Catalytic transformations of supercoiled DNA as studied by flow linear dichroism technique Alexander Gabibov 1,2,3, *, Elena Yakubovskaya 1 , Mark Lukin 4 , Peter Favorov 3 , Andrey Reshetnyak 5 and Michael Monastyrsky 2,6, * 1 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia 2 Max-Plank-Institute for Physics of Complex Systems, Dresden, Germany 3 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia 4 Institute of Experimental Cardiology, Cardiology Research Center, Moscow, Russia 5 Moscow State University, Chemical Department, Vorobjovy Gory, Moscow, Russia 6 Institute of Theoretical and Experimental Physics, Russian Academy of Sciences, Moscow, Russia The dynamic of supercoiled DNA (scDNA) transfor- mations is the key point for understanding the numer- ous processes taking place in the living cell [1]. DNA topology changes are vital in replication, transcription, recombination, chromosome condensation, and segre- gation. From the topological point of view DNA can be represented as a closed ribbon [2–4]. A study of the dynamic aspects of the DNA topology is closely associ- ated with a design of an adequate mathematical des- cription of the DNA polymeric molecule and methods for monitoring its properties [5–8]. The main topologi- cal changes of scDNA are catalyzed by DNA-topo- isomerases I and II, which induce single and double nicks in DNA strains, respectively. These correspond to changes in the linking number (Lk) of the polymer sub- strate by 1 or 2. From the chemical point of view, the substrates and products of DNA topoisomerization are identical and catalytic events result in only slight topo- logical changes. The product of the previous turnover acts as a substrate at the next stage, so an ensemble of topoisomers exists at each step of the reaction. This fact stipulates a description of scDNA biocatalytic Keywords anticancer drugs; flow linear dichroism; supercoiled DNA transformations; topoisomerases Correspondence A. Gabibov, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16 ⁄ 10 Mikluho- Maklaya str., 117871, Moscow, Russia Fax: +7095 3107007 Tel: +7095 4298269 E-mail: gabibov@ibch.ru *Note A. G. and M. M. were visiting scientists. (Received 28 February 2005, revised 13 September 2005, accepted 19 October 2005) doi:10.1111/j.1742-4658.2005.05027.x A catalytic turnover of supercoiled DNA (scDNA) transformation medi- ated by topoisomerases leads to changes in the linking number (Lk) of the polymeric substrate by 1 or 2 per cycle. As a substrate of the topoisomeri- zation reaction it is chemically identical to its product; even a single catalytic event results in the quantum leap in the scDNA topology. Non-intrusive continuous assay to measure the kinetics of the scDNA topoisomerization was performed. The development of such a technique was hindered because of multiple DNA species of intermediate topology present in the reaction mixture. The interrelation of DNA topology, its hydrodynamics, and optical anisotropy enable us to use the flow linear dichroism technique (FLD) for continuous monitoring of the scDNA topo- isomerization reaction. This approach permits us to study the kinetics of DNA transformation catalyzed by eukaryotic topoisomerases I and II, as well as mechanistic characteristics of these enzymes and their interactions with anticancer drugs. Moreover, FLD assay can be applied to any enzy- matic reaction that involves scDNA as a substrate. It also provides a new way of screening drugs dynamically and is likely to be potent in various biomedical applications. Abbreviations AMPPNP, adenosine-5¢-phosphate-b,c-iminodiphosphate; FLD, flow linear dichroism; Lk, linking number; scDNA, supercoiled DNA; Tw, twist; Wr, writhing; q, linear dichroism value; r, supercoiling density. 6336 FEBS Journal 272 (2005) 6336–6343 ª 2005 The Authors Journal compilation ª 2005 FEBS transformations within the DNA topology, as well as hydrodynamics and statistics of biopolymers. How- ever, lack of an adequate nonintrusive technique for quantitative estimation of enzyme-mediated scDNA transformations makes the above-mentioned considera- tion inapplicable. Time-dependent topological trans- formations of the scDNA can be experimentally investigated by electrophoretic separation of DNA in the reaction mixture probes at different stages. However, this technique cannot provide instant and nondisturbing quantitative kinetic analysis, especially in the case of tiny inhibitory effects. A recently pub- lished precise approach based on immobilization of a single DNA molecule demands a state-of-the-art tech- nique and cannot be utilized in routine quantitative monitoring [9]. We proposed to use flow linear dichroism (FLD) to monitor different nuclease reactions. This allowed us to estimate kinetic parameters of enzymes and abzymes using the flow-oriented scDNA molecule as a substrate [10–14]. The application of FLD to study nucleic acids has been extensively reviewed in [15]. This method is based on the fact that oriented DNA possesses the property of optical anisotropy. The polymer molecule changes its topology, by low-molecular weight effectors (such as benzpyrene) [16,17], influencing its hydrody- namics, which affects orientation and optical factors. Thus, FLD allowed characterization of the interaction of the DNA molecule with intercalators and drugs, and to estimate the interaction geometry [18,19]. This technique turned out to be adequate to discriminate intercalators by the mode of their interaction with DNA. A number of DNA–protein complexes, inclu- ding specific receptors, were studied by the FLD method [20,21]. These data initiated us to apply FLD in biocatalytic studies. In this paper, we attempt to substantiate the applica- bility of the FLD technique for an adequate monitor- ing of enzyme-induced changes in highly supercoiled DNA topology. We use this method for mechanistic studies of DNA topoisomerases and the mechanisms of drug targeting. Results and discussion Flow linear dichroism and dynamics of DNA supercoiling DNA topisomerases are considerably smaller than their natural substrates, large scDNAs. Consequently, the enzymes cannot recognize the global shape param- eters of DNA, i.e. their writhe or twist. The use of a length-independent parameter, the parameter reflecting a local alteration of DNA geometry, seems reasonable for the description of the substrate properties of scDNA caused by supercoiling which might be recog- nized by topoisomerases. A well-known parameter, superhelix density r, could be applied for this purpose. Thus, superhelix density may be represented as: r ¼ DLk=Lk 0 ¼ cDLk=N ð1Þ where N is the number of base pairs in the DNA mole- cule and c is the mean number of base pairs per turn of the double helix under the given conditions [8]. It is known [8] that the only energy source determin- ing both the rate and direction of the topoisomeriza- tion process catalyzed by topoisomerase types I and II is a steric strain in the scDNA. Thus the equation combining the free energy of DNA supercoiling, the DNA length and the superhelix density can be derived [22]. It is proportional to square r 2 : DG r =N ¼ 10RTr 2 ð2Þ As mentioned above, r, a single parameter independ- ent of DNA length, describes both normalized confor- mation properties of DNA (most probably, affecting enzyme binding) and normalized energy (driving topo- isomerization). Consequently, r can be regarded as a universal parameter reflecting the substrate properties of scDNA. The value of reduced linear dichroism, q, is defined as a ratio between DA, the difference in light absorp- tion, polarized parallel and perpendicular to the DNA orientation axis, and A, the optical density of a sample in nonpolarized light [23]. It depends on the optical anisotropy of an individual molecule and on the aver- age orientation of these molecules in the sample relat- ive to the laboratory axis. The q(r) function was used for monitoring topo- isomerase-induced transition in DNA molecules. Strong dependence of reduced FLD (q) on its superhe- lix density (r) was first observed for circular DNA. Titration of the sc DNA pIBI30 [24] or /X174 phage [16,17] with intercalating dyes, which unwind DNA and change its topological state, revealed the bell- shaped dependence of q on r, with the maximum value of q corresponding to the nonsupercoiled (relaxed) DNA. Our measurements demonstrated close dependence; however these differed from data pub- lished in [12,25]. The ethidium bromide titration curve of pUC19 plasmid according to those data had the maximum, corresponding to completely unwound DNA. However, at r ¼ )0.04, an FLD signal reached its minimum value, increasing thereafter (Fig. 1). Qual- itatively similar behavior was also demonstrated by other plasmids under study, including pTM and A. Gabibov et al. DNA topoisomerization studied by FLD FEBS Journal 272 (2005) 6336–6343 ª 2005 The Authors Journal compilation ª 2005 FEBS 6337 BlueScript (data not shown). To the best of our know- ledge, an increase in the FLD signal for highly negat- ive supercoiled circular DNA has not been previously reported. According to [15], the tertiary structure of DNA is represented as a superhelix with radius R and pitch P. For such an oriented DNA, linear dichroism value can be represented as product of orientation factor S and optical factor O q ¼ SO ¼ 1=2ð3 <Cos 2 H> À 1Þ1=2ð3 <Cos 2 b> À 1Þ ð3Þ where Q is an angle between the DNA axis and the unique laboratory axis, and b a pitch angle of the superhelix, b ¼ tan )1 (2pR ⁄ P) [15]. Reduction of |q| upon increase of |r| was primarily associated with a decrease of the optical factor, which for the pitch angle 50° was as low as 0.12. However, electron microscopy data established that if the superhelix density r of circular DNA molecules ranged from )0.019 to )0.12, then R and P decreased proportion- ally [26] As a result, the pitch angle b appeared to be almost constant ($56°) for all topological states studied. On the other hand, the FLD value and sedi- mentation coefficients for circular DNA in a wide range of topological states were in perfect accord- ance [27]. This correlation demonstrates that hydro- dynamics of circular DNA can explain a change in the FLD value upon increase of |r|. In contrast to the relaxed DNA, which represents a loose loop, well-aligned along a sheared flow, scDNA behaves as a dense, shortened and rigid rod with impaired ori- entation ability. In the case of highly supercoiled long DNA the orientation could be better than that for its relaxed form [28]. Experimental measurements of sedimentation coefficients of PM2 [29], and SV40 [30] DNA demonstrated that the latter appeared to be a minimum for the relaxed DNA, and reached its maximum at r ¼ )0.04, then dropped again at more negative r values. Monte-Carlo simulation for PM2 [8] reproduced experimental data and demonstrated that at r < )0.05 DNA had a less branched and more extended conformation. As this conformation facilitates DNA orientation in the shear flow, our observation agrees well with the above data. Interaction of scDNA with eukaryotic DNA-topoisomerases Let us consider a sample of the flow-oriented circular plasmid DNA undergoing enzyme-induced topoisome- rization. At time point ‘t’, the state of the reaction mixture could be described using the function S(s,t), which determines the total length of DNA segments (i.e. a fraction of circular DNA) with the given super- helix density s. At any time point ‘t’, this function pos- sesses a property: Z Sðr; tÞdr ¼ DNA½ ð4Þ An instant value of the reduced FLD at time t could be represented as: qðtÞ¼ Z Sðr; tÞqðrÞdr ð5Þ Thorough analysis of the function S(s,t) using the FLD time dependence is not a straightforward task. However, data analysis could be easily performed for at least two types of reaction conditions: (1) After binding the enzyme to the supercoiled DNA, fast and virtually complete relaxation of DNA has taken place, followed by dissociation of the result- ing enzyme–relaxed DNA complex (processive mech- anism). This process presumably occurs in eukaryotic topoisomerase I [31,32], and in topoisomerase II under optimum conditions. As a result, a bimodal distribu- tion of topoisomers is observed [S(s,t) has two max- ima]. The first maximum corresponds to an initial topological state of DNA and the second represents a fully relaxed form, so Eqn (5) can be rearranged to Eqn (6): Fig. 1. Correlation between supercoiling density of plasmid DNA and linear dichroism value. Here linear dichroism of the sample is presented as relative value q, which is zero for the reaction buffer and 1 for the original plasmid sample. Supercoiling density is altered by titration with ethidium bromide. DNA topoisomerization studied by FLD A. Gabibov et al. 6338 FEBS Journal 272 (2005) 6336–6343 ª 2005 The Authors Journal compilation ª 2005 FEBS qðtÞ¼ DNA½ f½ð1 À kðtÞqðs init ÞþkðtÞqðr fin Þg; ð6Þ where k(t) is a degree of DNA conversion. (2) When the enzyme changes the DLk of DNA strictly by l (for prokaryotic topoisomerase I) or by 2 (for eukaryotic topoisomerase II) with the following ligation of the gap formed, and dissociation from DNA, a distributive mechanism occurs. In this case, S(s,t) may have only one maximum at any time point, this maximum is drifting toward a state with s ¼ 0. If dispersion is low enough, S(s,t) could be approximated as a product: [DNA] <s(t)>, so the observed FLD sig- nal is expressed as: qðtÞ¼ DNA½q½<rðtÞ>ð7Þ In other words, the q(t) function will be close to the intercalating agent titration curve (Figs 1 and 2). Figure 3 displays kinetic curves q(t) for human topoisomerases I and II, obtained by monitoring of the FLD signal during the incubation of enzymes with pUC19. These enzymes display dramatically different kinetic curves. The pseudo-first order kinetics is observed for topoisomerase I. Similar observation was made by Pulleyblank and Ellison [33] in the experi- ment based on registration of the intercalating agent fluorescence during the DNA topoisomerization. For topoisomerase II the kinetic curve agrees well with the DNA titration curve by ethidium bromide (Fig. 1). The differences can be explained by a different enzyme mechanism of action. As in case of topoisom- erase I we observed only initial and final DNA forms present in the reaction mixture. On the other hand, for topoisomerase II under experimental conditions (rather a high enzyme ⁄ DNA ratio) the concentration of intermediate topoisomers is significant. When the enzyme ⁄ DNA ratio decreases, the local minimum of AB Fig. 2. Mechanisms of action of enzymes of DNA topoisomerization. (A) Topoisomerase I-3¢. (B) Topoisomerase II. Two possible ways of action of topoisomerase I are shown, see text, as well as distributive and processive characters of catalysis by topoisomerase II. Fig. 3. FLD monitoring of kinetics of topoizomerization pUC19 plas- mid DNA (8 lg) catalyzed by human topoisomerase I-3¢ (0.45 l g) and topoisomerase II (4 lg) in reaction buffer. A. Gabibov et al. DNA topoisomerization studied by FLD FEBS Journal 272 (2005) 6336–6343 ª 2005 The Authors Journal compilation ª 2005 FEBS 6339 q(t) is expressed less; at high excess of the substrate it never reaches the minimum value q min (data not shown). Dynamics of drug targeting The FLD technique proves very useful for studying the topoisomerases interactions with various inhibitors and poisons [34,35]. These effectors are of interest because of their high antitumor activity [36]. As an example, we used a number of relatively well-studied compounds, some of them applied earlier as anticancer drugs. For topoisomerase I, camptothecin (CPT) and its two analogues, 10,11-ethylenedioxycamptothecin (MCPT-10,11) and 7-ethyl-10-hydroxycamptothecin (SN-38) (see Fig. 4) were taken. These drugs are known to inhibit the process at the religation stage of the nicked DNA chain (see Fig. 2A) [37,38]. As shown in Fig. 4, kinetic curves retain their exponential character at increasing concentrations of CPT and MCPT-10,11. For these compounds I 50 can be estima- ted as 1 and 0.1 lm, respectively. On the contrary, SN-38 (Fig. 4) does not affect the reaction rate at the initial stage, but effectively inhibits it afterwards (I 50 for the second portion is 0.2 lm). We propose that the FLD technique here promotes to distinguish between the mechanisms of action of these closely related com- pounds. The three inhibitors, as previously reported, hinder the DNA religation; CPT and MCPT-10,11 also impede the initial enzyme–DNA interactions, while SN-38 lacks this additional activity (see Fig. 2A). For topoisomerase II, we chose two compounds with totally different modes of action: etoposide [39] and adenosine-5¢-phosphate- b,c-iminodiphosphate (AMPPNP) [40]. The first one-etoposide, now a wide- spread chemotherapeutic drug, is a classic topoiso- merase II poison, acting by binding to single-stranded DNA ends and inhibiting the religation of the hydro- lyzed DNA segment (see Fig.2B) [41]. This inhibitor does not dramatically change the kinetic curve; etopo- side makes the whole process slower. The local mini- mum of q(t) in this case is less expressed (Fig. 5A). Using the initial rates, we found the I 50 value for this compound to be equal to 10 lm. Other topoisomerase II effectors, such as AMPPNP, are non hydrolysable analogues of ATP [42,43]. AMP- PNP blocks the ATP-dependent turnover of the type II enzyme. If the AMPPNP molecule binds to one or both topoisomerase II subunits, the enzyme remains in the ‘closed clamp’ conformation, topologically bound to the closed DNA molecule, which is kinetically and irreversibly inactivated (Fig. 2B). If ATP and AMP- PNP are present in the reaction mixture, only a certain portion of enzyme molecules is inactivated at each catalytic step. As shown in Fig. 5B, the reaction under these conditions does not reach an equilibrium state, but kinetic curves achieve the plateau at the values of the FLD signal corresponding to r <0. Conclusion The intrinsic connection between DNA topology, and the hydrodynamic and optical properties dis- cussed in this paper enables us to study experiment- Fig. 4. The effect of increasing concentrations of several campothe- cine analogues on the kinetics of topoisomerization of pUC19 DNA (8 lg) by topoisomerase I (0.45 lg): (A) 0 (curve 1), 0.25 (2), 0.5 (3), 0.75 (4), 1.25 (5), and 12.5 (6) l M CPT; (B) 0 (curve 1), 0.05 (2), 0.075 (3), 0.125 (4), and 0.25 (5) l M MCPT-10,11; (C) 0 (curve 1), 0.075 (2), 0.15 (3), 0.2 (4), and 0.25 (5) l M SN-38. Inserts: enlarged initial portions of graphs and chemical formulas of each inhibitor. DNA topoisomerization studied by FLD A. Gabibov et al. 6340 FEBS Journal 272 (2005) 6336–6343 ª 2005 The Authors Journal compilation ª 2005 FEBS ally the dynamics of DNA relaxation. The major functional dependence of the FLD signal on the DNA topology makes possible continuous analysis of DNA topoisomerization. The proposed technique is a unique nondisturbing procedure for estimating kinetics in the enzyme-mediated changes of topologi- cal parameters of the supercoiled plasmid DNA. It allows display of the substrate properties directly from the optic flow cell without additional proce- dures with the sample, thus avoiding any other topo- logical alterations not induced during the main reaction. This technique provided us with more data on the topoisomerase I action. For the first time FLD allowed the study of real-time kinetics of topoiso- merase II. We believe the developed approach is promising for analysis of the dynamics of drug target- ing, which opens up various biomedical applications including vast quantitative screening of antibacterial and anticancer drugs. Experimental procedures Harvesting and purification of plasmid DNA Ecolab DH5a strain was used for pUC19 amplification. A standard method of lyses by alkali [44] was used for plas- mid harvesting and purification. Phenol–chloroform extrac- tion was used to separate plasmid DNA from protein contamination. The separation of supercoiled and relaxed form of pUC19 was performed by Cycle gradient (1.55 gÆmol )1 ) ultracentrifugation. Enzyme assay TopoGEN Inc. topoisomerase I and II were used. The reac- tion was carried out in the final volume of 220 lL. The buffer for topoisomerase I was 0.02 m Tris pH 7.5, 0.1 m NaCl, 0.5 mm EDTA and 75 lgÆml )1 bovine serum albu- min. Toboggan 1 · reaction buffer (0.05 m Tris pH 8.0, 120 mm KCL, 10 mm MgCl 2 , 0.5 mm ATP, and 0.5 mm dithiothreitol, 30 lgÆmol )1 bovine serum albumin) was used in assay with topoisomerase II, 1 · reaction buffer was obtained from 10 · stock solution. The reaction was ter- minated with a 0.25 volume of the stop solution buffer (5% sodium dodecyl sulfate, 0.0025% bromophenol blue, and 25% glycerol). FLD technique The signal of linear dichroism was measured on JASCO J715 dichrograph equipped with achromatic quarterwave prism (wavelength 260 nm). The gradient was formed in the flow-through quartz cell (volume 200 lL) by reciprocating pumping with frequency 100 min )1 , using the same tech- nique as described in [45]. After washing with Piranha solu- tion the quartz details were rinsed with water, then treated with 3-aminopropyltrietoxysilane (10% in ethanol, 10 min), and a mixture of lutidine, acetyl anhydride, 4-dimethyl- aminopyridine in tetrahydrofuran (5 : 5 : 1 : 50, 2 h), rinsed with acetone, ethanol and water. Titration of 2–5 lgof plasmid DNA by ethidium bromide was performed in 20 mm Tris ⁄ HCl (pH 7,5), with 100 mm NaCl and a 20 lm solution of ethidium bromide. Supercoiling density was calculated using standard unwinding value of ethidium bromide of )26° per molecule. All new plasmid prepara- tions were titrated with ethidium bromide before their use in kinetic experiments. The normalized FLD signal was used, where q of the reac- tion buffer was taken as q ¼ 0 and the FLD signal of the ini- tial supercoiled plasmid was taken as q ¼ 1. Experimentally obtained kinetic curves (Figs 1 and 2 in supplementary material section) were smoothed using Microsoft Excel. Acknowledgements We thank the late Professor Marat Karpeysky for stimulating discussions and dedicate this paper to his memory. We thank Mrs Tatyana Chernichko for assistance in the paper preparation. We thank the Russian Foundation for Fundamental Investigations (00-04-48378, 99-01-00123) and the Fig. 5. Inhibition of topoizomerase II activity measured by the FLD technique. Comparison of the kinetics of inhibition of reaction of 8 lg of pUC19 DNA with 4 lg of topoisomerase II by etoposide and AMPPNP. (A) 0 l M (curve 1), 5 lM (2), 10 lM (3), 25 lM (4), 50 l M (5) of etoposide; (B) ratio [AMPPNP] ⁄ ([ATP] + [AMPPNP]) ¼ 0 (curve 1), 0.005 (2), 0.01 (3), 0.02 (4). A. Gabibov et al. DNA topoisomerization studied by FLD FEBS Journal 272 (2005) 6336–6343 ª 2005 The Authors Journal compilation ª 2005 FEBS 6341 Russian National Program of Support of Scientific Schools (1800.22003.4). References 1 Wang JC (1996) DNA topoisomerases. 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Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 45 Makarov VL & Dimitrov SI (1982) [Study of structural changes in chromatin in the presence of mono- and divalent cations by means of flow linear dichroism]. Mol Biol (Mosk) 16, 1086–1096. Supplementary material The following supplementary material is available online: Fig. S1. Non smoothed kinetic curve of topoizomeriza- tion of pUC19 catalyzed by human topoisomerase I-3. Fig. S2. Non smoothed kinetic curve of topoizomeriza- tion of pUC19 catalyzed by topoisomerase II. A. Gabibov et al. DNA topoisomerization studied by FLD FEBS Journal 272 (2005) 6336–6343 ª 2005 The Authors Journal compilation ª 2005 FEBS 6343 . 5. Inhibition of topoizomerase II activity measured by the FLD technique. Comparison of the kinetics of inhibition of reaction of 8 lg of pUC19 DNA with 4 lg of topoisomerase II by etoposide and. Transformations of Supercoiled DNA as Studied by Flow Linear Dichroism Technique. , Russian Academy of Sciences, Moscow. 36 Andoh T (1998) Bis(2,6-dioxopiperazines), catalytic inhibitors of DNA topoisomerase. Catalytic transformations of supercoiled DNA as studied by flow linear dichroism technique Alexander Gabibov 1,2,3, *, Elena Yakubovskaya 1 ,