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Revealing the mode of action of DNA topoisomerase I and its inhibitors by atomic force microscopy
BBRC Biochemical and Biophysical Research Communications 301 (2003) 789–797 www.elsevier.com/locate/ybbrc
Revealing the mode of action of DNA topoisomerase I and its inhibitors by atomic force microscopy Miriam Argaman,a Sarit Bendetz-Nezer,a Sophie Matlis,b Shraga Segal,a and Esther Priela,* a
Department of Immunology and Microbiology, Faculty of Health Sciences, The Ben-Gurion Cancer Research Center, Ben-Gurion University, Beer-Sheva 84105, Israel b Chemical Services Department, Weizmann Institute of Science, Rehovot 76100, Israel Received 7 January 2003
Abstract In this study, we used, for the first time, atomic force microscope (AFM) images to investigate the mode of action of DNA topoisomerase I (topo I) in the presence and absence of its inhibitors: camptothecin (CPT) and tyrphostin AG-1387. The results revealed that in the absence of the inhibitors, the enzyme relaxed supercoiled DNA starting from a certain point in the DNA molecules and proceeded in one direction towards one of the edges of the DNA molecule. In addition, the relaxation of the supercoiled DNA is subsequently followed by a knotting event. In the presence of CPT, enzyme-supercoiled DNA complexes in which the enzyme is locked inside a relaxed region of the supercoiled DNA molecule were observed. Tyrphostin AG-1387 altered the DNA relaxation process of topo I producing unique shapes of DNA molecules. AFM images of the topo I protein provided a picture of the enzyme, which resembles its known crystallographic structure. Thus, AFM images provide new information on the mode of action of topo I in the absence and presence of its inhibitors. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: DNA topoisomerase type I; Atomic force microscope; Camptothecin; Tyrphostin
DNA topoisomerases are a family of enzymes responsible for the topological state of the DNA in living cells. Two major types of topoisomerases (I and II) were identified and are distinguished by the number of DNA strands that they cleave and the covalent linkage formed in the enzyme–DNA intermediate [1–3]. Topo I enzymes were isolated from various prokaryotic and eukaryotic cells including bacteria, yeast, viruses, mammalian and human cells. Type IB enzymes include the eukaryotic topo I which is the product of the TOPO1 gene [4–6]. It is predominantly associated with transcriptionally active sequences and relaxes the supercoiled DNA during DNA replication, transcription, and recombination [1,3]. Topo I is essential during embryogenesis in drosophila and mice [7,8]. Since topo I participates in several processes that are essential for cell division and viability, it is the target of anticancer agents [9,10]. Understanding the mechanism of action of this enzyme * Corresponding author. Fax: +972-8-647-7626. E-mail address: [email protected] (E. Priel).
in the presence of its inhibitors is a major requirement for future clinical development of important therapeutic agents. Biochemical [11,12], crystallographic [1,13,14], and electron microscopy [15,16] methods were used to investigate the mode of action of this enzyme, the DNA topoisomers produced by its enzymatic activity, and the mechanism of topo I inhibitors. Topo I relaxes supercoiled DNA by a nicking-closing activity in which it introduces a single DNA break forming a covalent DNA–enzyme intermediate. The helical duplex downstream of the cleavage site rotates to relieve any torsional stress within the substrate DNA, followed by religation of the DNA break [1]. In addition to the relaxation of supercoiled DNA, topo I can convert single stranded circles to knotted forms [3]. CPT, a known inhibitor of this enzyme, acts by stabilizing the enzyme– DNA complexes and thus prevents the religation step [3]. In our previous study, we showed that certain tyrphostin derivatives, known as protein tyrosine kinase antagonists, also inhibited the DNA relaxation activity of topo I. The mode of action of tyrphostins differed
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00025-1
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from that described for CPT. They probably interact with the topo I protein and prevent the binding of this enzyme to DNA [17]. Therefore, certain tyrphostin derivatives represent a new group of topo I inhibitors. The AFM technique enables the imaging of soft biological samples with minimal disruption of the sample. As opposed to electron microscopy analysis, in the AFM technique the examined biological samples, in their native form, are directly loaded onto the mica sample surface followed by AFM imaging [18]. In this technique, it is possible to image the shapes of single macromolecules such as, nucleic acids, proteins, and complexes of nucleic acids with proteins [19–23]. In addition, the AFM images may provide information on the mode of action of nucleic acid binding enzymes [24– 28]. In this study, for the first time we used the AFM images to analyze the mode of action of topo I in the absence and presence of its inhibitors. We used the Tapping Mode of AFM in which a sharp tip raster scans across the sample only briefly touching the surface of the soft biological sample [29]. The AFM images revealed new information that has not been demonstrated by other methods. The structures of the topoisomers produced by the DNA relaxation activity of topo I suggest that topo I is a processive enzyme. In addition, we found that the overall topo I action consists of two sequential steps: a relaxation of the supercoiled DNA, which is then followed by a knotting event. The imaging of the DNA–enzyme complexes in the presence of CPT revealed that the enzyme is located in a DNA relaxed bubble providing a new information for its mode of action in the presence of this drug. We also demonstrated unique shapes of DNA topoisomers in the presence of tyrphostin AG-1387. Interestingly, the AFM image of the topo I protein provided a picture of the shape of the enzyme, which resembled its known crystallographic structure [1,13].
Electrophoresis in the first dimension was performed for 16 h at 65 V in the absence of chloroquine. For the second dimension the gel was soaked in TBE buffer containing 1.4 lM chloroquine and equilibrated for 6 h. The gel was shifted 90 ° from its original position (in the first dimension) and electrophoresis in the presence of chloroquine for16 h at 65 V was performed. The gel was extensively washed with deionized water and subjected to Southern blot analysis using 32 P-labeled pUC 19 DNA as the probe. AFM sample preparation and imaging conditions. Red Ruby mica (EMS, Fort Washington, PA) bound to an AFM disk (Digital Instruments, Santa Barbara, CA) was freshly cleaved prior to use and non-coated or coated with 0.5–1 mM NiCl2 [31]. Samples (5 ll) were loaded directly on the mica, washed with 1ml doubled distilled water, and dried with a stream of compressed air. The samples were further dried in a desiccator in the presence of silica gel. Samples were imaged in a Tapping mode in air, using a MultiMode Nanoscope III with phase base and extender electronics (Digital Instruments, Santa Barbara, CA). Olympus silicon cantilevers with a spring constant of 20 N/ m were used. Samples were routinely imaged in parallel with the AFM height and phase imaging. Images were processed by flattening to remove background slope (nanoscope software). The 3-D plot in Fig. 3E was obtained using a quick surface plot of the nanoscope software. The measurements of height, width, and length of molecules imaged by AFM. Height and width of particles in the AFM images were measured by section analysis of the Nanoscope software. The full width was measured at half the maximum height in the cross-section profiles of the images as a compensation for the convolution between the tip and the sample [32]. The size of the pUC19 DNA is 2686 bp. The length of fully relaxed pUC19 molecules in AFM should be 895 nm (as calculated according to the equation that 3 bp equals 1 nm in AFM). Indeed, the measurements of the length of the fully relaxed molecules in our AFM images revealed similar values. The length value of Supercoiled pUC19 DNA in AFM should be half or less than the length of fully relaxed molecules (i.e., 448 nm) depending on the degree of supercoiling. The length of the partially relaxed pUC19 molecules varied between these values. The calculated height of the fully relaxed pUC19 molecules is 0:4 nm 0:1 nm and the supercoiled regions have a maximum height of 2.3 nm. The calculated widths of the supercoiled and relaxed molecules are 25:1 5 nm and 12:5 1:5 nm, respectively. Estimation of topoisomerase I size in AFM. The calf topo I enzyme has a molecular weight of 96 kDa. The mass of one molecule of the enzyme is: 96,000/Avogadro number ¼ 1:6 1019 g. Assuming there is a density of 1.4 g/ml, a molecular volume of 1:2 1019 ml is calculated (volume ¼ mass/density). Thus, for hemispherical shape of the protein, a radius of 3.8 nm and a diameter of 7.6 nm are obtained (v ¼ 2=3pr3 ).
Materials and methods
Results
Topo I DNA relaxation assay. Ten units of purified calf thymus topo IB (obtained from two companies: TopoGEN, Columbus, OH, USA or MBI Fermentance, Hanover, MD, USA) was added to a topo I reaction mixture with a final volume of 25 ll: 20 mM Tris–HCl (pH 8.0), 20 mM KCl, 10 mM MgCl2 , 0.5 mM DTT, and 350 ng pUC19 supercoiled DNA. Where indicated, 200 lM tyrphostin AG-1387 or CPT was added. Following incubation at 37 °C for 5 or 15 min, 5 ll of the sample was immediately loaded on the mica surface and AFM samples were prepared as described below. In parallel, stopping buffer (final concentration; 1% sodium dodecyl sulphate, 15% glycerol, 0.5% bromophenol blue, and 50 mM EDTA, pH 8.0) was added to the remaining reaction. The reaction products were analyzed by 1% agarose gel-electrophoresis followed by staining of the gel with ethidium bromide (1 lg/ml). The gel was photographed using a short wavelength UV lamp. Two-dimensional (2-D) gel electrophoresis. To detect knotted DNA molecules 5 ll from the topo I reaction products, obtained after 15 min incubation, was analyzed by 2-D gel electrophoresis as described [30].
AFM analysis of the topoisomerase I DNA relaxation products To determine the AFM images of topo I DNA relaxation products and topo I-DNA complexes, purified calf thymus topo I protein was added to the specific topo I reaction mixture containing supercoiled pUC-19 DNA plasmid as the substrate. Following incubation at 37 °C for 5 and 15 min, the reaction products were analyzed by agarose gel electrophoresis (Fig. 1A) and by AFM (Figs. 1B–F). The results depicted in Fig. 1B demonstrate the AFM images of supercoiled pUC-19 DNA derived from a sample containing only the DNA plasmid in a relaxation buffer (Fig. 1A, lane 1). Most of the DNA molecules were supercoiled showing a plectonemic structure
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Fig. 1. AFM analysis of topoisomerase I DNA relaxation products. (A) One-dimensional agarose gel elecrophoresis of topo I DNA relaxation products: lane 1, supercoiled pUC19 DNA; lanes 2 and 3, products of 5 and 15 min of topo I DNA relaxation assay (respectively). (B) AFM image of pUC19 plasmid. (C) AFM image of topo I DNA relaxation products following 5 min of topo I activity. (D–F) AFM close up images of relaxed DNA-topo I complexes. Symbols: S, supercoiled DNA; R, relaxed DNA. Scale bars: B,C, 500 nm; D–F, 100 nm.
as previously described for supercoiled plasmid DNA using the electron microscope [33], and only a few were found in a partially supercoiled form. Following 5 min of topo I activity (Fig. 1A, lane 2), the AFM images showed that all of the supercoiled pUC-19 DNA molecules were converted to the fully or partially relaxed forms (Fig. 1C is a typical AFM image of the reaction products). Interestingly, the partially relaxed DNA molecules had a ‘‘stem & loop’’ conformation in which the ‘‘stem’’ represented a supercoiled state and the ‘‘loop’’ the relaxed state of the DNA molecules. The partially relaxed DNA molecules vary in the length of the supercoiled ‘‘stem’’ structure, which is compatible with the ladder of the relaxed topoisomers seen by agarose gel electrophoresis. Only a few DNA–enzyme complexes were observed in the field shown in Fig. 1C. However, a variation in the number of DNA–enzyme complexes in different fields was detected. In any given field, the ratio between DNA–bound enzyme to free enzyme (bound to the mica) did not exceed 0.7. A close up on the DNA–enzyme complexes shown in Figs. 1D– F demonstrates the binding of topo I to a relaxed form of the plasmid DNA.
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When the topo I DNA relaxation assay was carried out for 15 min, the AFM images of the reaction products revealed a field of coiled and compact DNA molecules with different shapes (Figs. 2A and B are typical AFM images for this reaction products). A close up on one of these structures shows a typical knotted conformation of a DNA molecule (Fig. 2C). However, analysis of the reaction products by the 1-D agarose gel electrophoresis could not distinguish between the partially relaxed topoisomers and the different knotted topoisomers (Fig. 1A, compare lane 2 and lane 3). These DNA species can be resolved by 2-D gel electrophoresis in which the intercalating agent (i.e., chloroquine) is present during the second dimension. A sample of 5 ll from the 15 min topo I DNA relaxation assay was analyzed for the presence of knotted DNA molecules using a specific 2-D gel electrophoresis followed by Southern blot analysis as described by Rodriguez-Campos [30]. The results depicted in Fig. 2DI show the partially supercoiled DNA topoisomers which are resolved as discrete spots along a regular curve and additional spots (indicated by arrows in the scheme of Fig. 2DII), which represent knotted molecules [30]. These results are compatible with the AFM image of the topo I reaction products obtained after 15 min reaction (Figs. 2A–C), indicating the formation of knotted DNA molecules by topo I. AFM images of the topoisomers and DNA–enzyme complexes in the presence of camptothecin Camptothecin (CPT), a specific topo I inhibitor, is known to act by stabilizing the enzyme–DNA intermediate complexes [34–36]. Therefore, CPT (200 lM) was added to the topo I reaction mixture and following a 15 min incubation at 37 °C, the reaction products were analyzed by gel electrophoresis (Fig. 3A) and by AFM images (Figs. 3B–F). The results depicted in Fig. 3B demonstrated the AFM images of the topo I reaction products in the presence of CPT. The outcome of the inhibition of topo I DNA relaxation activity by CPT was that most of the DNA molecules remained in a supercoiled form (compare Fig. 3A lanes 2 and 3). Indeed the AFM images revealed branched supercoiled and stretched DNA molecules (Fig. 3B) that differed in their structure from the untreated supercoiled plasmid molecules (Fig. 1B). In the presence of CPT, the DNA molecules appeared to be less flexible and more branched than the structure of the pUC19 DNA in the absence of this drug. In addition, complexes of topo I with supercoiled DNA were observed (Fig. 3B). A close up of these complexes was performed and here we show the height (Fig. 3C) and phase (Fig. 3D) images and a 3-D plot (Fig. 3E) of one of the DNA–enzyme complexes (marked by arrow 1 in Fig. 3B). Surprisingly, topo I was locked inside an open region of the supercoiled DNA
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Fig. 2. AFM images of DNA topoisomers following 15 min of topo I activity. (A,B) AFM images (height and phase, respectively) of DNA topoisomers. (C) AFM close up of a phase image of one of the DNA topoisomers. Scale bars: (A,B), 1000 nm; (C) 100 nm. (D) Electrophoretic characterization of the topo I reaction products by the 2-D gel analysis. (I) following the 2-D gel electrophoresis, Southern blot analysis was performed and the membrane was hybridized with a labeled pUC19 probe. (II) The scheme of the blot in 2DI, in which the knotted molecules are shown by arrows.
molecules which may be considered as a ‘‘relaxed DNA bubble,’’ usually at the edges of the stretched supercoiled DNA molecules. An estimation of the width of the DNA surrounding the enzyme revealed that it was compatible with that of a relaxed double stranded DNA imaged by AFM [37]. The measurements of the height and the diameter of the topo I protein locked in the ‘‘relaxed DNA bubble’’ were 2:8 0:4 nm and 27 3:1 nm, respectively. These data are compatible with the AFM values obtained for a single topo I protein molecule. In addition, the phase image depicted in Fig. 3D provided a more detailed observation of the supercoiled turns of the pUC19 DNA molecule. A topo I-DNA complex which contains more than one enzyme molecule per one DNA molecule is demonstrated in Fig.
3B (see arrow 2). A close up phase image of that complex is shown in Fig. 3F. It is possible that this is a noncovalent topo I-DNA complex containing at least two topo I molecules. AFM images of the topoisomers formed in the presence of tyrphostin Certain tyrphostin derivatives, known as protein tyrosine kinase (PTK) antagonists, were shown to be catalytic inhibitors of topo I [17] which differed from CPT in their mode of action. Therefore, it was interesting to image the products of the inhibition of topo I relaxation activity by tyrphostin AG-1387 (Fig. 4A). The reaction products were loaded on a mica surface
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Fig. 3. AFM images of topoisomers and DNA–enzyme complexes formed in the presence of CPT, a topo I inhibitor. (A) Agarose gel elecrophoresis of topo I DNA relaxation assay in the absence and presence of CPT. (B) AFM image of the DNA molecules obtained following 15 min of topo I activity in the presence of CPT. Arrows 1 and 2 point to the DNA–enzyme complexes shown in C, D, and F, respectively. (C,D) AFM image (height and phase, respectively) of a representative topoisomerase I-DNA complex formed in the presence of CPT from the field shown in B. (E) A 3D plot of the complex shown in C. (F) A phase image of supercoiled pUC19 DNA plasmid in a complex with topo I molecules. Symbols: S, supercoiled DNA; R, relaxed DNA. Scale bars: B, 500 nm; C, D, and F, 100 nm.
coated or uncoated with 0.5mM Niþþ and analyzed by AFM. The AFM images revealed partially relaxed DNA molecules (Fig. 4B). The structures of the DNA topoisomers produced by topo I in the presence of tyrphostin differed from those obtained in the absence of this drug (compare Fig. 4B and 1C). In addition to the ‘‘stem & loop’’ structures, various different con-
formations in which combinations of relaxed ‘‘ bubbles’’ and supercoiled cord in the same molecule were observed. In this AFM sample, supercoiled and relaxed configurations may also be distinguished by their color, representing a different height in nanometers (see scale bar in Fig. 4B). These topoisomer shapes were also seen when the samples were loaded on mica
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Fig. 4. AFM images of topoisomers formed in the presence of tyrphostin AG-1387, a topo I inhibitor. (A) Agarose gel electrophoresis of topo I DNA relaxation assay (for 15 min) in the absence (lane 2) and presence (lane 3) of tyrphostin. (B) AFM image of the DNA molecules obtained following 15 min of topo I activity in the presence of tyrphostin. Symbols: S, supercoiled DNA, R, relaxed DNA. Scale bars: B, 500 nm.
coated with the Ni cations. In addition a field of supercoiled DNA molecules with a plectonemic structure was observed (data not shown), which was expected according to the results obtained using agarose gel electrophoresis analysis (Fig. 4A). DNA–enzyme complexes were not observed in these AFM images which is compatible with our previous data, indicating that tyrphostins alter the binding ability of topo I to the DNA [17]. AFM images of topo I protein AFM images of purified proteins and enzymes such as human topo II a [38], TATA binding protein [39], and immunoglobulins [40,39] were previously reported. In this study the AFM image of purified calf thymus topo I was performed under the following conditions: in topo I buffer, in the presence or absence of supercoiled DNA or in the presence of CPT. A clear field of topo I proteins could be observed in the presence of CPT (Fig. 5A) but
Fig. 5. AFM images of topoisomerase I enzyme. (A) An AFM height image of topo I protein. (B,C) AFM close up phase images of topo I molecules. Scale bars: A, 500 nm; B, 200 nm; C, 100 nm.
not in the other aforementioned conditions. The AFM height images at lower magnifications (5 lm scans) revealed circular molecules varying in sizes as depicted in Fig. 5A. Additional AFM phase images obtained by zooming into the smallest molecules in the field are shown in Fig. 5B (a field of 1.27 lm) and in Fig. 5C (a field of 332 nm). A crescent shape with four distinguishable domains differing in height, a deep socket, and a fifth cord domain were observed for different molecules in the field. The measurement of the protein size in height images was performed on single topo I molecules observed in different fields of topo I DNA relaxation assay (i.e., Fig. 1C) and the average size was calculated. According to the AFM measurements, topo I protein has a height of 2:8 0:4 nm and a diameter of 27 3:1 nm. This size is larger than that predicted for a protein of 96 kDa with a hemispherical shape, which should
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have a diameter of 7.6 nm (see Materials and methods). It is known that AFM measurements of proteins are affected by convolution between the tip and the sample, which adds width to the protein [32]. Therefore, a protein with a diameter of 7.6 nm imaged with an AFM tip of about 10 nm in radii of curvature will appear as a protein with a width of approximately 27.6 nm. Indeed, the size of topo I protein, as measured from the AFM images, is compatible with the calculated value.
Discussion The relaxation of supercoiled pUC 19 DNA by topo I produced DNA molecules that differed in their topological state and are usually seen as a ladder of bands in agarose gel electrophoresis [11]. The AFM images provide a single molecule analysis of the topoisomers with a minimal disruption of their native forms [37]. The visualization of supercoiled DNA with AFM was previously demonstrated [41–44]. The overall geometry of supercoiled DNA visualized by AFM depends on ionic conditions. At low ionic strength the DNA molecules are loosely interwound supercoils with an irregular shape while at high salt concentrations plectonemic superhelices are formed [41,42]. The AFM image of the supercoiled DNA plasmid depicted in Fig. 1B shows plectonemic supercoiled molecules since it was taken from a topo I reaction mixture which contained moderate salt concentrations and a fully supercoiled DNA plasmid. This plectonemic structure of the supercoiled DNA molecule was also observed by electron microscopy [33]. This AFM image actually represents the structure of the supercoiled DNA plasmid in the topo I reaction mixture prior to the addition of the enzyme. Following topo I DNA relaxation activity an AFM field of the topoisomers produced by topo I demonstrated two different structures of the DNA molecules: fully relaxed circular molecules and partially relaxed molecules with a ‘‘stem & loop’’ structure. One could expect to observe various conformations in which combinations of more than one relaxed ‘‘bubble’’ and more than one supercoiled cord are present in the same molecule. However, such DNA structures were not seen in the AFM images of the topo I relaxation products (Fig. 1C). Therefore, the ‘‘stem & loop’’ structures suggest that the enzyme performs the relaxation of the supercoiled DNA starting from a certain point on the supercoiled DNA molecule from which it further proceeds in one direction towards one of the edges of the DNA molecule, in a processive manner. A comparison between the topoisomers produced by topo I activity at different time intervals revealed similar DNA ladders at 5 and 15 min of topo I activity, when examined by gel electrophoresis in the absence of intercalating agents. The AFM analysis of these reaction products demonstrated a dramatic difference between the shapes
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of the topoisomers produced by topo I activity at the aforementioned time intervals. In addition to the partially and fully relaxed DNA molecules observed following 5 min of topo I activity (Figs. 1C–F), knotted DNA molecules were produced by the enzyme following 15 min of activity (Figs. 2A–C). The presence of knotted DNA molecules was confirmed by the 2-D gel electrophoresis analysis (Fig. 2D) in which chloroquine was present in the second dimension as previously described [30]. These data suggest that the overall topo I action consists of two sequential steps: a relaxation of the supercoiled DNA subsequently followed by a knotting event. In the presence of CPT the AFM images revealed branched supercoiled DNA molecules combining with each other (Fig. 3B) that differed in their structure from the untreated supercoiled plasmid molecules (Fig. 1B). It was recently shown that topotecan (a derivative of CPT) interacts with calf thymus DNA in solution and forms complexes of DNA molecules [45]. Indeed, the AFM image suggests that CPT interacts with the supercoiled DNA molecules and causes the formation of branched molecules combined with each other. This may suggest that CPT affects the activity of topo I not only by stabilizing the DNA–enzyme complexes but also by modulation of the supercoiled DNA and the formation of DNA–DNA complexes. Crystallographic studies with human topo I and 22 bp DNA duplexes suggested that topo I wraps completely around the DNA substrate [13]. The binding mode of CPT was proposed on the basis of chemical and biochemical information combined with the 3-D structure of topo I-DNA complexes [46]. However, crystallographic studies of topo I-supercoiled DNA complexes formed in the presence of CPT were not performed. Therefore, to the best of our knowledge, the present study is the first that provides the visualization of the topo I-supercoiled DNA complexes formed in the presence of CPT. In this study, we examined the complexes formed between DNA and topo I after the DNA relaxation assay in the presence and absence of CPT. The AFM analysis revealed that the position of the enzyme in the topo I-DNA covalent complexes (in the presence of CPT) differed from its position in the absence of this drug. Topo I was attached to the double stranded relaxed DNA molecules in the absence of CPT (Figs. 1D–F), while in the presence of this drug the enzyme was located inside a relaxed DNA bubble (Figs. 3C–E). Two possible models for the mechanism of DNA relaxation by topo I were suggested. The ‘‘controlled rotation’’ model suggests that the enzyme binds and wraps the DNA, and once the DNA has been cleaved, the helical duplex downstream of the cleavage site rotates to relieve torsional stress within the DNA. The rotating DNA shifts the position of both the cap and the linker domains of the enzyme hindering the rotation reaction [1,14]. An alternative model
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suggests that following the cleavage step, the enzyme opens up to allow rotation of the DNA [1]. Since the presence of CPT prevents the ligation step (which reseals the nick in the DNA), and the release of the enzyme, it is possible to image by AFM the location of the enzyme in the presence of the drug. The AFM image provides a new interpretation for the mode of action of the enzyme in the presence of CPT. The stabilization of the covalent DNA– enzyme complex by CPT allows the enzyme to rotate together with the DNA to 180° and as a result, it becomes located in the inner side of the ‘‘relaxed bubble.’’ This suggests that under these conditions, the rotation of the DNA downstream to the cleavage site also causes the rotation of the DNA–enzyme complex upstream to the cleavage. This interpretation is based solely on the AFM image of the topo I-DNA complexes formed in the presence of CPT and on the estimation of the width of the DNA and the size of the enzyme in these complexes. The validity of this interpretation should be proven by other means such as crystallographic studies of the ternary complex of topo I-DNA–CPT. In the presence of tyrphostin AG-1387, a catalytic inhibitor of topo I [17], the AFM analysis revealed various shapes of partially relaxed DNA molecules and the absence of DNA–enzyme complexes (Fig. 4B). These DNA structures differed from the ‘‘stem & loop’’ shapes of the topoisomers produced by topo I activity in the absence of drugs (as seen in Fig. 1C). These images suggest that tyrphostin alters the enzyme activity and may affect its DNA binding ability as it was previously suggested using biochemical assays [17]. Therefore the AFM imaging provides a comprehensive analysis of the mode of action of topo I in the presence of two different inhibitors. Usually the structure of a protein is deduced from its crystallography. Using AFM imaging Nettikadan et al [47] demonstrated a low resolution image of human topoisomerase II a. In this study in the presence of CPT, we were able to image a close up picture of the topo I protein (Figs. 5B and C). One may suggest that the interactions between the protein, CPT, and the mica surface cause the protein to spread on the mica, allowing the observation of the shape of the enzyme. This AFM image of the topo I enzyme resembles the 3-D structure of topo I which was suggested by crystallographic studies [13,14]. It should be noted that here we show for the first time that it is possible to image details of the shape of a protein under specific conditions, using a close up of a phase image of AFM. This study indicates that AFM images not only provide a close and new insight into the topological structure of DNA topoisomers but, more important, they render a comprehensive perspective regarding the mode of action of topo I in either the absence or presence of its inhibitors. This approach is important for pharmacological and regulatory investigations of the interaction between DNA binding enzymes and their inhibitors.
Acknowledgments This work was supported by the Joan Baker and Rosalind Hendwood Fund and by the Center for Absorbency in Science of the Ministry of Absorption of the state of Israel. We thank Dr. N.H. Thomson from University of Leeds, UK, for the theoretical calculation of the expected size of topo I protein to be imaged by AFM.
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Revealing the mode of action of DNA topoisomerase I and its inhibitors by atomic force microscopy Miriam Argaman,a Sarit Bendetz-Nezer,a Sophie Matlis,b Shraga Segal,a and Esther Priela,* a
Department of Immunology and Microbiology, Faculty of Health Sciences, The Ben-Gurion Cancer Research Center, Ben-Gurion University, Beer-Sheva 84105, Israel b Chemical Services Department, Weizmann Institute of Science, Rehovot 76100, Israel Received 7 January 2003
Abstract In this study, we used, for the first time, atomic force microscope (AFM) images to investigate the mode of action of DNA topoisomerase I (topo I) in the presence and absence of its inhibitors: camptothecin (CPT) and tyrphostin AG-1387. The results revealed that in the absence of the inhibitors, the enzyme relaxed supercoiled DNA starting from a certain point in the DNA molecules and proceeded in one direction towards one of the edges of the DNA molecule. In addition, the relaxation of the supercoiled DNA is subsequently followed by a knotting event. In the presence of CPT, enzyme-supercoiled DNA complexes in which the enzyme is locked inside a relaxed region of the supercoiled DNA molecule were observed. Tyrphostin AG-1387 altered the DNA relaxation process of topo I producing unique shapes of DNA molecules. AFM images of the topo I protein provided a picture of the enzyme, which resembles its known crystallographic structure. Thus, AFM images provide new information on the mode of action of topo I in the absence and presence of its inhibitors. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: DNA topoisomerase type I; Atomic force microscope; Camptothecin; Tyrphostin
DNA topoisomerases are a family of enzymes responsible for the topological state of the DNA in living cells. Two major types of topoisomerases (I and II) were identified and are distinguished by the number of DNA strands that they cleave and the covalent linkage formed in the enzyme–DNA intermediate [1–3]. Topo I enzymes were isolated from various prokaryotic and eukaryotic cells including bacteria, yeast, viruses, mammalian and human cells. Type IB enzymes include the eukaryotic topo I which is the product of the TOPO1 gene [4–6]. It is predominantly associated with transcriptionally active sequences and relaxes the supercoiled DNA during DNA replication, transcription, and recombination [1,3]. Topo I is essential during embryogenesis in drosophila and mice [7,8]. Since topo I participates in several processes that are essential for cell division and viability, it is the target of anticancer agents [9,10]. Understanding the mechanism of action of this enzyme * Corresponding author. Fax: +972-8-647-7626. E-mail address: [email protected] (E. Priel).
in the presence of its inhibitors is a major requirement for future clinical development of important therapeutic agents. Biochemical [11,12], crystallographic [1,13,14], and electron microscopy [15,16] methods were used to investigate the mode of action of this enzyme, the DNA topoisomers produced by its enzymatic activity, and the mechanism of topo I inhibitors. Topo I relaxes supercoiled DNA by a nicking-closing activity in which it introduces a single DNA break forming a covalent DNA–enzyme intermediate. The helical duplex downstream of the cleavage site rotates to relieve any torsional stress within the substrate DNA, followed by religation of the DNA break [1]. In addition to the relaxation of supercoiled DNA, topo I can convert single stranded circles to knotted forms [3]. CPT, a known inhibitor of this enzyme, acts by stabilizing the enzyme– DNA complexes and thus prevents the religation step [3]. In our previous study, we showed that certain tyrphostin derivatives, known as protein tyrosine kinase antagonists, also inhibited the DNA relaxation activity of topo I. The mode of action of tyrphostins differed
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00025-1
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from that described for CPT. They probably interact with the topo I protein and prevent the binding of this enzyme to DNA [17]. Therefore, certain tyrphostin derivatives represent a new group of topo I inhibitors. The AFM technique enables the imaging of soft biological samples with minimal disruption of the sample. As opposed to electron microscopy analysis, in the AFM technique the examined biological samples, in their native form, are directly loaded onto the mica sample surface followed by AFM imaging [18]. In this technique, it is possible to image the shapes of single macromolecules such as, nucleic acids, proteins, and complexes of nucleic acids with proteins [19–23]. In addition, the AFM images may provide information on the mode of action of nucleic acid binding enzymes [24– 28]. In this study, for the first time we used the AFM images to analyze the mode of action of topo I in the absence and presence of its inhibitors. We used the Tapping Mode of AFM in which a sharp tip raster scans across the sample only briefly touching the surface of the soft biological sample [29]. The AFM images revealed new information that has not been demonstrated by other methods. The structures of the topoisomers produced by the DNA relaxation activity of topo I suggest that topo I is a processive enzyme. In addition, we found that the overall topo I action consists of two sequential steps: a relaxation of the supercoiled DNA, which is then followed by a knotting event. The imaging of the DNA–enzyme complexes in the presence of CPT revealed that the enzyme is located in a DNA relaxed bubble providing a new information for its mode of action in the presence of this drug. We also demonstrated unique shapes of DNA topoisomers in the presence of tyrphostin AG-1387. Interestingly, the AFM image of the topo I protein provided a picture of the shape of the enzyme, which resembled its known crystallographic structure [1,13].
Electrophoresis in the first dimension was performed for 16 h at 65 V in the absence of chloroquine. For the second dimension the gel was soaked in TBE buffer containing 1.4 lM chloroquine and equilibrated for 6 h. The gel was shifted 90 ° from its original position (in the first dimension) and electrophoresis in the presence of chloroquine for16 h at 65 V was performed. The gel was extensively washed with deionized water and subjected to Southern blot analysis using 32 P-labeled pUC 19 DNA as the probe. AFM sample preparation and imaging conditions. Red Ruby mica (EMS, Fort Washington, PA) bound to an AFM disk (Digital Instruments, Santa Barbara, CA) was freshly cleaved prior to use and non-coated or coated with 0.5–1 mM NiCl2 [31]. Samples (5 ll) were loaded directly on the mica, washed with 1ml doubled distilled water, and dried with a stream of compressed air. The samples were further dried in a desiccator in the presence of silica gel. Samples were imaged in a Tapping mode in air, using a MultiMode Nanoscope III with phase base and extender electronics (Digital Instruments, Santa Barbara, CA). Olympus silicon cantilevers with a spring constant of 20 N/ m were used. Samples were routinely imaged in parallel with the AFM height and phase imaging. Images were processed by flattening to remove background slope (nanoscope software). The 3-D plot in Fig. 3E was obtained using a quick surface plot of the nanoscope software. The measurements of height, width, and length of molecules imaged by AFM. Height and width of particles in the AFM images were measured by section analysis of the Nanoscope software. The full width was measured at half the maximum height in the cross-section profiles of the images as a compensation for the convolution between the tip and the sample [32]. The size of the pUC19 DNA is 2686 bp. The length of fully relaxed pUC19 molecules in AFM should be 895 nm (as calculated according to the equation that 3 bp equals 1 nm in AFM). Indeed, the measurements of the length of the fully relaxed molecules in our AFM images revealed similar values. The length value of Supercoiled pUC19 DNA in AFM should be half or less than the length of fully relaxed molecules (i.e., 448 nm) depending on the degree of supercoiling. The length of the partially relaxed pUC19 molecules varied between these values. The calculated height of the fully relaxed pUC19 molecules is 0:4 nm 0:1 nm and the supercoiled regions have a maximum height of 2.3 nm. The calculated widths of the supercoiled and relaxed molecules are 25:1 5 nm and 12:5 1:5 nm, respectively. Estimation of topoisomerase I size in AFM. The calf topo I enzyme has a molecular weight of 96 kDa. The mass of one molecule of the enzyme is: 96,000/Avogadro number ¼ 1:6 1019 g. Assuming there is a density of 1.4 g/ml, a molecular volume of 1:2 1019 ml is calculated (volume ¼ mass/density). Thus, for hemispherical shape of the protein, a radius of 3.8 nm and a diameter of 7.6 nm are obtained (v ¼ 2=3pr3 ).
Materials and methods
Results
Topo I DNA relaxation assay. Ten units of purified calf thymus topo IB (obtained from two companies: TopoGEN, Columbus, OH, USA or MBI Fermentance, Hanover, MD, USA) was added to a topo I reaction mixture with a final volume of 25 ll: 20 mM Tris–HCl (pH 8.0), 20 mM KCl, 10 mM MgCl2 , 0.5 mM DTT, and 350 ng pUC19 supercoiled DNA. Where indicated, 200 lM tyrphostin AG-1387 or CPT was added. Following incubation at 37 °C for 5 or 15 min, 5 ll of the sample was immediately loaded on the mica surface and AFM samples were prepared as described below. In parallel, stopping buffer (final concentration; 1% sodium dodecyl sulphate, 15% glycerol, 0.5% bromophenol blue, and 50 mM EDTA, pH 8.0) was added to the remaining reaction. The reaction products were analyzed by 1% agarose gel-electrophoresis followed by staining of the gel with ethidium bromide (1 lg/ml). The gel was photographed using a short wavelength UV lamp. Two-dimensional (2-D) gel electrophoresis. To detect knotted DNA molecules 5 ll from the topo I reaction products, obtained after 15 min incubation, was analyzed by 2-D gel electrophoresis as described [30].
AFM analysis of the topoisomerase I DNA relaxation products To determine the AFM images of topo I DNA relaxation products and topo I-DNA complexes, purified calf thymus topo I protein was added to the specific topo I reaction mixture containing supercoiled pUC-19 DNA plasmid as the substrate. Following incubation at 37 °C for 5 and 15 min, the reaction products were analyzed by agarose gel electrophoresis (Fig. 1A) and by AFM (Figs. 1B–F). The results depicted in Fig. 1B demonstrate the AFM images of supercoiled pUC-19 DNA derived from a sample containing only the DNA plasmid in a relaxation buffer (Fig. 1A, lane 1). Most of the DNA molecules were supercoiled showing a plectonemic structure
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Fig. 1. AFM analysis of topoisomerase I DNA relaxation products. (A) One-dimensional agarose gel elecrophoresis of topo I DNA relaxation products: lane 1, supercoiled pUC19 DNA; lanes 2 and 3, products of 5 and 15 min of topo I DNA relaxation assay (respectively). (B) AFM image of pUC19 plasmid. (C) AFM image of topo I DNA relaxation products following 5 min of topo I activity. (D–F) AFM close up images of relaxed DNA-topo I complexes. Symbols: S, supercoiled DNA; R, relaxed DNA. Scale bars: B,C, 500 nm; D–F, 100 nm.
as previously described for supercoiled plasmid DNA using the electron microscope [33], and only a few were found in a partially supercoiled form. Following 5 min of topo I activity (Fig. 1A, lane 2), the AFM images showed that all of the supercoiled pUC-19 DNA molecules were converted to the fully or partially relaxed forms (Fig. 1C is a typical AFM image of the reaction products). Interestingly, the partially relaxed DNA molecules had a ‘‘stem & loop’’ conformation in which the ‘‘stem’’ represented a supercoiled state and the ‘‘loop’’ the relaxed state of the DNA molecules. The partially relaxed DNA molecules vary in the length of the supercoiled ‘‘stem’’ structure, which is compatible with the ladder of the relaxed topoisomers seen by agarose gel electrophoresis. Only a few DNA–enzyme complexes were observed in the field shown in Fig. 1C. However, a variation in the number of DNA–enzyme complexes in different fields was detected. In any given field, the ratio between DNA–bound enzyme to free enzyme (bound to the mica) did not exceed 0.7. A close up on the DNA–enzyme complexes shown in Figs. 1D– F demonstrates the binding of topo I to a relaxed form of the plasmid DNA.
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When the topo I DNA relaxation assay was carried out for 15 min, the AFM images of the reaction products revealed a field of coiled and compact DNA molecules with different shapes (Figs. 2A and B are typical AFM images for this reaction products). A close up on one of these structures shows a typical knotted conformation of a DNA molecule (Fig. 2C). However, analysis of the reaction products by the 1-D agarose gel electrophoresis could not distinguish between the partially relaxed topoisomers and the different knotted topoisomers (Fig. 1A, compare lane 2 and lane 3). These DNA species can be resolved by 2-D gel electrophoresis in which the intercalating agent (i.e., chloroquine) is present during the second dimension. A sample of 5 ll from the 15 min topo I DNA relaxation assay was analyzed for the presence of knotted DNA molecules using a specific 2-D gel electrophoresis followed by Southern blot analysis as described by Rodriguez-Campos [30]. The results depicted in Fig. 2DI show the partially supercoiled DNA topoisomers which are resolved as discrete spots along a regular curve and additional spots (indicated by arrows in the scheme of Fig. 2DII), which represent knotted molecules [30]. These results are compatible with the AFM image of the topo I reaction products obtained after 15 min reaction (Figs. 2A–C), indicating the formation of knotted DNA molecules by topo I. AFM images of the topoisomers and DNA–enzyme complexes in the presence of camptothecin Camptothecin (CPT), a specific topo I inhibitor, is known to act by stabilizing the enzyme–DNA intermediate complexes [34–36]. Therefore, CPT (200 lM) was added to the topo I reaction mixture and following a 15 min incubation at 37 °C, the reaction products were analyzed by gel electrophoresis (Fig. 3A) and by AFM images (Figs. 3B–F). The results depicted in Fig. 3B demonstrated the AFM images of the topo I reaction products in the presence of CPT. The outcome of the inhibition of topo I DNA relaxation activity by CPT was that most of the DNA molecules remained in a supercoiled form (compare Fig. 3A lanes 2 and 3). Indeed the AFM images revealed branched supercoiled and stretched DNA molecules (Fig. 3B) that differed in their structure from the untreated supercoiled plasmid molecules (Fig. 1B). In the presence of CPT, the DNA molecules appeared to be less flexible and more branched than the structure of the pUC19 DNA in the absence of this drug. In addition, complexes of topo I with supercoiled DNA were observed (Fig. 3B). A close up of these complexes was performed and here we show the height (Fig. 3C) and phase (Fig. 3D) images and a 3-D plot (Fig. 3E) of one of the DNA–enzyme complexes (marked by arrow 1 in Fig. 3B). Surprisingly, topo I was locked inside an open region of the supercoiled DNA
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Fig. 2. AFM images of DNA topoisomers following 15 min of topo I activity. (A,B) AFM images (height and phase, respectively) of DNA topoisomers. (C) AFM close up of a phase image of one of the DNA topoisomers. Scale bars: (A,B), 1000 nm; (C) 100 nm. (D) Electrophoretic characterization of the topo I reaction products by the 2-D gel analysis. (I) following the 2-D gel electrophoresis, Southern blot analysis was performed and the membrane was hybridized with a labeled pUC19 probe. (II) The scheme of the blot in 2DI, in which the knotted molecules are shown by arrows.
molecules which may be considered as a ‘‘relaxed DNA bubble,’’ usually at the edges of the stretched supercoiled DNA molecules. An estimation of the width of the DNA surrounding the enzyme revealed that it was compatible with that of a relaxed double stranded DNA imaged by AFM [37]. The measurements of the height and the diameter of the topo I protein locked in the ‘‘relaxed DNA bubble’’ were 2:8 0:4 nm and 27 3:1 nm, respectively. These data are compatible with the AFM values obtained for a single topo I protein molecule. In addition, the phase image depicted in Fig. 3D provided a more detailed observation of the supercoiled turns of the pUC19 DNA molecule. A topo I-DNA complex which contains more than one enzyme molecule per one DNA molecule is demonstrated in Fig.
3B (see arrow 2). A close up phase image of that complex is shown in Fig. 3F. It is possible that this is a noncovalent topo I-DNA complex containing at least two topo I molecules. AFM images of the topoisomers formed in the presence of tyrphostin Certain tyrphostin derivatives, known as protein tyrosine kinase (PTK) antagonists, were shown to be catalytic inhibitors of topo I [17] which differed from CPT in their mode of action. Therefore, it was interesting to image the products of the inhibition of topo I relaxation activity by tyrphostin AG-1387 (Fig. 4A). The reaction products were loaded on a mica surface
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Fig. 3. AFM images of topoisomers and DNA–enzyme complexes formed in the presence of CPT, a topo I inhibitor. (A) Agarose gel elecrophoresis of topo I DNA relaxation assay in the absence and presence of CPT. (B) AFM image of the DNA molecules obtained following 15 min of topo I activity in the presence of CPT. Arrows 1 and 2 point to the DNA–enzyme complexes shown in C, D, and F, respectively. (C,D) AFM image (height and phase, respectively) of a representative topoisomerase I-DNA complex formed in the presence of CPT from the field shown in B. (E) A 3D plot of the complex shown in C. (F) A phase image of supercoiled pUC19 DNA plasmid in a complex with topo I molecules. Symbols: S, supercoiled DNA; R, relaxed DNA. Scale bars: B, 500 nm; C, D, and F, 100 nm.
coated or uncoated with 0.5mM Niþþ and analyzed by AFM. The AFM images revealed partially relaxed DNA molecules (Fig. 4B). The structures of the DNA topoisomers produced by topo I in the presence of tyrphostin differed from those obtained in the absence of this drug (compare Fig. 4B and 1C). In addition to the ‘‘stem & loop’’ structures, various different con-
formations in which combinations of relaxed ‘‘ bubbles’’ and supercoiled cord in the same molecule were observed. In this AFM sample, supercoiled and relaxed configurations may also be distinguished by their color, representing a different height in nanometers (see scale bar in Fig. 4B). These topoisomer shapes were also seen when the samples were loaded on mica
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Fig. 4. AFM images of topoisomers formed in the presence of tyrphostin AG-1387, a topo I inhibitor. (A) Agarose gel electrophoresis of topo I DNA relaxation assay (for 15 min) in the absence (lane 2) and presence (lane 3) of tyrphostin. (B) AFM image of the DNA molecules obtained following 15 min of topo I activity in the presence of tyrphostin. Symbols: S, supercoiled DNA, R, relaxed DNA. Scale bars: B, 500 nm.
coated with the Ni cations. In addition a field of supercoiled DNA molecules with a plectonemic structure was observed (data not shown), which was expected according to the results obtained using agarose gel electrophoresis analysis (Fig. 4A). DNA–enzyme complexes were not observed in these AFM images which is compatible with our previous data, indicating that tyrphostins alter the binding ability of topo I to the DNA [17]. AFM images of topo I protein AFM images of purified proteins and enzymes such as human topo II a [38], TATA binding protein [39], and immunoglobulins [40,39] were previously reported. In this study the AFM image of purified calf thymus topo I was performed under the following conditions: in topo I buffer, in the presence or absence of supercoiled DNA or in the presence of CPT. A clear field of topo I proteins could be observed in the presence of CPT (Fig. 5A) but
Fig. 5. AFM images of topoisomerase I enzyme. (A) An AFM height image of topo I protein. (B,C) AFM close up phase images of topo I molecules. Scale bars: A, 500 nm; B, 200 nm; C, 100 nm.
not in the other aforementioned conditions. The AFM height images at lower magnifications (5 lm scans) revealed circular molecules varying in sizes as depicted in Fig. 5A. Additional AFM phase images obtained by zooming into the smallest molecules in the field are shown in Fig. 5B (a field of 1.27 lm) and in Fig. 5C (a field of 332 nm). A crescent shape with four distinguishable domains differing in height, a deep socket, and a fifth cord domain were observed for different molecules in the field. The measurement of the protein size in height images was performed on single topo I molecules observed in different fields of topo I DNA relaxation assay (i.e., Fig. 1C) and the average size was calculated. According to the AFM measurements, topo I protein has a height of 2:8 0:4 nm and a diameter of 27 3:1 nm. This size is larger than that predicted for a protein of 96 kDa with a hemispherical shape, which should
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have a diameter of 7.6 nm (see Materials and methods). It is known that AFM measurements of proteins are affected by convolution between the tip and the sample, which adds width to the protein [32]. Therefore, a protein with a diameter of 7.6 nm imaged with an AFM tip of about 10 nm in radii of curvature will appear as a protein with a width of approximately 27.6 nm. Indeed, the size of topo I protein, as measured from the AFM images, is compatible with the calculated value.
Discussion The relaxation of supercoiled pUC 19 DNA by topo I produced DNA molecules that differed in their topological state and are usually seen as a ladder of bands in agarose gel electrophoresis [11]. The AFM images provide a single molecule analysis of the topoisomers with a minimal disruption of their native forms [37]. The visualization of supercoiled DNA with AFM was previously demonstrated [41–44]. The overall geometry of supercoiled DNA visualized by AFM depends on ionic conditions. At low ionic strength the DNA molecules are loosely interwound supercoils with an irregular shape while at high salt concentrations plectonemic superhelices are formed [41,42]. The AFM image of the supercoiled DNA plasmid depicted in Fig. 1B shows plectonemic supercoiled molecules since it was taken from a topo I reaction mixture which contained moderate salt concentrations and a fully supercoiled DNA plasmid. This plectonemic structure of the supercoiled DNA molecule was also observed by electron microscopy [33]. This AFM image actually represents the structure of the supercoiled DNA plasmid in the topo I reaction mixture prior to the addition of the enzyme. Following topo I DNA relaxation activity an AFM field of the topoisomers produced by topo I demonstrated two different structures of the DNA molecules: fully relaxed circular molecules and partially relaxed molecules with a ‘‘stem & loop’’ structure. One could expect to observe various conformations in which combinations of more than one relaxed ‘‘bubble’’ and more than one supercoiled cord are present in the same molecule. However, such DNA structures were not seen in the AFM images of the topo I relaxation products (Fig. 1C). Therefore, the ‘‘stem & loop’’ structures suggest that the enzyme performs the relaxation of the supercoiled DNA starting from a certain point on the supercoiled DNA molecule from which it further proceeds in one direction towards one of the edges of the DNA molecule, in a processive manner. A comparison between the topoisomers produced by topo I activity at different time intervals revealed similar DNA ladders at 5 and 15 min of topo I activity, when examined by gel electrophoresis in the absence of intercalating agents. The AFM analysis of these reaction products demonstrated a dramatic difference between the shapes
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of the topoisomers produced by topo I activity at the aforementioned time intervals. In addition to the partially and fully relaxed DNA molecules observed following 5 min of topo I activity (Figs. 1C–F), knotted DNA molecules were produced by the enzyme following 15 min of activity (Figs. 2A–C). The presence of knotted DNA molecules was confirmed by the 2-D gel electrophoresis analysis (Fig. 2D) in which chloroquine was present in the second dimension as previously described [30]. These data suggest that the overall topo I action consists of two sequential steps: a relaxation of the supercoiled DNA subsequently followed by a knotting event. In the presence of CPT the AFM images revealed branched supercoiled DNA molecules combining with each other (Fig. 3B) that differed in their structure from the untreated supercoiled plasmid molecules (Fig. 1B). It was recently shown that topotecan (a derivative of CPT) interacts with calf thymus DNA in solution and forms complexes of DNA molecules [45]. Indeed, the AFM image suggests that CPT interacts with the supercoiled DNA molecules and causes the formation of branched molecules combined with each other. This may suggest that CPT affects the activity of topo I not only by stabilizing the DNA–enzyme complexes but also by modulation of the supercoiled DNA and the formation of DNA–DNA complexes. Crystallographic studies with human topo I and 22 bp DNA duplexes suggested that topo I wraps completely around the DNA substrate [13]. The binding mode of CPT was proposed on the basis of chemical and biochemical information combined with the 3-D structure of topo I-DNA complexes [46]. However, crystallographic studies of topo I-supercoiled DNA complexes formed in the presence of CPT were not performed. Therefore, to the best of our knowledge, the present study is the first that provides the visualization of the topo I-supercoiled DNA complexes formed in the presence of CPT. In this study, we examined the complexes formed between DNA and topo I after the DNA relaxation assay in the presence and absence of CPT. The AFM analysis revealed that the position of the enzyme in the topo I-DNA covalent complexes (in the presence of CPT) differed from its position in the absence of this drug. Topo I was attached to the double stranded relaxed DNA molecules in the absence of CPT (Figs. 1D–F), while in the presence of this drug the enzyme was located inside a relaxed DNA bubble (Figs. 3C–E). Two possible models for the mechanism of DNA relaxation by topo I were suggested. The ‘‘controlled rotation’’ model suggests that the enzyme binds and wraps the DNA, and once the DNA has been cleaved, the helical duplex downstream of the cleavage site rotates to relieve torsional stress within the DNA. The rotating DNA shifts the position of both the cap and the linker domains of the enzyme hindering the rotation reaction [1,14]. An alternative model
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suggests that following the cleavage step, the enzyme opens up to allow rotation of the DNA [1]. Since the presence of CPT prevents the ligation step (which reseals the nick in the DNA), and the release of the enzyme, it is possible to image by AFM the location of the enzyme in the presence of the drug. The AFM image provides a new interpretation for the mode of action of the enzyme in the presence of CPT. The stabilization of the covalent DNA– enzyme complex by CPT allows the enzyme to rotate together with the DNA to 180° and as a result, it becomes located in the inner side of the ‘‘relaxed bubble.’’ This suggests that under these conditions, the rotation of the DNA downstream to the cleavage site also causes the rotation of the DNA–enzyme complex upstream to the cleavage. This interpretation is based solely on the AFM image of the topo I-DNA complexes formed in the presence of CPT and on the estimation of the width of the DNA and the size of the enzyme in these complexes. The validity of this interpretation should be proven by other means such as crystallographic studies of the ternary complex of topo I-DNA–CPT. In the presence of tyrphostin AG-1387, a catalytic inhibitor of topo I [17], the AFM analysis revealed various shapes of partially relaxed DNA molecules and the absence of DNA–enzyme complexes (Fig. 4B). These DNA structures differed from the ‘‘stem & loop’’ shapes of the topoisomers produced by topo I activity in the absence of drugs (as seen in Fig. 1C). These images suggest that tyrphostin alters the enzyme activity and may affect its DNA binding ability as it was previously suggested using biochemical assays [17]. Therefore the AFM imaging provides a comprehensive analysis of the mode of action of topo I in the presence of two different inhibitors. Usually the structure of a protein is deduced from its crystallography. Using AFM imaging Nettikadan et al [47] demonstrated a low resolution image of human topoisomerase II a. In this study in the presence of CPT, we were able to image a close up picture of the topo I protein (Figs. 5B and C). One may suggest that the interactions between the protein, CPT, and the mica surface cause the protein to spread on the mica, allowing the observation of the shape of the enzyme. This AFM image of the topo I enzyme resembles the 3-D structure of topo I which was suggested by crystallographic studies [13,14]. It should be noted that here we show for the first time that it is possible to image details of the shape of a protein under specific conditions, using a close up of a phase image of AFM. This study indicates that AFM images not only provide a close and new insight into the topological structure of DNA topoisomers but, more important, they render a comprehensive perspective regarding the mode of action of topo I in either the absence or presence of its inhibitors. This approach is important for pharmacological and regulatory investigations of the interaction between DNA binding enzymes and their inhibitors.
Acknowledgments This work was supported by the Joan Baker and Rosalind Hendwood Fund and by the Center for Absorbency in Science of the Ministry of Absorption of the state of Israel. We thank Dr. N.H. Thomson from University of Leeds, UK, for the theoretical calculation of the expected size of topo I protein to be imaged by AFM.
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