Ascorbic acid induces a marked conformational change in long duplex DNA Yuko Yoshikawa 1,5 , Mari Suzuki 1 , Ning Chen 2 , Anatoly A. Zinchenko 2 , Shizuaki Murata 2,5 , Toshio Kanbe 3 , Tonau Nakai 4 , Hidehiro Oana 4,5 and Kenichi Yoshikawa 4,5 1 Department of Food and Nutrition, Nagoya Bunri College, Japan; 2 Graduate School of Environmental Studies, Nagoya University, Japan; 3 Laboratory of Medical Mycology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Japan; 4 Department of Physics, Kyoto University, Japan; 5 CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation Ascorbic acid is often regarded as an antioxidant in vivo, where it protects against cancer by scavenging DNA-dam- aging reactive oxygen species. However, the detailed mech- anism of the action of ascorbic acid on genetic DNA is still unclear. We examined the effect of ascorbic acid on the higher-order structure of DNA through real-time observa- tion by fluorescence microscopy. We found that ascorbic acid generates a pearling structure in single giant DNA molecules, with elongated and compact regions coexisting along a molecular chain. Results from electron microscopy and atomic force microscopy indicate that the compact regions assume a loosely packed conformation. A possible mechanism for the induction of this conformational change is discussed in relation to the interplay between the higher- order and second-order structures of DNA. Keywords: ascorbic acid; higher order structure of DNA; pearling structure; single molecular observation. Vitamin C (ascorbic acid) is ubiquitous and fundamental in living cells, where it acts as a water-soluble antioxidant and an essential cofactor for many enzymes involved in diverse metabolic pathways. Several epidemiological and experi- mental studies have shown that the consumption of foods rich in vitamin C is associated with a decreased risk of several chronic diseases, including cardiovascular disease and cancer [1–4]. However, the extent to which vitamin C contributes to these effects is still unclear [5]. There is evidence that vitamin C inhibits oxidative DNA damage in isolated and cultured cells exposed to reactive oxygen species and UV/visible light [5–9]. On the other hand, several studies have shown that vitamin C sometimes increases DNA damage in humans [5,10,11]. These studies suggest that vitamin C may have anti-oxidative or pro- oxidative properties depending on the conditions in the cell. Thus, it may be useful to clarify the mechanism of the action of vitamin C on DNA [12,13]. It is well known that genomic DNA molecules are very long, e.g. of the order of 1 cm in human cells. Recently, it has become clear, from single-chain observation using fluorescence microscopy together with electron microscopy, that long DNA exhibits unique responses to different condensing chemicals [14]. Polyamines, metal cations, neutral polymers, polypeptides and basic proteins have all been found to be efficient condensing agents [15]. A variety of higher-order structures can be generated from the same long DNA molecule: e.g. toroid, rod, spherical, spool and intrachain segregated structures [14]. We performed a single-molecule observation of giant DNA molecules to examine the effect of ascorbic acid at physiological pH. Surprisingly, we found that ascorbic acid generates a pearling structure in a giant DNA molecule, in which elongated and compact parts coexist along a single molecular chain. A possible mechanism is discussed in relation to the interplay between the higher-order and second-order structures of DNA. Experimental procedures Materials T4 phage DNA, 166 kbp with a contour length of 57 lm, was purchased from Nippon Gene (Toyama, Japan). The fluorescent dye YOYO-1 was obtained from Molecular Probes, Inc. (Portland, Oregon, USA). An antioxidant, 2-mercaptoethanol, and L -ascorbic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan). Fluorescence microscopic observations T4 phage DNA was dissolved in 10 m M Tris/HCl buffer with 0.1 l M YOYO-1 (nucleic acid staining) and 4% (v/v) 2-mercaptoethanol at pH 7.4. Various concentrations of L -ascorbic acid (50 l M to 10 m M ) were added. To avoid intermolecular DNA aggregation, measurements were con- ducted at a low DNA concentration, 0.3 l M in nucleotide units. Fluorescent DNA images were obtained using a microscope (Axiovert 135 TV; Carl Zeiss, Jena, Germany) equipped with a 100 · oil-immersion objective lens and a Correspondence to Y. Yoshikawa, Department of Food and Nutrition, Nagoya Bunri College, Nagoya, 451-0077, Japan. Fax: + 81 52 521 2259, Tel.: + 81 52 521 2259, E-mail: yuko@chem.scphys.kyoto-u.ac.jp Abbreviations: AFM, atomic force microscopy; TEM, transmission electron microscopy. (Received 18 March 2003, revised 4 May 2003, accepted 2 June 2003) Eur. J. Biochem. 270, 3101–3106 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03699.x highly sensitive Hamamatsu SIT TV camera, which allowed recording of images on video tapes. The video image was analyzed with an image processor (Argus 20; Hamamatsu Photonics, Hamamatsu, Japan). Imaging by atomic force microscopy (AFM) A DNA solution containing ascorbic acid was prepared as described above, and 5 lL was adsorbed on to freshly cleaved mica for 1 min. The mica surface was washed with Milli-Q-purified water, and dried in N 2 gas. An NVB 100 (Olympus, Tokyo, Japan) operated in trapping mode was used. Images were displayed without modification except for flattening to remove the background curvature of the mica surface. Electron microscopic observations Samples used for electron microscopy were mounted on carbon-coated copper grids (No. 200), negative-stained with 1% uranyl acetate, and observed with a transmission electron microscope (Jeol 1200EX, Tokyo, Japan) at 100 kV. CD spectroscopic measurements Measurements were performed at a T4 phage DNA concentration of 30 l M in 20 m M Tris/HCl, pH 7.4. Vari- ous concentrations of L -ascorbic acid (10 l M to 150 l M ) were added to the DNA solution. CD spectra were recorded on a Jasco J-720 spectropolarimeter in a 1.0 · 1.0 · 5.0 cm quartz cell at room temperature. Results Figure 1 shows fluorescence microscopic images of individ- ual T4 DNA molecules in aqueous solution at pH 7.4 in the absence and presence of ascorbic acid. Depending on the concentration of ascorbic acid, DNA molecules exhibit intrachain and translational Brownian motion with differ- ent conformations. Without ascorbic acid (Fig. 1A), DNA molecules assume an elongated coil conformation. At 200 l M ascorbic acid, DNA remains in a coiled state (Fig. 1B). At 5 m M , folded compact and elongated regions coexist along a single molecular chain, i.e. intrachain segregation is observed (Fig. 1C). This segregated confor- mation appears at concentrations of ascorbic acid of 1 m M and above. At 1 m M , about 10% of the DNA molecules show segregated structures, the majority exhibiting the elongated coil conformation. Above 3 m M , most of the DNA molecules (>80%) are in the segregated state. To examine the segregated conformation, we used fluorescence microscopic observation of fixed DNA mole- cules on a glass surface. Figure 2 shows the fluorescence images of T4 DNA molecules on a glass slide in 5 m M ascorbic acid solution. The DNA molecule was extended on a glass slide by introducing shear to the solution with a cover slide. Mini-globules are observed along an extended DNA molecule on a 2D glass plate. On the other hand, under the same conditions, the segregated DNA molecules in the bulk solution show only a few compact regions (Fig. 1C). As the effective resolution becomes higher on the fixed DNA, the small mini-globules became visible. From these observations and the following results obtained by AFM, it is expected that such small mini-globules observed A B C 0s 3s 5s 0s 3.2s 7s 0s 0.7s 1s 5 µm Fig. 1. Fluorescence microscopic images of T4 DNA moving freely in aqueous solution at different ascorbic acid concentrations. (A) Buffer solution; (B) 200 l M ascorbic acid; (C) 5 m M ascorbic acid. 5 µm Fig. 2. Fluorescence microscopic images of T4 DNA fixed on a glass surface in the presence of 5 m M ascorbic acid. 3102 Y. Yoshikawa et al.(Eur. J. Biochem. 270) Ó FEBS 2003 on the fixed DNA may also exist on the segregated DNA in bulk solution. Figure 3A,B shows the detailed morphological features of DNA molecules observed by AFM in the presence of 3m M ascorbic acid. The intrachain segregated state is observed with much higher resolution than those observed by fluorescence microscopy. It is clear that the mini-globule part assumes a loosely packed conformation. This AFM picture corresponds well to the segregated structure observed by fluorescence microscopy with a lower resolu- tion (Figs 1 and 2). Figure 3B shows a magnified view of a condensed part from Fig. 3A. Interestingly, the condensed part shows the irregular packing of DNA segments. Figure 3C shows the transmission electron microscopy 1 µm 0.2 µm 0.1 µm A B C Fig. 3. AFM and TEM images of T4 DNA molecules in the presence of ascorbic acid. (A) and (B) AFM images of T4 DNA molecules in the presence of 3 m M ascorbic acid, where (B) is a magnified view of (A). (C) TEM image of T4 DNA in the presence of 5 m M ascorbic acid. Ó FEBS 2003 Ascorbic acid induces conformational change in DNA (Eur. J. Biochem. 270) 3103 (TEM) image of T4 DNA in the presence of 5 m M ascorbic acid, and indicates that a loosely packed structure is formed. The morphological features of the condensate observed by TEM are similar to those observed by AFM, although with TEM it was difficult to judge whether an elongated coil region existed in the DNA chain. It has been well established that single DNA molecules are packed into a compact toroidal structure with a diameter of 50–100 nm on the addition of condensing agents such as polyamines or tervalent metal cations [16,17]. Compared with such a tightly packed state, the folded state of DNA induced by ascorbic acid is rather swollen. Next, we measured the changes in the CD spectra of DNA. Figure 4 shows that the positive Cotton sign at the % 280 nm band almost disappears when the ascorbic acid concentration is above 100 l M . The change in the negative band at 246 nm to a positive value is mostly attributable to the contribution from the added ascorbic acid (see Fig. 4B). Figure 4C shows the differential CD spectrum [(30 l M DNA + 100 l M ascorbic acid) ) (100 l M ascor- bic acid)]. A decrease in the plus Cotton band is observed at 280 nm, whereas the minus band at 246 nm remains essentially constant. Interestingly, the band shape in the difference spectrum resembles that of the C-form of DNA [18,19]. Discussion Ascorbic acid induces substantial changes both in the higher-order and second-order structures of DNA It is clear that ascorbic acid induces a significant change in the higher-order structure of DNA. We confirmed the generation of a segregated structure with multiple mini- globules using different experimental tools: fluorescence microscopy and AFM. The mini-globule shows irregular packing and is very different from previously observed regular conformations, such as toroid and rod [14,15]. A similar pearling structure was generated from a long DNA molecule complexed with histone H1 [20]. However, the action of ascorbic acid on the pearling structure is thought to be very different from that of histone H1, as the former is anionic and the latter is cationic. As ascorbic acid is negatively charged, it cannot neutralize the charges on the negative phosphate groups of DNA. Instead, it may interact with the bases inside the double-stranded structure. Such an interaction may cause distortion in the double-stranded structure. Neault et al. [12] performed a Raman and infrared spectroscopic study on the effect of ascorbic acid on DNA. They suggested that the OH and C-O groups of ascorbic acid interact directly with DNA bases. We shall now discuss the mechanism of the large conformational change in DNA induced by ascorbic acid, in relation to the change in second-order structure. Figure 3C shows the presence of a gnarled conformation in the condensed part of the chain. This result suggests that torsional stress is generated along the double-stranded DNA. It is known that the C-form-like structure of DNA is found on nucleosome particles [18,21–23], where DNA is wound around the histone core proteins with high curvature; the radius is % 5 nm. The similarity of the CD spectrum to the C-form in Fig. 4C may be explained by the formation of such an over-wound double-stranded con- formation, as in a nucleosome. To interpret the CD spectra, we need to consider the possible effect of aggregates [24]. It is known that aggregates induce distortion of the absorption spectrum, as well as the CD spectrum, through scattering of light. We have confirmed that, when the concentration of ascorbic acid is increased, the UV spectra remain on the null level for the region above 310 nm where both DNA and ascorbic acid exhibit no light absorption. This indicates that there will be negligible contribution from aggregates on the CD band. The above consideration suggests that the unique features observed by AFM and TEM are closely related to the change in the secondary structure of DNA. Theoretical consideration of the stability of the segregated structure We now consider the stability of the intrachain segregated structure induced by ascorbic acid. In general, the free energy of a condensed object is the result of two different contributions: bulk and surface energies. For the conden- sation of DNA induced by ascorbic acid, we have to take into account the effect of the surviving negative charge, because negatively charged ascorbate cannot neutralize the negative charge of DNA. 0 2 220 320240 260 280 300 220 320 240 260 280 300 0 -4 2 -2 4 6 -2 -4 220 320240 260 280 300 0 4 8 CD[mdeg] CD[mdeg] CD[mdeg] Wave length[nm] Wave length[nm] Wave length[nm] A B C 0 µM 20 40 60 100 150 Control Difference Fig. 4. CD spectra. (A) 30 l M T4 DNA in the presence of different concentrations of ascorbic acid. (B) 100 l M ascorbic acid. (C) Solid line, difference spectrum [(30 l M T4 DNA + 100 l M ascorbic acid) solution ) (100 l M ascorbic acid) solution]. Broken line, 30 l M T4 DNA in the absence of ascorbic acid. 3104 Y. Yoshikawa et al.(Eur. J. Biochem. 270) Ó FEBS 2003 It is well established that, when DNA is folded into a tightly packed structure accompanied by parallel ordering of the segments, the negative charge on the DNA molecule almost completely disappears. This is similar to the com- paction by spermidine(3+) at low salt concentrations [25]. On the other hand, when DNA is compacted with spermidine(3+) at high salt concentration, its volume becomes one order larger than that of the tightly packed and ordered state, suggesting the survival of negative charge in the volume part of compact DNA [26]. Thus, the remaining negative charge would make a significant contri- bution to the stability of the compact state when the packing is less dense, as in the case of DNA complexed with ascorbic acid. In the situation of less dense compaction, the free energy of a single giant DNA in the compact globular state with respect to the elongated state is given as F ¼ÀaN þ bN 2=3 þ cQ 2 =R ð1Þ where N is the number of Kuhn segments on the DNA. It is well established that a single Kuhn segment in double- stranded DNA is composed of 300 base pairs [14,27,28]. In eqn (1), the first and second terms correspond to the volume and surface energy of a globular compact state, respectively. The third term represents the instability due to the remaining charge in the globule. Q and R are the remaining electronic charge and radius of the condensed particle. The constants a, b, and c depend on the manner of compaction or condensation, and are all positive. It is reasonable to expect that Q$N and R$N 1/3 .Insuchaframework,the following relationship is deduced [14,27,28], where c 1 is a constant. F ¼ÀaN þ bN 2=3 þ c 1 N 5=3 ð2Þ Equation (2) implies that a condensate with a surviving electronic charge in the bulk should be destabilized above a critical number N c of segments. When N is larger than N c , the single-globule conformation is destabilized and, as a result, multiple mini-globules are formed. Thus, the fully compact state becomes unstable when the residual charge becomes large, which may correspond to the present case. It has been found that polycations, such as histone H1 [20] and aminated poly(ethylene glycol) [29], induce a similar intrachain segregated structure in giant DNA molecules. The stability of a segregated structure induced by poly- cations has been interpreted in terms of the incomplete charge neutralization [28,29]. Although the mechanism of the compaction induced by ascorbic acid is quite different from that caused by polycations, the contribution of the surviving negative charge to the stability of the segregated state will be the same. It has been reported that giant DNA molecules are folded into a compact state by the addition of a negatively charged polymer, polyglutamic acid [30]. In this case, DNA com- paction is induced by the Ôcrowding effectÕ, similar to the mechanism of compaction induced by neutral hydrophilic polymers such as poly(ethylene glycol) [15]. The concentra- tion of polyglutamic acid required in monomer units to induce compaction is very high, of the order of 1 M . Thus, ascorbic acid induces compaction of DNA in a very different way from the negatively charged polymer. Biological significance of the action of ascorbic acid on DNA Ascorbic acid is present in human blood at a concentration of % 50 l M [31,32]. Moreover, the concentration of ascorbic acid in human cells and tissues can exceed that in the blood by one order of magnitude [31–33]. In particular, human circulating immune cells, such as neutrophils, monocytes and lymphocytes, accumulate ascorbic acid in millimolar concentrations [32,33]. Therefore, the ascorbic acid concen- tration that induced the large conformational change in DNA in this study may be of physiological significance. It is thought that the folded compact state of DNA is resistant to external stimuli, such as reactive oxygen species and restriction enzymes. For example, it has been reported that the tight and ordered DNA packing in the bacterium Deinococcus radiodurans promotes resistance to environ- mental stress [34]. It has also been shown that compacted DNA is highly resistant to the action of a restriction enzyme [35]. It is possible that ascorbic acid may reduce oxidative damage by changing the higher-order structure of DNA. To our knowledge, this possible biological effect of ascorbic acid has not previously been considered. 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Imaging by atomic force microscopy (AFM) A DNA solution containing ascorbic acid was prepared as described above, and 5 lL was adsorbed