24 In relation to this, it was reported that the interaction of the restriction enzyme Nae I with DNA was regulated by spermidine; high spermidine turns off enzyme activity and low spermidine turns it on.25 In the present work together with the current studies in our laboratory, we showed the collapse/decollapse processes of DNA induced by spermidine. Further experimental and theoretical work will be useful to account for these remarkable physical/ biological processes. Acknowledgment. The present study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports in Japan. LA981159G (22) Vasilevskaya, V. V.; Khokhlov, A. R.; Kidoaki, S.; Yoshikawa, K. Biopolymers 1997, 41, 51. (23) Takahashi, M.; Yoshikawa, K.; Vasilevskaya, V. V.; Khokhlov, A. R. J. Phys. Chem. 1997, 101, 9396. (24) Tabor, C. W.; Tabor, H. Annu. Rev. Biochem. 1984, 53, 749. (25) Conrad, M.; Topal, M. D. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9707.
