Plant Molecular Biology 19: 265-276, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

265

In vitro analysis of a type I DNA topoisomerase activity from cultured

tobacco cells Allyson D. Cole 1, Sharon Heath-Pagliuso 1, Annette Baich 2 and Eric B. Kmiec 1.

1Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, 233 S. lOth Street, Philadelphia, PA 19107, USA (*author for correspondence); 2Department of Biology, Southern Ilinois University, Edwardsville, IL 62026, USA Received 25 February 1991; accepted in revised form 14 January 1992

Key words."D N A topoisomerase I, D N A relaxation, tobacco cells

Abstract

The role of D N A topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled D N A in the presence of MgC12, and to a lesser extent in the presence of KC1. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H 1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified D N A molecules unless destabilized by the addition of KC1. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.

Introduction

Topoisomerases are fundamentally important enzymes that regulate the topological status of DNA [27]. By doing so, these enzymes influence many metabolic processes including replication and transcription [9, 21] by providing a molecular swivel so that topological blocks along the chromosome can be avoided. Mechanistically, topoisomerases transiently break the D N A strands, allow for a helical rotation and then reseal the broken strand(s). Detailed studies have revealed that the nicking-closing reaction proceeds through an intermediate in which the protein is covalently attached to the nicked D N A site [2, 5]. These

reaction intermediates have been isolated and in most cases consist of a phosphodiester linkage between the DNA and a tyrosine residue of the protein [ 5, 20 ]. Based on their mode of action, topoisomerases are divided into two classes. Type I topoisomerases catalyze DNA relaxation by topological steps of one, while type II topoisomerases promote D N A relaxation in steps of two. Type I enzymes are relatively abundant in many cell types and have been purified from numerous sources [17]. Although many biochemical and molecular studies have helped elucidate the mechanism of topoisomerase activity, by and large these studies were conducted in animal tis-

266 sue. Because of peculiar biochemical restrictions, plant cell extracts have yielded only a few pure proteins. The elegant work of Champoux and his colleagues [1] using a wheat germ system has provided much of the information about plant cell topoisomerases. Topoisomerases from carrot cells [6], pea chloroplast [18] and wheat embryo mitochondria [8] have also been helpful in understanding the role of these enzymes in plant cell metabolism. The work in the pea chloroplast system demonstrated an important relationship between topoisomerase activity and D N A replication. We are interested in understanding the roles that D N A topoisomerases play in the chromatin assembly pathway in tobacco cells. As an initial step in this direction, we have developed a purification scheme for a type I topoisomerase from cultured tobacco cells, and successfully isolated the nuclear, type I activity [11]. In this manuscript, we describe some of the biochemical characteristics of this enzyme, including the capacity to catalyze D N A relaxation and D N A breakage.

Materials and methods

Purification of iopoisomerase I and assay conditions The topoisomerase was purified according to Heath-Pagliuso et al. [11] except that the active protein from the phosphocellulose column was loaded onto a 2 m l (bed volume) Heparinsepharose column. The fractions were eluted stepwise from 0.25 M, 0.5 M, 0.75 M and 1 M KC1 concentrations respectively. D N A relaxation activity was found only in the 0.5 M bump. D N A relaxation assays were carried out in 20/~1 volumes at pH 7.8 and usually containing 5 m M MgC12 unless otherwise noted. The superhelical D N A substrate was pUC-18 D N A which had been purified by banding in CsC1 density gradients. Reactions were terminated by addition of 1 ~o SDS and deproteinized by treatment with Proteinase K (10 mg/ml). Tracking dye (0.25~o bromophenol blue, 0.25 ~o xylene cyanol in 50 ~o glycerol) v)as added and the D N A topology an-

alyzed in two ways. First, the samples were loaded onto a 1 ~o agarose gel (Tris-glycine) and electrophoresed at 40 V for 16 h. The D N A was visualized after staining in ethidium bromide (10/~g/ ml) by photography. Secondly, in the experiments where camptothecin (50 #M) was employed, the gel system described by Aronoff and Champoux [1] was used. In these cases, it was desirable to separate the nicked circular D N A from the covalently closed, relaxed circular DNA.

Chromatin assembly and protein modification reactions The indirect labeling of topoisomerase I was carried out as described [3] and phosphorylation or dephosphorylation was carried out as described by Kaisermann etal. [13]. Histones H1 and H5 were a kind gift of S. Leuba (University of Oregon) and were pure as judged by SDS-PAGE. Chromatin assembly reactions were carried out as described in Kmiec et al. [14] and the minichromosomes were purified by either sucrose gradient centrifugation or gel filtration. For this assembly process, the Xenopus oocyte S-150 cellfree extract was employed at a D N A to extract ratio (ng/#l) of 4 [22]. Alternatively, purified core histones (H2A, H2B, H3, H4) were isolated from the smut fungus Ustilago maydis or chicken erythrocytes according to Morse [ 18] and loaded onto a [32p]-labeled circular template (pUC-18) at a 0.7:1 w/w ratio by a reverse dialysis method. Nucleosome formation was assessed by the appearance of negative supercoils after deproteinization after electrophoresis through agarose gels.

Results

In a previous manuscript, we reported the extensive purification of a type I topoisomerase activity from cultured tobacco cells (cv. Xanthi) [ 11 ]. Our most purified fraction contained one major band and several minor, but persistent, bands after phosphocellulose chromatography as visualized by silver staining of an SDS-PAGE pro-

267 tein gel. The phosphocellulose fraction, containing topoisomerase activity, was loaded directly onto a 1.5 ml (bed volume) Heparin-Sepharose column and proteins fractionated by gradually increasing the KC1 in the elution buffer. At a salt concentration of 0.5 M, topoisomerase I activity was recovered. The phosphocellulose fraction had a specific activity of 8.3 x 105 with a protein concentration of 10 #g/ml. One unit of activity corresponds to the amount of enzyme required to relax 50 ng of negatively supercoiled D N A in 30min at 31 °C. The Heparin-Sepharose fraction (2.5 #g/ml) has a specific activity of 8.0 x 106 and thus this final step further purifies the enzyme eight-fold. The protein composition of this fraction was visualized by S D S-PAGE [15] followed by silver staining (Fig. 1A) A unique major protein band appears at a denatured molecular mass of ca. 30 kDa. An important enzymatic feature of type I topoisomerases is their ability to bind DNA covalently and produce a DNA-protein cleavage intermediate. The Heparin-Sepharose fraction was mixed with [32P]-labeled nick translated pBR-322 D N A and following the procedure of Brougham et al. [3], we detected radioactive labeling of the 30 kDa protein after SDS-PAGE (Fig. 1B). The Heparin-Sepharose fraction was also size-fractionated by glycerol gradient centrifugation, and the relaxation activity was localized at a position corresponding to ca. 7 0 k D a (Fig. 1C). No other DNA relaxation activity was observed even with extended (10 h) of incubation time. These results are consistent with our previous observations regarding the native molecular weight of the phosphocellulose fraction containing the D N A relaxation activity [11 ]. The molecular mass of this topoisomerase was assessed using Sephacryl (S-200) sizing column under various salt conditions. Previously Rowe et aI. [20] used a similar strategy to determine the hydrodynamic distribution coefficient (Kd) of a Ustilago topoisomerase. Following their protocol closely, we size-fractionated the tobacco cell topoisomerase similarly and our results are presented as Table 1. Based on the assumption that the tobacco enzyme is globular in nature, once again we found that fractions corresponding to

Table 1. Estimation of size of the topoisomerase.

KC1

Activity peaks

Kal/3

Molecular weight

Recovery (%)

0.2 M

1 2 1 2

0.60 0.91 0.61 0.91

72 30 73 30

26% 21% 33% 23%

0.05 M

000 000 000 000

A Sephacryl S-200 (1.4 c m x 56 cm) was equilibrated with 50 mM Tris-HC1 (pH 7.8), 0.5 mM DTT, 10% glycerol, 0.5 mM PMS and the indicated salt concentrations. The excluded and included volumes were determined using [3H] M 13 phage and [3Zp] orthophosphate respectively. Marker proteins used to calibrate the column included ferritin, catalase, bovine serum albumin, carbonic anhydrase and lysozyme. Based on previous experimentation, we assume a globular nature for the topoisomerase and determined the hydrodynamic distribution coefficient. This was related linearly to molecular weight. The column was run at 10 ml/h and 1 ml fractions were collected after loading 5000 units. DNA relaxation activity was determined by agarose gel electrophoresis and visually inspected after staining with ethidium bromide.

molecular masses of ca. 72 kDa and 30 kDa contain D N A relaxation activity. At higher concentrations of KC1 (0.5 M, 1.0 M) no D N A relaxation activity was observed as expected, hence no definitive correlations between active enzyme and molecular weight could be made. The D N A relaxation activity of the Heparinsepharose purified topoisomerase is displayed in Fig. 1D. A gradual increase in the population of relaxed topoisomers appears as the number of enzyme units are increased. Once again, the presence of MgCI2 in the reaction mixture was found to stimulate activity significantly. Even in the presence of 2 units of enzyme, a minor proportion of the D N A is relaxed completely in the absence of MgC12. The dependence on MgC12 is in all likelihood a reflection of the low concentration of KC1 in the reaction mix and this supposition is examined below. The D N A relaxation activity can be titrated by increasing the concentration of the supercoiled template. As seen in Fig. 1E, 50 ng of negatively supercoiled D N A is completely relaxed by 1 unit of the tobacco topoisomerase I. A gradual increase in substrate concentration from 75 ng to 200 ng) results in a reduction of fully relaxed D N A

268 Fig. 1. Purification and characterization of the type I topoisomerase. A. SDS-PAGE analysis of the Heparin-Sepharose fraction was carried out according to Laemmli [ 15] and silverstained. M, molecular weight standards of BSA, 67 kDa, and carbonic anhydrase, 29.4 kDa; 1.2 #g of heparin sepharose fraction. B. Reaction mixtures (50 #1) containing 5 #g or 10 #g of the Heparin-Sepharose-purified fraction and nick-translated DNA were incubated for 15 rain at 31 ° C in the presence of 5 mM MgC12 followed by denaturation with 50 mM NaOH. After neutralization, digestion with 0.3 units of DNAse I was carried out in tile presence of 3 mM CaC12. The protein-DNA complexes were extracted by phenol six times, precipitated with acetone and subjected to SDS-PAGE (12.5 % resolving gel). The labeled protein band is visualized by autoradiography. Lane 2, 5 #g protein; lane 3, 10 #g protein. C. Glycerol gradient analysis of heparin-purified fraction was carried out by loading 20 #g of the fraction onto a 5 - 2 0 ~ glycerol gradient made in Buffer A [ 11]. The gradients were centrifuged at 40000 rpm for 48 h at 4 °C and 120 aliquots collected by drop. DNA relaxation activity was measured by incubating 10 #1 of the gradient sample with 100 ng of superhelical DNA (pUC-18) in the presence of 5 mM MgC12 for 1 h at 31 °C and the percentage relaxation judged visually after 1% agarose gel electrophoresis. Molecular weight indicators are in daltons and are based on purified controls processed in the same fashion. D. Reaction mixtures (20 #i) containing the indicated amount of topoisomerase I units and 100 ng of supercoiled D N A in the absence or presence of 5 mM MgCI 2 were incubated for 30 min at 31 °C and changes in DNA topology visualized by electrophoresis through 1% agarose and staining in ethidium bromide (10 #g/ml). Sc, supercoiled DNA; R, relaxed DNA; C, no topoisomerase added. E. Reaction mixtures were identical to those described in D except that the topoisomerase units were fixed at 1 and the indicated amount of DNA was present in the reaction mixture.

269 produced by the action of the enzyme. Still, many topoisomers with a higher linking number are generated even at a high D N A concentration, reflecting the high inherent activity of this enzyme. Coupled with our previous observations [ 11 ], the results reflect the processive nature of the enzyme in the presence of low concentrations of MgC12. One intriguing feature of eukaryotic topoisomerases is their capacity to relax positively supercoiled D N A [26]. We next tested the tobacco topoisomerase for relaxation of positive supercoils. The generation of positive supercoils is carried out by incubating negatively supercoiled DNA/with ethidium bromide (EtBr) [23]. At a certain concentration of EtBr, the node of supercoiling changes from negative to positive. This template was incubated with increasing levels of either calf thymus topoisomerase I (BRL) or with the tobacco topoisomerase. After 1 h of incubation the reaction was terminated, the samples deproteinized, but the EtBr was not extracted. Hence, relaxed templates still appear as a slower moving species after gel electrophoresis. As displayed in Fig. 2, both enzymes catalyze D N A relaxation. Specifically, 5 units of the tobacco topoisomerase I are required to relax fully the positive supercoils. The product of this reaction is primarily covalently closed circles as judged by chloroquine-agarose gel eletrophoresis (data not shown). Rowe et al. [20] demonstrated that histone H1 enhanced D N A relaxation catalyzed by the UstiIago maydis topoisomerase I. These results

were rather interesting because only histone H 1, not the core histones, stimulated the topoisomerase. Hence, correlations between DNA, packaged into nucleosomes, and topoisomerases were made. We incubated purified histone H 1 or purified histone H5 with the D N A for 15 min at different protein/DNA ratios (0.05, 0.1, 0.15, 0.2) and then added 0.5 units of the tobacco topoisomerase. It is within this range of histone/DNA ratios that H 1 was found to stimulate relaxation; Fig. 3 illustrates the results. We observed only a slight stimulation of D N A relaxation by the addition of H1 or H5 to the reaction mixture at neutral pH (7.0). Although we specifically used a lower amount of enzyme in order to better assess any enhancement of relaxation, higher amounts of enzyme also reveal no stimulation of activity. In addition, no stimulation is seen at lower stoichometry or at various pH values (A. Baich, unpublished observations). The purified topoisomerase I from Xenopus laevis is regulated in its ability to promote D N A relaxation by protein phosphorylation [ 13 ]; treatment of the enzyme with alkaline phosphatase reduces the D N A relaxation activity. We next treated the purified tobacco topoisomerase with either immobilized alkaline phosphatase or casein kinase II and monitored the activity of these modified enzymes. As illustrated in Fig. 4A, treatment of the topoisomerase with phosphatase does inhibit slightly the capacity of this enzyme to relax negatively supercoiled DNA. The shift is small but very reproducible; the unit requirement for

Fig. 2. Relaxation of positive supercoils by calf thymus topoisomerase I and tobacco cell topoisomerase I. Reaction mixtures (20/~1) containing 100 ng of positively supercoiled pUC-18 DNA, 5-raM MgC12 and the indicated amounts of either calf thymus topoisomerase I (BRL) or tobacco cell topoisomerase were incubated at 31 °C for 1 h. The reactions were then processed as described in Materials and methods and visualized by 1% agarose gel electrophoresis. SC, supercoiled DNA; R, relaxed DNA; C, no topoisomerase added; CT, calf thymus topoisomerase; T, tobacco cell topoisomerase.

270

Fig. 3. Stimulation of DNA relaxation by the addition of histones H1 or H5. Reaction mixtures (20 ~tl) containing 100 ng of superhelical DNA, 0.25 units of topoisomerase, 5 mM MgC12 and the indicated amounts of histone H 1 or H5 were incubated for 1 h at 31 °C. Changes in DNA topology were analyzed by electrophoresis through a 1% agarose gel and staining with EtBr (10/~g/ml). SC, supercoiled DNA; R; relaxed DNA; protein-DNA, protein/DNA ratio (w/w); C, no topoisomerase added.

DNA relaxation is doubled. In control experiments, inactivated alkaline phosphatase was shown not to inhibit DNA relaxation catalyzed by either the tobacco topoisomerase or the calf thymus enzyme (data not shown). In the reverse experiment, we treated the purified topoisomerase preparation with casein kinase II using the procedure of Kaiserman et al. [13] in order to phosphorylate the topoisomerase. Figure 4B displays the phosphorylation of the enzyme. To assess the efficiency of labeling, we carried out SDS-PAGE on the labeled protein preparation. In lane 1, the reaction mixture lacks the topoisomerase and does reveal a small amount of protein labeling at a position of 30-31 kDa. This is consistent with the autophosphorylation of the fl subunit of casein kinase, but is not substantial. In lane 2 (Fig. 4B), a clear distinct band at 31 kDa is observed and corresponds to the molecular weight of the tobacco topoisomerase under SDS-PAGE conditions. Surprisingly, a faint but distinct labeled protein (?) band appears at a slightly higher molecular weight. This band is not visible when the purified enzyme preparations are visualized by silver staining of protein gels. Nevertheless, the bands were extracted from the gel and repurified according to Kaiserman etal. [13]. Only the lower-molecular-weight band contained DNA relaxation activity and as revealed in Fig. 4C, phosphorylation of the enzyme did not influence DNA relaxation catalyzed by this topoisomerase. As expected, additional units of the S D S gel-purified enzyme were required to catalyze DNA relax-

ation. These results do reveal, though, that the 31 kDa protein band does contain DNA relaxation activity. The use of cAMP-dependent kinase to phosphorylate the enzyme produced similar results. The drug camptothecin has been used extensively to unbalance the equilibrium between noncleavable and cleavable complexes during topoisomerase I activity [12]. The addition of SDS immobilizes the covalently bound protein-DNA complex. This methodology is used to identify the sequence specificity of many type I topoisomerase break sites [9, 26]. Recently, Aronoff and Champoux [1] demonstrated that although camptothecin enhances covalent complex formation, it has minimal effect on sequence specificity. These authors also used a simple, but elegant, agarose gel assay system in which nicked circles and relaxed covalently closed circles can be differentiated. Next, we analyzed the effect of camptothecin on DNA cleavage promoted by the tobacco topoisomerase using this gel assay. One unit of enzyme in the phosphorylated or unphosphorylated state was incubated with supercoiled D N A in the presence of several concentrations of camptothecin. In addition, we reacted an increasing number of enzyme units with the D N A in the absence of the drug. The reactions were terminated after 1 hour by the addition of SDS and the samples were then deproteinized. As illustrated in Fig. 5A, camptothecin does enhance the formation or stabilization of the nicked D N A intermediate (OC) (compare lanes 2 and 8). No difference is ob-

271

Fig. 4. The influence of dephosphorylation or phosphorylation on the capacity of the topoisomerase to catalyze DNA relaxation. A. Reaction mixtures (20 #1) containing 100 nanograms of supercoiled pUC-18 DNA, 5 mM MgC12 and 1 unit of the tobacco topoisomerase, modified as indicated, were incubated at 31 °C for the indicated times. Changes in DNA topology were analyzed by electrophoresis through a 1% agarose gel. C, no topoisomerase added; SC, supercoiled DNA; R, relaxed DNA; o, untreated topoisomerase I; + / - , heat-inactivated alkaline phosphatase-treated topoisomerase I; +, alkaline phosphatase-treated topoisomerase I. B. SDS-PAGE (12.5 % resolving gel) analysis of casein-kinase treated tobacco topoisomerase I. The enzyme was phosphorylated using [32p]-labelled 7-ATP. Control, no topoisomerase I; Topo I, 10 gg of topoisomerase I. C. Reaction mixtures were identical to those described in A, except that either the phosphorylated topoisomerase ( + ) or unphosphorylated topoisomerase ( - ) was used and the time of incubation is indicated. SC, supercoiled DNA; R, relaxed DNA.

served between the phosphorylated or unphosphorylated forms of the enzyme. In addition, as predicted, a substantial level of nicked D N A accumulates when an excessive level of enzyme is

used in the absence of camptothecin and the reaction terminated by the addition of SDS (lanes 8, 9). The D N A relaxation activity of many eukary-

272

Fig. 5. Camptothecin enhances D N A breakage by the tobacco cell topoisomerase. A. Reaction mixtures (20/~1) containing 100 ng of superhelical pUC-18 DNA, 5 mM MgC12 and 1, 2 or 3 units of either the phosphorylated or unphosphorylated topoisomerase were incubated at 31 ° C for 1 h in the absence or presence of 50 #M camptothecin. The reactions were terminated by the addition of 1% SDS and deproteinized by treatment with Proteinase K (10 mg/ml). Gel electrophorcsis through 1% agarose was carried out as described in Materials and methods. SC, supercoiled DNA; OC, open circular DNA; CC, covalently closed DNA; C, no topoisomerase added; lanes 1, 2, 3 correspond to 1, 2 or 3 units of the phosphorylated topoisomerase; lanes 4, 5, 6 correspond to the untreated enzyme; lanes 7, 8, 9 correspond to untreated enzyme incubated in the absence of camptothecin. B. Reaction mixtures (20/~1) containing 100 ng of supercoiled pUC-18 DNA, 1 unit oftopoisomerase 5 mM MgC12 were incubated at 31 °C for 1 hour with the following concentrations of KCI: 1, 0 mM; 2, 1 raM; 3, 2 mM; 4, 5 mM; 5, 10 mM; 6, 25 mM; 7, 35 mM; 8, 50 raM; 9, 75 mM; 10, 100 mM; 11,200 mM; 12, 300 raM; 13, 400 mM; 14, 500 raM; 15, 750 mM. The change in D N A topology was analyzed by agarose gel electrophoresis. C. Re-

otic topoisomerases is influenced by monovalent cation concentration of the reaction mix. In some cases, KC1 is used as the salt and partial inhibition is seen at concentrations of ca. 200 mM [5]. We wanted to examine the influence of KC1 on the camptothecin enhanced D N A breakage. First, we analyzed the effect of increasing concentrations of KC1 on D N A relaxation promoted by the tobacco enzyme. Figure 5B illustrates this effect. One unit of relaxation activity was incubated with the D N A template in the presence of KC1 for one hour. At a concentration of 50 mM, D N A relaxation is inhibited 50~o and completely abolished at a concentration of 200 mM KC1. This low salt sensitivity is somewhat unusual and may reveal an important difference between the biochemical characteristics of plant and animal cell topoisomerases. The wheat germ topo I however is not significantly inhibited until the KC1 concentration reaches 200 mM and so correlations must be made with caution. The experiments presented above (5A and 5B) were carried out in the presence of MgC12. Hence, the inhibition of relaxation by KC1 may be a result of protein-DNA complex destabilization. Since most eukaryotic type I topoisomerases can utilize KCI in place of MgC12, we analyzed camptothecin-induced D N A nicking in the absence of MgC12 but in the presence of KC1. From previous results, we knew that a large number of topoisomerase units would be required in the absence of MgC12, hence 10 units of the enzyme was used. The presence of 100 mM KC1 was found to enhance relaxation but not the formation of a cleavable complex (Fig. 5C). This lack of cleavable complex formation could be due to complex instability in the presence of KC1. Hence, under these reaction conditions, camptothecin does not stimulate formation of the cleavable DNA-protein

action mixtures (20/~1) containing 100 ng of pUC-18 superhelical DNA, 1 unit of type I topoisomerase, 50/~M camptothecin and the indicated concentration of KC1 were incubated for 1 h at 31 °C. Changes in DNA topology were analyzed as described in Materials and methods. SC, supercoiled DNA; OC, open circular DNA, CC, covalently closed DNA.

273 complex [12] and may reflect a different type of enzymatic behavior. DNA relaxation is stimulated under certain reaction conditions that include a high concentration of topoisomerase I. Next, we wondered whether the protein complex, formed in the presence of camptothecin, could be dissociated by the addition of salt. To carry out this reaction, we incubated a small amount of the tobacco topoisomerase I with the D N A template in the presence of MgC12 and camptothecin. Under these conditions, a significant amount of topoisomerase-promoted DNA nicking is observed (see above). This is confirmed by the appearance of nicked D N A as a function of time (Fig. 6A). If KC1 is added at any point during the incubation (60 rain), the amount of

Fig. 6. Destabilization of the cleavable complex promoted by KC1. Reaction mixtures (20 #1) containing 100 ng of superhelical pUC-18 DNA, 5 mM MgC12, 50 # M camptothecin and 1 unit of topoisomerase were incubated at 31 oC. To one series of tubes (Stopped at), 1% SDS and Proteinase K (10 mg/ml) were added at the indicated times. To another series (KC1 added at), KC1 was added to a final concentration of 100 mM and all the reactions terminated by SDS and Proteinase K addition at 1 h. D N A topology was analyzed as described in Materials and methods. SC, supercoiled DNA; OC, open circular DNA; CC, covalently closed DNA.

nicked (OC) DNA is reduced, while the level of relaxed D N A (CC) is maximized (Fig. 6B). These results suggest that KC1, in all likelihood, disrupts the camptothecin-induced protein-DNA complexation and caused the topoisomerase to dissociate. The reversal of camptothecin-induced DNA cleavage by salt was reported previously by Hsiang etal. [12] using a mammalian type I topoisomerase. Recently, a series of reports have established that camptothecin can induce topoisomerase I to catalyze single-strand breaks in vivo [see 7, and references therein]. Because eukaryotic D N A is packaged into chromatin, presumably the type I topoisomerase must be causing single-stranded scissons on a chromatin template. We wanted to test the capacity of the tobacco enzyme to cleave DNA, assembled into chromatin in vitro. 32p_ labelled pUC-18 D N A was incubated with the Xenopus S-150 cell-free extract [10] for 12 h. This in vitro system catalyzes the formation of nucleosomes on circular D N A templates with regular periodicity and under physiological conditions [14]. We utilized a sufficient amount of extract to assemble all of the D N A molecules fully into chromatin [21]. The minichromosomes were isolated and purified by sucrose gradient centrifugation [14]. These templates were then incubated with the purified tobacco enzyme in the presence of MgC12 and camptothecin and the reactions were terminated by the addition of SDS. Under these conditions, the assembled chromatin templates will appear as supercoiled DNA molecules after deproteinization. The samples were electrophoresed using the procedure of Aronoff and Champoux [ 1]. As displayed in Fig. 7A, the chromatin templates did not become nicked or relaxed by the action of the tobacco topoisomerase regardless of the concentration of MgC12 or camptothecin. The DNA remains fully supercoiled (SC) in sharp contrast to results illustrated in Fig. 5A. Hence, the tobacco topoisomerase was unable to cleave D N A packaged into chromatin under these reaction conditions. We next explored the role of the tobacco topoisomerase in the formation of stable nucleosomes on a circular D N A template. Radioactively la-

274

Fig. 7. Activity of topoisomerase I on chromatin templates. A. Reaction mixtures (20 #1) containing 100 ngofapreformed and purified minichromosome template (see Materials and methods), 10 units oftopoisomerase, 50 #M camptothecin and 5 mM MgC12 were incubated for the indicated times at 31 °C. The reactions were terminated by addition of SDS and deproteinized by treatment with Proteinase K (10 mg/ml). DNA topology was analyzed as described in Materials and methods. SC, superhelical DNA; OC, open circular DNA; CC, covalently closed DNA; C, no topoisomerase I added; o, topoisomerase I added concurrent with SDS at time zero. B. Reaction mixtures (20 #1) containing 1 ng of assembled nucleosomal templates (weight ratio 0.7:1) were incubated with 5, 10, 15, 20 units of tobacco cell topoisomerase I. Some of the reaction mixtures (lanes 1, 2, 3, 4) were incubated with histone H 1 prior to the addition of the topoisomerase. Incubation was for 4 h at 31 °C after which the samples were deproteinized by addition of SDS and proteinase K. DNA topology was analyzed by agarose gel eletrophoresis followed by autoradiography. Lanes 1, 2, 3, 4 contain H1 and 5, 10, 15 or 20 units oftopoisomerase I respectively. Lanes 5, 6, 7 and 8 contain no H1 and 5, 10, 15, 20 units of topoisomerase I. C, control, no topoisomerase I added. SC, supercoiled DNA; R, relaxed DNA.

beled circular DNA templates were incubated with purified core histones following the protocol of Morse [ 18]. Nucleosomes were tamed by using the reverse dialysis of salt concentrations with a final concentration of 50 mM KC1. To one set of tubes, purified histone H 1 from calf thymus (gift of S. Leuba, University of Oregon) was incubated with a [32p]-labeled relaxed D N A template (Fig. 7B, lane C) at a molecular weight ratio of 10. Then, increasing amounts (units) of the tobacco topoisomerase I were added and the reaction allowed to proceed for 4 h (Lanes 1-4). After deproteinization, the D N A topology was analyzed by agarose gel electrophoresis and autoradiography. The results, depicted in Fig. 7B, illustrate that the tobacco topoisomerase catalyzes the formation of stable nucleosomes in both the absence and presence of histone H1. For example, the addition of 20 units of topoisomerase catalyzes the formation of fully supercoiled D N A (lane 8). The existence of supercoiled DNA, after deproteinization, reveals that the circular DNA was in fact, assembled into a nucleosomal template. The addition of histone H 1 to the assembled template however, reduces the extent of stable nucleosomal structures after the addition of topoisomerase I. The influence of histone H1 on the assembly process is currently under further investigation in our laboratory.

Discussion

DNA topology is an important parameter surrounding many metabolic reactions that occur along the chromosome. The enzymes that control the degree of superhelicity, present at any time in the life of the cell, are known as DNA topoisomerases. Although a wealth of information has accumulated regarding the behavior of these enzymes in animal cells [27] relatively little information has been gathered describing the activity of these enzymes in plant cells. Previously, we reported the purification of a type I topoisomerase from tobacco cells and in the present manuscript we describe its biochemical characterization.

275 An intriguing characteristic of the tobacco cell topoisomerase is the apparent molecular weight of 30 kDa as judged by SDS-PAGE. Since we have been able to extract a slice from the gel at the 30 kDa position and recover D N A relaxation activity, we are confident that this protein band has biochemical traits consistent with those of D N A topoisomerases. It is, however, somewhat puzzling that in glycerol gradient and gel filtration analysis some D N A relaxation activity is found in fractions corresponding to a molecular weight of ca. 70 kDa. This data suggests that the protein may be, in native state, a dimer of two 30 kDa subunits, but such a supposition is hard to reconcile with previous studies demonstrating that single subunits of eukaryotic topoisomerase I usually have a molecular weight of 70-100 kDa [27]. There are some major exceptions to this rule. First, the Vaccinia virus topoisomerase I has a molecular weight of 32 kDa [24, 25]. Second, Rowe et al. [20] observe a similar type of behavior with the Ustilago topoisomerase; a series of proteins (peptides) eminate from a sizing column. These investigators suggest that the multitude of protein bands of low molecular weight present in their purified protein sample are the result of proteolytic breakdown. This is consistent with the suggestions of Lui and Miller [16] that topoisomerases are particularly labile to proteases. For the present study, it is possible that the 30 kDa band is proteolytic fragment of the native topoisomerase. We have attempted unsuccessfully to cleave the isolated 70 kDa protein population with a variety of endopeptides in the hope of generating the 30 kDa fragment. Hence, the true molecular weight of the tobacco topoisomerase, in native form, must await the cloning of the gene currently underway in our lab. Biochemically, D N A relaxation, catalyzed by the tobacco topoisomerase, is enhanced by MgC12 and less so by KC1 unless a large excess oftopoisomerase I is used. DNA relaxation by other eukaryotic topoisomerases is less dependent on divalent cations and is active up to approximately 200 mM monovalent cation [4]. This property difference could be a reflection of an altered mechanism of relaxation or a reflection of enzyme

stability. In the presence of camptothecin, D N A breakage is enhanced by MgC12 while there is little DNA nicking in the presence of KC1. The addition of KC1 to a reaction mixture containing camptothecin, topoisomerase I and MgCI2 reduced significantly the amount of nicked DNA product. Those results suggest that KC1 disrupts the stability of protein-DNA interaction, similar to the results reported by Hsiang et al. [12]. In agreement with the elegant studies of Aronoff and Champoux [ 1], we believe that MgC12 drives the tobacco enzyme to behave in a processive fashion while KC1 induces a distributive behavior. Since topoisomerases are an integral component of the chromatin assembly pathway, we carried out several experiments addressing possible roles for topoisomerase I in this process. It had previously been reported that phosphorylation of the enzyme changed its behavior [13]. The tobacco enzyme is marginally affected by phosphorylation or dephosphorylation with regard to DNA relaxation activity. Although we have been careful in optimizing the phosphorylation conditions, it is always possible that technical shortcomings may contribute to these types of negative conclusions. The enzyme is not stimulated by histones H1 and H5. This may not be surprising since these histones are not part of the initial nucleosome assembly pathway. The core histones, however, were also not stimulatory. The ability of the enzyme to relax positively supercoiled DNA however is consistent with its potential participation in the nucleosome assembly process. When a stable nucleosome is formed, the D N A responds by coiling in a positive fashion. As demonstrated in Fig. 7B, the tobacco topoisomerase catalyzes the formation of a stable nucleosomal structure through the relaxation of these positive supercoils. After deproteinization a negatively supercoiled molecule is generated only if the topoisomerase I has acted upon the nucleosomal template. Our long-term goal is to reconstitute native plant chromatin using a minichromosome model and decipher the positioning of nucleosomes. The demonstration that the isolated topoisomerase I participates in this process facilitates future studies.

276

Acknowledgements We are grateful to the members of the Kmiec laboratory for helpful discussions and to the Council for Tobacco Research (2821) for support.

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