Plant MolecularBiology6: 137-144, 1986 © 1986 Martinus NijhoffPublishers, Dordrecht - Printed in the Netherlands
Isolation and characterization of DNA topoisomerase II from cauliflower inflorescences Hideki Fukata, Kazue Ohgami & Hirosuke Fukasawa
Biochemical Laboratory, Kobe Women's University, Suma-ku, Kobe 654, Japan
Keywords: cauliflower, DNA catenation, DNA cleavage, DNA supercoil, DNA topoisomerase
Summary Type II DNA topoisomerase has been isolated from inflorescences of cauliflower (Brassica oleracea var. botrytis) through a sequence of polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex, hydroxyapatite and phosphocellulose. The molecular weight of the native enzyme, based on sedimentation coefficient (9S) and gel filtration analysis (Stokes radius, 60 A), was estimated to be 223000. This enzyme was able to catalyze fully the relaxation of supercoiled DNA by breaking and then rejoining the double-stranded DNA. The breaking reaction was reversible by a change in salt concentrations. When an antitumor drug, 4'-(9-acridinylamino)-methanesulfon-m-anisidide, was added to the topoisomerase reaction, DNA cleavage fragments were accumulated; and this suggested that the drug interfered with the reaction at the rejoining step. This enzyme also catalyzed the formation of DNA catenanes in the presence of 8°7o polyethylene glycol or histone H1, while few catenanes were formed in the presence of spermidine, which was highly effective on a bacterial enzyme.
plants, there is no information so far on type II topoisomerase other than in cauliflower, although an ATP-dependent topoisomerase from a lower eukaryotic unicellular algae, Chlamydomonas reinhardii, has been recently reported (36), and the presence of type I enzyme has been described in wheat germ (4) and in spinach chloroplasts (32). The inflorescence of cauliflower exhibits hypertrophic differentiation with rapid growth and frequent mitotic divisions. The apical portion of the inflorescence consists of small undifferentiated cells with much higher DNA content than the cells in the underlying axial tissues (5). It is interesting to investigate the presence or absence of interrelationships between the rapid nuclear divisions and the DNA topoisomerase II activity in the hypertrophic tissues. In previous papers, we reported on the isolation and partial characterization of two distinct DNA topoisomerases from cauliflower inflorescences (6-8). Here, we further characterized DNA topoisomerase II from cauliflower, especially
Introduction DNA topoisomerases, the nomenclature coined by Wang & Liu (38), are enzymes able to interconvert various topological forms of DNA without altering the primary structure of DNA. Topoisomerases have been found in both pr~karyotic and eukaryotic cells, and mechanistically, these enzymes fall into two classes (2, 20). The type I enzyme are able to relieve torsional constraints in DNA by breaking and rejoining one strand of the helix, changing the linking number by one in each step. The type II topoisomerases break and rejoin both strands of duplex DNA, changing the linking number by two in each steps (12, 15, 20, 25, 29). The importance of type II topoisomerases in the process and mechanism of DNA replication, recombination and transcription in bacteria is evident (9, 14, 16, 38). It is suggested that type II topoisomerases in mammalian cells are part of the DNA replication machinery (23, 27). In higher 137
138 with respect to the analysis of reaction products, size of the native enzyme, transient breakage of DNA in relation to an antitumor drug, and catenation of DNA by this enzyme.
Materials and methods
Materials Fresh cauliflower heads, weighing 350-500 g, were purchased from a local market. The apical portion of the head were dissected with a shears into small pieces (about 2 mm in thickness from the surface), and these were stored at -70 °C until use. The following chemicals were purchased: CMSephadex (C-25) from Pharmacia Fine Chemicals; hydroxyapatite from Japan Seikagaku Kogyo Co.; Phosphocellulose P-11 from Whatman Co.; agarose powder (H-14) and T4 DNA ligase from Takara Shuzo Co.; Eco RI (restriction endonuclease) from Nippon Gene Co.; Toyopearl HW-60S (Fractogel) from Toyo-Soda Kogyo Co.; Deoxyribonuclease I and chloroquine from Sigma Co.; 4'-(9-acridinyamino)-methanesulfon-m-anisidide (mAMSA) from National Cancer Institute, USA; and ethidium bromide from Nakarai Chemicals, Japan. Supercoiled pBR322 DNA was isolated from Escherichia coli (HB 101) and was purified by centrifugation to the equilibrium in two successive CsCl-ethidium bromide gradients. Histone H1 was repurified from calf thymus fraction 1 histone (Sigma Co. Type V-S) by gel filtration on a column (130xl.5 cm) of Toyopearl HW-50F in 0.01 N HC1 saturated with chloroform. Histone H1 peak was monitored by the absorbance at 230 nm and by gel electrophoresis, and the pooled histone H1 was collected by centrifugation after precipitation with 6 volumes of acetone.
Preparation of enzyme fractions Frozen cauliflower tissues (300 g) were homogenized with 600 ml of TEM-buffer (50 mM TrisHCI at pH 8.0, 1 mM EDTA, 5 mM mercaptoethanol). Nucleic acids were precipitated with 10% polyethylene glycol-6000 (PEG) containing 2 M NaCI and removed by centrifugation at 12000×g for 20 min. The supernatant was treated with 0.224 g/ml of ammonium sulfate, and a proteincake accumulated in the middle phase between
PEG and ammonium sulfate phases was centrifuged at 12000xg for 15 min. The precipitate was dissolved in 1000 ml of TGD-buffer (50 mM TrisHCI at pH 8.0, 20% glycerol, and 0.5 mM dithiothreitol), and the solution was centrifuged at 15000×g for 20 min. The clear supernatant was loaded on a CM-Sephadex column (2 x 35 cm) previously equilibrated with TGD-buffer. The enzyme was eluted with a 500 ml of a linear gradient of 0 to 1.8 M KC1 in the buffer.
Assay for DNA topoisomerase activity The activity was assayed based on the relaxation of negatively supercoiled pBR322 DNA and monitored by agarose gel electrophoresis. The standard reaction mixture (25 #1) was composed of 50 mM Tris-HC1 at pH 7.5, 10 mM MgC12, 0.5 mM ATP, 1 mM dithiothreitol, 160/~g/ml bovine serum albumin, 0.5/~g pBR322 DNA, and 5/~1 enzyme solution. Incubation was done at 35 °C for 60 min and were stopped by adding 2/A of 13.5% sodium dodecyl sulfate (SDS) containing 10% glycerol and 0.5 mg/ml of bromophenol blue. Agarose gel electrophoresis and photography were done as described previously (6). One unit of topoisomerase activity is defined as the amount of enzyme required to fully relax 0.5/~g of supercoiled pBR322 DNA in 60 min at 35 °C.
Two dimensional gel electrophoresis Reaction product DNAs were subjected to agarose gel electrophoresis in the absence of chloroquine in the first dimension. The gel strip was cut from the gel and soaked for 30 min in TBE-buffer (89 mM Tris base, 89 mM borate, 2.5 mM EDTA) containing 0.6 #g/ml chloroquine. The gel slice was then attached precisely to a gel containing the same buffer and was subjected to electrophoresis in the second dimension.
Preparation of fully-relaxed DNA Nicked DNA was prepared by a treatment with DNase I according to the procedures of Hsieh & Wang (13). Ligation was carried out with T4 ligase under the same conditions for the relaxation assay of topoisomerase, and the DNA was purified by phenol extraction and ethanol precipitation.
139
Results and discussion
1. Separation of three different topoisomerase fractions F r a c t i o n s eluted f r o m C M - S e p h a d e x c o l u m n were assayed by the r e a c t i o n mixture with or witho u t A T P (Fig. 1). W h e n A T P was a b s e n t in the r e a c t i o n mixture, two distinct fractions o f D N A relaxing activity were recognized, one eluting at 0 . 3 - 0 . 6 M KCI a n d o t h e r at 0 . 8 - 1 . 0 M KC1. W h e n A T P was present, a n o t h e r activity fraction eluting at 0 . 1 - 0 . 2 M KC1 was o b s e r v e d a l o n g with the
s a m e activity fractions which were o b s e r v e d in the absence o f A T E T h e early eluting fraction was d e s i g n a t e d as A T P - d e p e n d e n t t o p o i s o m e r a s e , which was shown to be type II t o p o i s o m e r a s e in a previous r e p o r t (6). T h e late eluting fractions are designated as t o p o i s o m e r a s e Ia a n d Ib. The t o p o i s o m e r a s e II activity fractions taken f r o m C M - S e p h a d e x c o l u m n were l o a d e d on a hyd r o x y a p a t i t e c o l u m n a n d eluted with a linear gradient o f 0 . 1 - 0 . 7 5 M p o t a s s i u m p h o s p h a t e in 20°7o glycerol a n d 0.5 m M dithiothreitol. T h e fractions with an activity were a p p l i e d to a p h o s p h o c e l l u l o s e c o l u m n a n d were eluted with a linear gradient o f
Fig. 1. CM-Sephadex column chromatography of DNA-relaxing activity from cauliflower inflorescences. The extract from 300 g of cauliflower tissues was chromatographed on CM-Sephadex column. For each of the l0 ml-fractions, DNA topoisomerase activity was assayed as described in the text, except that 2/A of enzyme solution was used. (A): Assay for topoisomerase in the absence of ATE (B): Similar assay in the presence of ATE The positions of initial supercoiled plasmid material (I) and the relaxed form (II) are indicated at the left. (C): Absorbance at 280 nm and KCl concentrations in various fractions. Fractions with topoisomerase activity (shown with I I) were pooled, and were subsequently subjected to further chromatography.
140 0.1-0.9 M KC1 in the TGD-buffer. The active fractions were pooled and dialyzed against a buffer (50 mM Tris-HC1 at pH 8.0, 1 mM EDTA, 0.5 mM dithiothreitol and 50% glycerol), and these fractions were stored at - 2 0 ° C . The specific activity of the enzyme used in the present study was 27000 units/mg protein in the standard reaction mixture and 135000 units/mg protein in the optimum condition (exclusively for relaxation) in the presence of 60 mM KCI and 5 mM MgCI2 (8).
2. Analysis of products of topoisomerase H reaction Cauliflower topoisomerase II converts supercoiled pBR322 DNA to a relaxed form of low electrophoretic mobility. As described in a previous paper, the enzyme simultaneously catalyzes the breaking and rejoining of duplex DNA, and consequently, the linking number of DNA nucleotides in a molecule changes by two in a step, from n to n + 2, n + 4 and so on (7). No relaxation is detected when ATP and/or MgC12 is absent from the reaction mixture. It seems that the products of topoisomerase II reaction, as appears in Fig. 1B, are electrophoretically not fully relaxed. Furthermore, it is not possible to determine whether the DNA which is not fully relaxed has positive or negative supercoils in this gel system. Therefore, the products of topoisomerase II reaction were compared with the fullyrelaxed DNA (prepared by DNase I and ligase), using two-dimensional gel electrophoresis (18) (Fig. 2). The DNA was subjected to gel electrophoresis in the first (vertical) dimension without chloroquine in the buffer, followed with the second electrophoresis at horizontal dimension with chloroquine in the buffer. During the second electrophoresis, the originally positive superhelical DNA in the first dimension binds with chloroquine and gains positive superhelicity so that it migrates faster, while the originally negative superhelical DNA loses negative superhelicity and migrates slower. As shown in Fig. 2, the reaction products by topoisomerase II and fully-relaxed DNA (ligasejoined DNA) showed similar electrophoretic patterns. And the DNA bands which migrated relatively fast in the first dimension migrated faster in the second dimension. Therefore, it is considered that the former was fully relaxed in the reaction mixture
Fig. 2. Two dimensional gel electrophoresis of fully-relaxed DNA (A) and the reaction products catalyzed by cauliflower DNA topoisomeraseII (B). The DNA was subjectedto gel electrophoresis in the absence of chloroquine in the first (vertical) dimension (left panel). A gel strip was cut out and soaked in TBE-buffercontaining 0.6 ~tg/ml of chloroquine, and was subjected to electrophoresisin the second (horizontal)dimension in the same buffer for 150 min at 80 V (right panel). I: Relaxed form, but positively supercoiled in the chloroquine gel. II: Nicked form, mobility of this form is not affected with chloroquine.
like the latter, but these were positively supercoiled in the gel without chloroquine.
3. Molecular weight of cauliflower topoisomerase H native system The native molecular weight of cauliflower topoisomerase II was estimated by combining the results of sedimentation studies and gel filtration chromatography, according to the method of Siegel & Monty (33). The enzyme activity sedimented as a single peak with a sedimentation coefficient of approximately 9.0S in glycerol gradient containing 0.2 M KC1 (Fig. 3A). The Stokes radius of the enzyme analyzed by a gel filtration column, which had been calibrated with various proteins with known stokes radii (Fig. 3B), was 60 A. Taken
141
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Fig. 3. Sedimentation coefficient and Stokes radius of topoisomerase II. (A): The enzyme was applied to 20-40% glycerol gradient in the buffer (50 mM Tris-HC1at pH 8.0, 1 mM EDTA, 0.5 mM dithiothreitol), and was centrifuged at 2°C for 6 h at 45000 rpm (194000×g) in a Hitachi vertical RPV 65T rotor. As marker proleins, catalase (CAT, 11.3S), alcohol dehydrogenase (ADH, 7.4S), and bovine serum albumin (BSA, 4.3S) were added to the sample. The arrow indicates the position of cauliflower topoisomerase II activity. (B): The enzymewas applied to Toyopearl HW-60S(2.6×60 cm) and was eluted by YGD-buffer with 0.2 M KCI.As marker proteins, myoglobin(MYO, 11.5/~), bovine serum albumin (BSA, 35.5 A), ferritin (FER, 61.0 ~.), and thyroglobin (TG, 85.0/k) were used.
together, the sedimentation and gel filtration results can be used to calculated a molecular weight of approximately 223000. This value is smaller than 260000 o f Drosophila melanogaster topoisomerase II (30) and 320000 of H e L a cell topoisomerase II (24), but rather resembles the 220000 of E. coli gyrase (11).
4. Double-stranded D N A cleavage by topoisomerase H and the effect of the antitumor drug, m-AMSA E. coli gyrase can cleave double-stranded D N A when a inhibitor oxolinic acid is included in the reaction mixture (35). In eukaryotic D N A topoisomerase II, it is known that double-stranded cleavage fragments are produced as an intermediate at the early stage of the enzyme reaction (19, 29).
At high concentrations o f cauliflower topoisomerase II, D N A was cleaved and found mostly in linear form, occurring when the reaction was stopped within 10 min by the addition of 1°70 SDS (Fig. 4A) to make immediately inactive the enzyme activity. The appearance of linear forms suggested that the D N A cleavage by topoisomerase II was the result of SDS treatment which trapped a putative intermediate of the enzyme reaction. However, addition of high concentration o f NaC1 (0.1-0.2 M) to the reaction mixture after 10 min-incubation resulted in rapid conversion of the linear form DNA to supercoiled form (Fig. 4A). The fact that linear D N A molecules were converted to supercoiled form but not to relaxed form further suggested that the broken D N A ends were held tightly together by the enzyme (putative DNA-enzyme complex) without untwisting. Furthermore, it was found in these experiments that ATP was not required for the above cleavage reaction by the enzyme and also for the reversal reaction as type I enzyme. These suggested that ATP is required for this enzyme to pass double strands changing the linking numbers. An antitumor drug, m-AMSA, stimulates the formation o f a topoisomerase I I - D N A complex and double-stranded D N A breaks are produced (26). Addition of m-AMSA to the cauliflower topoisomerase reaction mixture resulted in the stimulation of D N A cleavage. Figure 4B shows the results of treating supercoiled plasmid pBR322 D N A with topoisomerase II in the presence of various concentrations m-AMSA. As the concentration of the drug increases, there is greater conversion of supercoiled form to linear form. Treatment of the reaction mixture with high concentration of salt after the first incubation, joins most of the cleavage products in supercoiled form without ATP (data not shown). These results suggest that m-AMSA inhibits the D N A rejoining step of enzyme-DNA complex at the DNA-relaxing reaction (cleavage and rejoining), similar to that for H e L a cell topoisomerase II postulated by Nelson et al. (26). When the topoisomerase II-cleavaged D N A was digested with Eco RI, several bands were observed (Fig. 4C), suggesting that topoisomerase II can introduce double-stranded D N A breakage at many specific sites.
142
Fig. 4. Cleavage of a duplex DNA by cauliflower topoisomerase II. (A): Topoisomerase II was incubated in the standard reaction mixture at 35 °C for 10 rain in the absence of m-AMSA. After a 10 min-incubation, various concentrations of NaCI were added to the mixture and the incubation was continued for 10 rain. Lane 1; the control without enzyme. Lane 2; the reaction was stopped with 1°70SDS and then treated with 0.1 mg/ml of proteinase K. Final concentrations of samples in lane 3 to 7 were 0, 25, 50, 100 and 200 mM, respectively. (B): Effects of concentration of m-AMSA on the topoisomerase II reaction, m-AMSA concentrations of the sample in lane 1 to 7 were 0, 5, 10, 20, 50, 100 and 200 #g/ml, respectively. (C): Eco Rl-digestion of the reaction product in the presence of m-AMSA. Lane 1; the control without enzyme. Lane 2; linear form (unit length pBR322) from Eco RI-digestion. Lane 3; topoisomerase IIreaction product obtained in the presence of m-AMSA digested with Eco RI. I, II and III indicated at left and right show supercoiled, nicked and linear forms, respectively.
5. Catenation and decatenation o f D N A rings by topoisomerase H D N A t o p o i s o m e r a s e catalyzes the f o r m a t i o n o f catenanes in the presence o f D N A - c o n d e n s i n g or crowding agents such as p o l y v a l e n t cations (spermidine etc.), hydrophilic p o l y m e r (polyethylene glycol etc.) o r D N A c o n d e n s i n g proteins (histone H1 etc.) (1, 15, 17, 21, 28, 30). We tested w h e t h e r or n o t c a t e n a t i o n occurs by cauliflower t o p o i s o m e r a s e II when these agents are present. E x p e r i m e n t s with s p e r m i d i n e resulted in little c a t e n a t i o n even at 5 - 6 m M which is also the o p t i m u m c o n c e n t r a t i o n for a bacterial e n z y m e (15). E x p e r i m e n t s with polyethylene glycol-6000, as seen in Fig. 5A, showed a r e m a r k a b l e c a t e n a t i o n in the s t a n d a r d r e a c t i o n mixture, except t h a t the c o n c e n t r a t i o n o f D N A was 2.5/zg a n d c o n t a i n i n g 8°70 P E G . C a t e n a n e s thus f o r m e d were n o t simple aggregates with the enzyme
protein, because the D N A catenanes r e m a i n e d after p h e n o l t r e a t m e n t (Fig. 5B-2). A l s o Eco RI-treatm e n t p r o d u c e d linear u n i f o r m D N A s f r o m the catenanes (Fig. 5B-3). C a u l i f l o w e r t o p o i s o m e r ase II requires A T P a n d MgC12 for c a t e n a t i o n as well as for relaxation. W h e t h e r the e n z y m e catalyzes r e l a x a t i o n o r c a t e n a t i o n d e p e n d s on P E G a n d the c o n c e n t r a t i o n o f MgC12. N o activity was observed w i t h o u t MgC12, a n d c a t e n a t i o n a n d a little relaxation activity were f o u n d at 5 m M MgCI2, while o n l y c a t e n a t i o n activity was f o u n d over 10 m M M g C I 2 ( o p t i m u m 10 raM). E x p e r i m e n t s with histone H1 showed t h a t the f o r m a t i o n o f D N A catenanes occurs in the r e a c t i o n mixture w i t h o u t P E G c o n t a i n i n g histone in a weight ratio to D N A a p p r o x i m a t e l y at 0.1 ( d a t a n o t shown). D N A d e c a t e n a t i o n activities have been r e p o r t e d in various systems including E. coli gyrase, t y p e II t o p o i s o m e r a s e s from T4 infected ceils, f r o m Droso-
143
Fig. 5. Catenation and decatenation of pBR322 D N A by cauliflower D N A topoisomerase II. (A): 60 units enzyme and 2.5 ~g of D N A were incubated in the standard reaction mixture in the presence of 8°70 PEG. Lane 1 to 5; products obtained from the incubation of 0, 15, 30, 45 and 60 min, respectively. (B): 15 units enzyme and 2.5 p.g of D N A were incubated in the same condition as A. Lane 1; reaction products obtained from 60 rain-incubation. Lane 2; the same reaction products after phenol treatment. Lane 3; reaction products after Eco RI-digestion. (C): Decatenation of D N A obtained from the above reactions. Lane 1; D N A catenanes obtained from the lane 2 of B. Lane 2; reaction products obtained from catenanes incubated with topoisomerase II for 60 min in the same reaction mixture without PEG. I, II, III, IV and V indicated at left show supercoiled form, nicked form, linear form, small catenanes (dimer and trimer) and large catenanes, respectively.
phila melanogaster, from Xenopus laevis, and from yeast (1, 10, 12, 22, 37). Our experiments on the cauliflower topoisomerase II showed that the enzyme was also capable of decatenating DNA in the same reaction mixture required for catenation but without DNA-condensing or crowding agents (Fig. 5C). Type II topoisomerase (gyrase) from E. coli is one of the essential enzymes for the initiation of replication at the replication origin of the chromosome (14), and also it participates in the separation of bacterial genomes (34). Topoisomerase II of Saccharomyces cerevisiae is involved in DNA replication or in the separation of daughter chromosomes at the termination of DNA replication (3). Chromosome replication accompanying the condensation and decondensation of chromosomes in the rapidly growing tissues of cauliflower is considered to be intimately regulated by topoisomerase II. Further comparative studies with axial tissues, not so rapidly growing portion of the cauliflower head, may provide additional information on the DNA metabolic events involved in hypertrophic differentiation.
Acknowledgements This research was supported in part by grants from the Ministry of Education, Science and Culture of Japan and from the Research Council, Ministry of Agriculture, Forestry and Fisheries Japan, original and creative research project on biotechnology.
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Isolation and characterization of DNA topoisomerase II from cauliflower inflorescences Hideki Fukata, Kazue Ohgami & Hirosuke Fukasawa
Biochemical Laboratory, Kobe Women's University, Suma-ku, Kobe 654, Japan
Keywords: cauliflower, DNA catenation, DNA cleavage, DNA supercoil, DNA topoisomerase
Summary Type II DNA topoisomerase has been isolated from inflorescences of cauliflower (Brassica oleracea var. botrytis) through a sequence of polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex, hydroxyapatite and phosphocellulose. The molecular weight of the native enzyme, based on sedimentation coefficient (9S) and gel filtration analysis (Stokes radius, 60 A), was estimated to be 223000. This enzyme was able to catalyze fully the relaxation of supercoiled DNA by breaking and then rejoining the double-stranded DNA. The breaking reaction was reversible by a change in salt concentrations. When an antitumor drug, 4'-(9-acridinylamino)-methanesulfon-m-anisidide, was added to the topoisomerase reaction, DNA cleavage fragments were accumulated; and this suggested that the drug interfered with the reaction at the rejoining step. This enzyme also catalyzed the formation of DNA catenanes in the presence of 8°7o polyethylene glycol or histone H1, while few catenanes were formed in the presence of spermidine, which was highly effective on a bacterial enzyme.
plants, there is no information so far on type II topoisomerase other than in cauliflower, although an ATP-dependent topoisomerase from a lower eukaryotic unicellular algae, Chlamydomonas reinhardii, has been recently reported (36), and the presence of type I enzyme has been described in wheat germ (4) and in spinach chloroplasts (32). The inflorescence of cauliflower exhibits hypertrophic differentiation with rapid growth and frequent mitotic divisions. The apical portion of the inflorescence consists of small undifferentiated cells with much higher DNA content than the cells in the underlying axial tissues (5). It is interesting to investigate the presence or absence of interrelationships between the rapid nuclear divisions and the DNA topoisomerase II activity in the hypertrophic tissues. In previous papers, we reported on the isolation and partial characterization of two distinct DNA topoisomerases from cauliflower inflorescences (6-8). Here, we further characterized DNA topoisomerase II from cauliflower, especially
Introduction DNA topoisomerases, the nomenclature coined by Wang & Liu (38), are enzymes able to interconvert various topological forms of DNA without altering the primary structure of DNA. Topoisomerases have been found in both pr~karyotic and eukaryotic cells, and mechanistically, these enzymes fall into two classes (2, 20). The type I enzyme are able to relieve torsional constraints in DNA by breaking and rejoining one strand of the helix, changing the linking number by one in each step. The type II topoisomerases break and rejoin both strands of duplex DNA, changing the linking number by two in each steps (12, 15, 20, 25, 29). The importance of type II topoisomerases in the process and mechanism of DNA replication, recombination and transcription in bacteria is evident (9, 14, 16, 38). It is suggested that type II topoisomerases in mammalian cells are part of the DNA replication machinery (23, 27). In higher 137
138 with respect to the analysis of reaction products, size of the native enzyme, transient breakage of DNA in relation to an antitumor drug, and catenation of DNA by this enzyme.
Materials and methods
Materials Fresh cauliflower heads, weighing 350-500 g, were purchased from a local market. The apical portion of the head were dissected with a shears into small pieces (about 2 mm in thickness from the surface), and these were stored at -70 °C until use. The following chemicals were purchased: CMSephadex (C-25) from Pharmacia Fine Chemicals; hydroxyapatite from Japan Seikagaku Kogyo Co.; Phosphocellulose P-11 from Whatman Co.; agarose powder (H-14) and T4 DNA ligase from Takara Shuzo Co.; Eco RI (restriction endonuclease) from Nippon Gene Co.; Toyopearl HW-60S (Fractogel) from Toyo-Soda Kogyo Co.; Deoxyribonuclease I and chloroquine from Sigma Co.; 4'-(9-acridinyamino)-methanesulfon-m-anisidide (mAMSA) from National Cancer Institute, USA; and ethidium bromide from Nakarai Chemicals, Japan. Supercoiled pBR322 DNA was isolated from Escherichia coli (HB 101) and was purified by centrifugation to the equilibrium in two successive CsCl-ethidium bromide gradients. Histone H1 was repurified from calf thymus fraction 1 histone (Sigma Co. Type V-S) by gel filtration on a column (130xl.5 cm) of Toyopearl HW-50F in 0.01 N HC1 saturated with chloroform. Histone H1 peak was monitored by the absorbance at 230 nm and by gel electrophoresis, and the pooled histone H1 was collected by centrifugation after precipitation with 6 volumes of acetone.
Preparation of enzyme fractions Frozen cauliflower tissues (300 g) were homogenized with 600 ml of TEM-buffer (50 mM TrisHCI at pH 8.0, 1 mM EDTA, 5 mM mercaptoethanol). Nucleic acids were precipitated with 10% polyethylene glycol-6000 (PEG) containing 2 M NaCI and removed by centrifugation at 12000×g for 20 min. The supernatant was treated with 0.224 g/ml of ammonium sulfate, and a proteincake accumulated in the middle phase between
PEG and ammonium sulfate phases was centrifuged at 12000xg for 15 min. The precipitate was dissolved in 1000 ml of TGD-buffer (50 mM TrisHCI at pH 8.0, 20% glycerol, and 0.5 mM dithiothreitol), and the solution was centrifuged at 15000×g for 20 min. The clear supernatant was loaded on a CM-Sephadex column (2 x 35 cm) previously equilibrated with TGD-buffer. The enzyme was eluted with a 500 ml of a linear gradient of 0 to 1.8 M KC1 in the buffer.
Assay for DNA topoisomerase activity The activity was assayed based on the relaxation of negatively supercoiled pBR322 DNA and monitored by agarose gel electrophoresis. The standard reaction mixture (25 #1) was composed of 50 mM Tris-HC1 at pH 7.5, 10 mM MgC12, 0.5 mM ATP, 1 mM dithiothreitol, 160/~g/ml bovine serum albumin, 0.5/~g pBR322 DNA, and 5/~1 enzyme solution. Incubation was done at 35 °C for 60 min and were stopped by adding 2/A of 13.5% sodium dodecyl sulfate (SDS) containing 10% glycerol and 0.5 mg/ml of bromophenol blue. Agarose gel electrophoresis and photography were done as described previously (6). One unit of topoisomerase activity is defined as the amount of enzyme required to fully relax 0.5/~g of supercoiled pBR322 DNA in 60 min at 35 °C.
Two dimensional gel electrophoresis Reaction product DNAs were subjected to agarose gel electrophoresis in the absence of chloroquine in the first dimension. The gel strip was cut from the gel and soaked for 30 min in TBE-buffer (89 mM Tris base, 89 mM borate, 2.5 mM EDTA) containing 0.6 #g/ml chloroquine. The gel slice was then attached precisely to a gel containing the same buffer and was subjected to electrophoresis in the second dimension.
Preparation of fully-relaxed DNA Nicked DNA was prepared by a treatment with DNase I according to the procedures of Hsieh & Wang (13). Ligation was carried out with T4 ligase under the same conditions for the relaxation assay of topoisomerase, and the DNA was purified by phenol extraction and ethanol precipitation.
139
Results and discussion
1. Separation of three different topoisomerase fractions F r a c t i o n s eluted f r o m C M - S e p h a d e x c o l u m n were assayed by the r e a c t i o n mixture with or witho u t A T P (Fig. 1). W h e n A T P was a b s e n t in the r e a c t i o n mixture, two distinct fractions o f D N A relaxing activity were recognized, one eluting at 0 . 3 - 0 . 6 M KCI a n d o t h e r at 0 . 8 - 1 . 0 M KC1. W h e n A T P was present, a n o t h e r activity fraction eluting at 0 . 1 - 0 . 2 M KC1 was o b s e r v e d a l o n g with the
s a m e activity fractions which were o b s e r v e d in the absence o f A T E T h e early eluting fraction was d e s i g n a t e d as A T P - d e p e n d e n t t o p o i s o m e r a s e , which was shown to be type II t o p o i s o m e r a s e in a previous r e p o r t (6). T h e late eluting fractions are designated as t o p o i s o m e r a s e Ia a n d Ib. The t o p o i s o m e r a s e II activity fractions taken f r o m C M - S e p h a d e x c o l u m n were l o a d e d on a hyd r o x y a p a t i t e c o l u m n a n d eluted with a linear gradient o f 0 . 1 - 0 . 7 5 M p o t a s s i u m p h o s p h a t e in 20°7o glycerol a n d 0.5 m M dithiothreitol. T h e fractions with an activity were a p p l i e d to a p h o s p h o c e l l u l o s e c o l u m n a n d were eluted with a linear gradient o f
Fig. 1. CM-Sephadex column chromatography of DNA-relaxing activity from cauliflower inflorescences. The extract from 300 g of cauliflower tissues was chromatographed on CM-Sephadex column. For each of the l0 ml-fractions, DNA topoisomerase activity was assayed as described in the text, except that 2/A of enzyme solution was used. (A): Assay for topoisomerase in the absence of ATE (B): Similar assay in the presence of ATE The positions of initial supercoiled plasmid material (I) and the relaxed form (II) are indicated at the left. (C): Absorbance at 280 nm and KCl concentrations in various fractions. Fractions with topoisomerase activity (shown with I I) were pooled, and were subsequently subjected to further chromatography.
140 0.1-0.9 M KC1 in the TGD-buffer. The active fractions were pooled and dialyzed against a buffer (50 mM Tris-HC1 at pH 8.0, 1 mM EDTA, 0.5 mM dithiothreitol and 50% glycerol), and these fractions were stored at - 2 0 ° C . The specific activity of the enzyme used in the present study was 27000 units/mg protein in the standard reaction mixture and 135000 units/mg protein in the optimum condition (exclusively for relaxation) in the presence of 60 mM KCI and 5 mM MgCI2 (8).
2. Analysis of products of topoisomerase H reaction Cauliflower topoisomerase II converts supercoiled pBR322 DNA to a relaxed form of low electrophoretic mobility. As described in a previous paper, the enzyme simultaneously catalyzes the breaking and rejoining of duplex DNA, and consequently, the linking number of DNA nucleotides in a molecule changes by two in a step, from n to n + 2, n + 4 and so on (7). No relaxation is detected when ATP and/or MgC12 is absent from the reaction mixture. It seems that the products of topoisomerase II reaction, as appears in Fig. 1B, are electrophoretically not fully relaxed. Furthermore, it is not possible to determine whether the DNA which is not fully relaxed has positive or negative supercoils in this gel system. Therefore, the products of topoisomerase II reaction were compared with the fullyrelaxed DNA (prepared by DNase I and ligase), using two-dimensional gel electrophoresis (18) (Fig. 2). The DNA was subjected to gel electrophoresis in the first (vertical) dimension without chloroquine in the buffer, followed with the second electrophoresis at horizontal dimension with chloroquine in the buffer. During the second electrophoresis, the originally positive superhelical DNA in the first dimension binds with chloroquine and gains positive superhelicity so that it migrates faster, while the originally negative superhelical DNA loses negative superhelicity and migrates slower. As shown in Fig. 2, the reaction products by topoisomerase II and fully-relaxed DNA (ligasejoined DNA) showed similar electrophoretic patterns. And the DNA bands which migrated relatively fast in the first dimension migrated faster in the second dimension. Therefore, it is considered that the former was fully relaxed in the reaction mixture
Fig. 2. Two dimensional gel electrophoresis of fully-relaxed DNA (A) and the reaction products catalyzed by cauliflower DNA topoisomeraseII (B). The DNA was subjectedto gel electrophoresis in the absence of chloroquine in the first (vertical) dimension (left panel). A gel strip was cut out and soaked in TBE-buffercontaining 0.6 ~tg/ml of chloroquine, and was subjected to electrophoresisin the second (horizontal)dimension in the same buffer for 150 min at 80 V (right panel). I: Relaxed form, but positively supercoiled in the chloroquine gel. II: Nicked form, mobility of this form is not affected with chloroquine.
like the latter, but these were positively supercoiled in the gel without chloroquine.
3. Molecular weight of cauliflower topoisomerase H native system The native molecular weight of cauliflower topoisomerase II was estimated by combining the results of sedimentation studies and gel filtration chromatography, according to the method of Siegel & Monty (33). The enzyme activity sedimented as a single peak with a sedimentation coefficient of approximately 9.0S in glycerol gradient containing 0.2 M KC1 (Fig. 3A). The Stokes radius of the enzyme analyzed by a gel filtration column, which had been calibrated with various proteins with known stokes radii (Fig. 3B), was 60 A. Taken
141
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Fig. 3. Sedimentation coefficient and Stokes radius of topoisomerase II. (A): The enzyme was applied to 20-40% glycerol gradient in the buffer (50 mM Tris-HC1at pH 8.0, 1 mM EDTA, 0.5 mM dithiothreitol), and was centrifuged at 2°C for 6 h at 45000 rpm (194000×g) in a Hitachi vertical RPV 65T rotor. As marker proleins, catalase (CAT, 11.3S), alcohol dehydrogenase (ADH, 7.4S), and bovine serum albumin (BSA, 4.3S) were added to the sample. The arrow indicates the position of cauliflower topoisomerase II activity. (B): The enzymewas applied to Toyopearl HW-60S(2.6×60 cm) and was eluted by YGD-buffer with 0.2 M KCI.As marker proteins, myoglobin(MYO, 11.5/~), bovine serum albumin (BSA, 35.5 A), ferritin (FER, 61.0 ~.), and thyroglobin (TG, 85.0/k) were used.
together, the sedimentation and gel filtration results can be used to calculated a molecular weight of approximately 223000. This value is smaller than 260000 o f Drosophila melanogaster topoisomerase II (30) and 320000 of H e L a cell topoisomerase II (24), but rather resembles the 220000 of E. coli gyrase (11).
4. Double-stranded D N A cleavage by topoisomerase H and the effect of the antitumor drug, m-AMSA E. coli gyrase can cleave double-stranded D N A when a inhibitor oxolinic acid is included in the reaction mixture (35). In eukaryotic D N A topoisomerase II, it is known that double-stranded cleavage fragments are produced as an intermediate at the early stage of the enzyme reaction (19, 29).
At high concentrations o f cauliflower topoisomerase II, D N A was cleaved and found mostly in linear form, occurring when the reaction was stopped within 10 min by the addition of 1°70 SDS (Fig. 4A) to make immediately inactive the enzyme activity. The appearance of linear forms suggested that the D N A cleavage by topoisomerase II was the result of SDS treatment which trapped a putative intermediate of the enzyme reaction. However, addition of high concentration o f NaC1 (0.1-0.2 M) to the reaction mixture after 10 min-incubation resulted in rapid conversion of the linear form DNA to supercoiled form (Fig. 4A). The fact that linear D N A molecules were converted to supercoiled form but not to relaxed form further suggested that the broken D N A ends were held tightly together by the enzyme (putative DNA-enzyme complex) without untwisting. Furthermore, it was found in these experiments that ATP was not required for the above cleavage reaction by the enzyme and also for the reversal reaction as type I enzyme. These suggested that ATP is required for this enzyme to pass double strands changing the linking numbers. An antitumor drug, m-AMSA, stimulates the formation o f a topoisomerase I I - D N A complex and double-stranded D N A breaks are produced (26). Addition of m-AMSA to the cauliflower topoisomerase reaction mixture resulted in the stimulation of D N A cleavage. Figure 4B shows the results of treating supercoiled plasmid pBR322 D N A with topoisomerase II in the presence of various concentrations m-AMSA. As the concentration of the drug increases, there is greater conversion of supercoiled form to linear form. Treatment of the reaction mixture with high concentration of salt after the first incubation, joins most of the cleavage products in supercoiled form without ATP (data not shown). These results suggest that m-AMSA inhibits the D N A rejoining step of enzyme-DNA complex at the DNA-relaxing reaction (cleavage and rejoining), similar to that for H e L a cell topoisomerase II postulated by Nelson et al. (26). When the topoisomerase II-cleavaged D N A was digested with Eco RI, several bands were observed (Fig. 4C), suggesting that topoisomerase II can introduce double-stranded D N A breakage at many specific sites.
142
Fig. 4. Cleavage of a duplex DNA by cauliflower topoisomerase II. (A): Topoisomerase II was incubated in the standard reaction mixture at 35 °C for 10 rain in the absence of m-AMSA. After a 10 min-incubation, various concentrations of NaCI were added to the mixture and the incubation was continued for 10 rain. Lane 1; the control without enzyme. Lane 2; the reaction was stopped with 1°70SDS and then treated with 0.1 mg/ml of proteinase K. Final concentrations of samples in lane 3 to 7 were 0, 25, 50, 100 and 200 mM, respectively. (B): Effects of concentration of m-AMSA on the topoisomerase II reaction, m-AMSA concentrations of the sample in lane 1 to 7 were 0, 5, 10, 20, 50, 100 and 200 #g/ml, respectively. (C): Eco Rl-digestion of the reaction product in the presence of m-AMSA. Lane 1; the control without enzyme. Lane 2; linear form (unit length pBR322) from Eco RI-digestion. Lane 3; topoisomerase IIreaction product obtained in the presence of m-AMSA digested with Eco RI. I, II and III indicated at left and right show supercoiled, nicked and linear forms, respectively.
5. Catenation and decatenation o f D N A rings by topoisomerase H D N A t o p o i s o m e r a s e catalyzes the f o r m a t i o n o f catenanes in the presence o f D N A - c o n d e n s i n g or crowding agents such as p o l y v a l e n t cations (spermidine etc.), hydrophilic p o l y m e r (polyethylene glycol etc.) o r D N A c o n d e n s i n g proteins (histone H1 etc.) (1, 15, 17, 21, 28, 30). We tested w h e t h e r or n o t c a t e n a t i o n occurs by cauliflower t o p o i s o m e r a s e II when these agents are present. E x p e r i m e n t s with s p e r m i d i n e resulted in little c a t e n a t i o n even at 5 - 6 m M which is also the o p t i m u m c o n c e n t r a t i o n for a bacterial e n z y m e (15). E x p e r i m e n t s with polyethylene glycol-6000, as seen in Fig. 5A, showed a r e m a r k a b l e c a t e n a t i o n in the s t a n d a r d r e a c t i o n mixture, except t h a t the c o n c e n t r a t i o n o f D N A was 2.5/zg a n d c o n t a i n i n g 8°70 P E G . C a t e n a n e s thus f o r m e d were n o t simple aggregates with the enzyme
protein, because the D N A catenanes r e m a i n e d after p h e n o l t r e a t m e n t (Fig. 5B-2). A l s o Eco RI-treatm e n t p r o d u c e d linear u n i f o r m D N A s f r o m the catenanes (Fig. 5B-3). C a u l i f l o w e r t o p o i s o m e r ase II requires A T P a n d MgC12 for c a t e n a t i o n as well as for relaxation. W h e t h e r the e n z y m e catalyzes r e l a x a t i o n o r c a t e n a t i o n d e p e n d s on P E G a n d the c o n c e n t r a t i o n o f MgC12. N o activity was observed w i t h o u t MgC12, a n d c a t e n a t i o n a n d a little relaxation activity were f o u n d at 5 m M MgCI2, while o n l y c a t e n a t i o n activity was f o u n d over 10 m M M g C I 2 ( o p t i m u m 10 raM). E x p e r i m e n t s with histone H1 showed t h a t the f o r m a t i o n o f D N A catenanes occurs in the r e a c t i o n mixture w i t h o u t P E G c o n t a i n i n g histone in a weight ratio to D N A a p p r o x i m a t e l y at 0.1 ( d a t a n o t shown). D N A d e c a t e n a t i o n activities have been r e p o r t e d in various systems including E. coli gyrase, t y p e II t o p o i s o m e r a s e s from T4 infected ceils, f r o m Droso-
143
Fig. 5. Catenation and decatenation of pBR322 D N A by cauliflower D N A topoisomerase II. (A): 60 units enzyme and 2.5 ~g of D N A were incubated in the standard reaction mixture in the presence of 8°70 PEG. Lane 1 to 5; products obtained from the incubation of 0, 15, 30, 45 and 60 min, respectively. (B): 15 units enzyme and 2.5 p.g of D N A were incubated in the same condition as A. Lane 1; reaction products obtained from 60 rain-incubation. Lane 2; the same reaction products after phenol treatment. Lane 3; reaction products after Eco RI-digestion. (C): Decatenation of D N A obtained from the above reactions. Lane 1; D N A catenanes obtained from the lane 2 of B. Lane 2; reaction products obtained from catenanes incubated with topoisomerase II for 60 min in the same reaction mixture without PEG. I, II, III, IV and V indicated at left show supercoiled form, nicked form, linear form, small catenanes (dimer and trimer) and large catenanes, respectively.
phila melanogaster, from Xenopus laevis, and from yeast (1, 10, 12, 22, 37). Our experiments on the cauliflower topoisomerase II showed that the enzyme was also capable of decatenating DNA in the same reaction mixture required for catenation but without DNA-condensing or crowding agents (Fig. 5C). Type II topoisomerase (gyrase) from E. coli is one of the essential enzymes for the initiation of replication at the replication origin of the chromosome (14), and also it participates in the separation of bacterial genomes (34). Topoisomerase II of Saccharomyces cerevisiae is involved in DNA replication or in the separation of daughter chromosomes at the termination of DNA replication (3). Chromosome replication accompanying the condensation and decondensation of chromosomes in the rapidly growing tissues of cauliflower is considered to be intimately regulated by topoisomerase II. Further comparative studies with axial tissues, not so rapidly growing portion of the cauliflower head, may provide additional information on the DNA metabolic events involved in hypertrophic differentiation.
Acknowledgements This research was supported in part by grants from the Ministry of Education, Science and Culture of Japan and from the Research Council, Ministry of Agriculture, Forestry and Fisheries Japan, original and creative research project on biotechnology.
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