Biochimica e" Bioph.vsica Acta, 1088 (1991) 36-40 ~e)1991 Elsevier Science Publishers B.V. (Biomedical Division) 0167-4781/91/$03.50 ADONIS 016747819100055R
36
BBAEXP 92184
Topoisomerase I in actively growing plasmodia and during differentiation of the slime mold Physarumpolycephalum K r z y s z t o f Staron, B a r b a r a K o w a l s k a - L o t h , R o b e r t M. C z e r w i n s k i , J o a n n a B a n d o r o w s k a and Joanna Guberska Department of Biochemistry, Warsaw University, Warszawa (Poland) (Received 15 May 1990)
Key words: DNA topoisomerase !; Enzyme purification; Spherulation; (P, polycephalum)
A type ! topoisomerase has been purified from nuclei of a slime mold Physarumpolycephalum and its activity was tested during spherulation. The final preparation contained a single polypeptide of about 100 kDa. Basic properties of Physarum tupoisomerase ! (substrate specificity, ionic requirement, sensitivity to inhibiturs) were similar to those of tupoisomerases from higher eukaryotes. Specific features of Pkvsarum enzyme were that it was rapidly inactivated at 45 °C and did not react with antibodies against human tupoisomerase !. The activity of topoisomerase ! in developed dormant spherules decreased approx. 2-fold, as compared with a 4-fold decrease of RNA and a 10-fold decrease of DNA synthesis. Basic properties of the enzyme remained unchanged during spherulation. Introduction Topoisomerases catalyse interconversions between topoisomers of circular or looped DNA and thus allow DNA replication, transcription and recombination (for reviews see Refs. 1-3). Of the two distinct classes of topoisomerases type I and type II enzymes act by a transient breaking of either single or double DNA strands, respectively. In eukaryotic cells topoisomerase I is preferentially associated with transcriptionally active chromatin [4-7]. It suggests that the main role of the enzyme is to relax the torsional stress resulting from a movement of the RNA polymerase complex [8]. A possible regulatory link between RNA synthesis and the activity of topoisomerase 1 remains obscure, altough it has been shown that the activity of topoisomerase I decreases precisely in parallel with transcriptional activity during differentiation of chicken spermatids [9]. On the other hand, a slight increase of topoisomerase I activity has been also observed in some cells stimulated to proliferation [10], infected with virus [11] or in several types of human cancer [12,13]. It suggests a link between topoisomerase I activity and DNA replication. Abbreviations: PEG, poly(ethyleneglycol); HAP, hydroxyapatite; PMSF, phenyimethylsulfonyl fluoride; DMSO, dimethylsulfoxide; NEM, N-cthylmaleimide. Correspondence: K. Staron, Department of Biochemistry, Warsaw University, AI. Zwirki i Wigury 93, 02-089 Warszawa, Poland.
However, in numerous other cells the activity of topoisomerase I has been found to be independent of proliferative capacity [14]. In this paper we present a study on topoisomerase I from Physarum polycephalum. Under unfavorable conditions (e.g., starvation, elevated temperature) Physarum plasmodia undergo a simple differentiation which leads to dormant spherules [15]. This is accompanied by the overall drop in RNA synthesis and halting of DNA replication [16]. We report here the purification and characterization of Physarum topoisomerase I and the pattern of changes of its activity during spherulation. Materials and Methods
pBR 322 pBR322 was purified from Escherichia coil host, HB 101, as described [17]. Positively supercoiled pBR322 was prepared by addition of ethidium bromide to the previously relaxed plasmid DNA to the concentration of 30/~g/ml.
Culture Microplasmodia of P. polycephalum (strain M3CIV) were grown in shaken cultures at 23°C in a semi-defined medium [18]. The cultures used for spherulation were grown in a culture medium for 48 h and were then transferred into starvation medium [18] and shaken up to 72 h. Formation of spherules was controlled microscopically.
37 Microplasmodia or spherules were labelled for 1 h at 23°C in the presence of 5 # C i / m l of [14C]thymidine (spec. act. 0.48 Ci/mmol) or [3H]uridine (spec. act. 25 Ci/mmol).
Purification of topoisomerase Nuclei were prepared from microplasmodia according to the detergent method [19]. Spherules were disrupted by grinding in liquid nitrogen. The nuclei were lysed in 1 M NaCI and treated with 6~ (w/v) PEG, according to Liu [20]. The PEG supernatant was purified in the following steps: (1) 1/20 volume of HAP was added and the suspension was stirred for 10 rain to adsorb the enzyme. HAP was washed 5-times with 1 mM potassium phosphate/10~ (v/v) glycerol/1 M NaCI/10 mM mercaptoethanol/50 mM Tris-HCI (pH 7.5) and the enzyme was eluted with the same medium but containing 200 mM potassium phosphate; (2) The preparation was dialysed against 5 mM KCI/10~ (v/v) glycerol/1 mM E D T A / 1 0 mM mercaptoethanol/25 mM Tris-HC! (pH 7.5) (column buffer). MgCI 2 was added to 5 mM and the preparation was chromatographed on CM-Sephadex column using KCI gradient in the column buffer ranging from 5 to 200 mM KCI. Topoisomerase ehited between 90-110 mM KCI; (3) Active fractions were combined, dialysed against the column buffer containing 5 mM MgCI 2 and loaded on DEAE-Sephadex column equilibrated in the same buffer. The column was developed with KC! gradient ranging from 5 to 200 mM KCI. The enzyme was eluted in the 50 mM KCI fraction; and (4) The final preparation was dialysed against 50~ (v/v) glycerol/10 mM mercaptoethanol/0.1 mM E D T A / 7 0 mM potassium phosphate (pH 7.0). The preparation was stored below -20°C. PMSF was present in all media at the concentration of 1 raM. Topoisomerase I from rabbit liver was isolated according to Champoux and McConaughy [21].
Assays Topoisomerase I assay measured the extent of relaxation of supercoiled pBR322 and was performed as described [20] except that the incubation was at 25 °C. 1 unit of activity relaxed 505 of the substrate DNA in 30 rain. lnbibitors, salts or buffers were mixed with the enzyme in the assay cocktail in the absence of substrate and incubated for 10 min at 25 o C. The exception was ethidium bromide which was added directly to the substrate. Camptothecin (lactone form) was dissolved in DMSO. For temperature dependence experiments the samples were incubated for 5 min at required temperatures. Then an equal volume of pBR322 was added to initiate the reaction which was performed at 25 ° C. Protein was determined by the method of Bradford [22]. DNA in nuclei was determined by the method of
Burton [23] after removal of RNA by alkaline hydrolysis.
Electrophoresis DNA electrophoresis was performed on 0.75~ agarose in 2 mM EDTA/40 mM Tris-acetate (pH 7.8), according to Maniatis et al. [24] or on 1.2~ agarose containing 7 5 / t g / m l chloroquine as described by Shure et ai. [25]. DNA bands were visualised by staining in 5 #M ethidium bromide and photographed under ultraviolet illumination. Proteins were analysed on 7.5~ SDS-polyacrylamide gels according to Laemmli [26].
A ntisera Sera containing antibodies to Scl 70 antigen were obtained from scleroderma-positive patients. IgG fraction was purified on Protein A-Sepharose according to Smith and Rollins [27]. Results
Purification procedure The major problem during the purification of P.
polycephalum topoisomerase I is a charged polymer, presumably polymalate [28], which is present in the extract of lysed nuclei, if not removed at the first stages of the procedure, it complexed the majority of proteins and affected further purification. We found that the polymer isolated from culture medium did not adsorb on HAP even at low concentration of phosphlte. Therefore, we used HAP as the first stage of the purification. The high ionic strength used (1 M NaCI) prevented the polymer from binding to the enzyme. The further purification protocol included CM- and DEAE-Sephadex chromatography. The final preparation migrated in SDS electrophoresis as a single band of the molecular weight of about 100 kDa with traces of low molecular ~eight material (Fig. 1). It was active in the relaxation assay under conditions specific for topoisomerase I (Fig. 2). The average activity of the final preparation was about 3.5 • l0 T U / r a g protein.
Basic properties of Physarum topoisomerase Physarum topoisomerase I shares several properties with type I enzymes from higher eukaryotes. They are listed below: (a) Ionic requirement and pH optimum. A slight stimulation of the activity occurred at 50-100 mM KC! and 5-10 mM MgCI 2. Higher concentrations of both KC! and MgCI 2 inhibited the enzyme. The enzyme was active above pH 6 and below pH 9 with the optimum at pH 8, which means, however, that it shifted slightly into the alkaline region as compared with mammalian topoisomerases [29]: (b) lnhibitors. The activity of the enzyme was inhibited by 1-5 mM NEM, 1-5 mM ATP and 20~ DMSO. It was also inhibited by
38 CH
HAP
DEAE
M
1
2
3
4
$
~;
7
IR,II'
-
e
~
-
94,000
67,000
Fig. 3. Effect of temperature cat Pkysarum (lanes 2-4) and rabbit (lanes 5-7) topoisomerases. Samples were preincubated 5 m/n at followingtemperatures: 2, 5 - 35°C: 3, 6 - 40°C; 4, 7 - 45°C and the reaction was performed for 30 rain at 25°C. Lane I - control pBR322. Indicationsas on Fig. 2.
Fig. l. SDS-polyacrylamidegel eleetrophoresis of preparations of Physarum topoisomerase ! at various stages of purification. HAP, hydroxyapat/te; CM, CM-Sephadex; DEAE, DEAE-Sephadex fractions; M, markersof the molecularweight.
concentration of about 1 m g / m l . The above values concern 50% inhibition of the activity; (c) Substrate specificity. The enzyme relaxed negative supercoiis a s well as positive ones introduced into relaxed plasmic D N A by the addition of ethidium bromide; a n d (d) Limited proteolysis. Limited proteolysis led to the active polypeptide of the molecular weight of 70 kDa.
D N A at about 1 /~g of double-stranded or 0.1 /zg of single-stranded DNA per 1 /tg of pBR322 DNA. The enzyme was inhibited by a topoisomerase i-specific alkaloid camptothecin [30] at the concentration of 2-10 /tg/ml. On the other hand, topoisomerase If-specific drug novoliocin [31] inhibited the enzyme only at the
Specific features of Physarum topoisomerase 1 Two features of Physarum topoisomerase were different from those of enzymes from higher eukaryotes:
43,000
1
2
II
thermal stabifity and immunological properties. Physarum topoisomerase is a heat-sensitive enzyme. It was completely inactivated after 5 min incubation at 4 5 ° C . Under the same conditions the enzyme from rabbit liver remained fully active (Fig. 3). Topoisomerase ! from Physarum was not recognized by antibodies against human enzyme. It has been shown that human topoisomerase l is responsible for immunological properties of Scl 70 antigen which is recognized by antibodies present in the sera from scleroderma-positive patients [32]. lgG isolated from such a serum inhibited the reaction catalysed by rabbit topoisomerase when 15 /Lg/ml or more lgG was used (Fig. 4). The activity of Physarum topoisomerase was not affected by similar concentrations of anti-Scl 70 antibodies (Fig. 4). Neither the rabbit nor the Physarum topoisomerase was inhibited by IgG isolated from control sera.
IR Fig. 2. Relaxation of topolsomers of pBR.~.2 by DEAE-Sephadex fractionof PhysarumtOlmhomerase.Lane l: ControlpBR322; lane 2: enzyme-treated plasmid. Electrophoresiswas performed in the presence of 75/~g/ml of chiomquine to allow for distinction between relaxed (la) and nicked (!1) form of the plasmid, i, supereoiledform of the plasm/d.
Activity of topoisomerase ! during spherulation Microscopic examination of Physarum cultured in the starvation medium revealed that the transition from plasmodia to spherules occurred between 24 and 48 h. Therefore, we tested the activity of topoisomerase I at both points and additionally after 72 h of starvation.
39 1
2
3
4
S
6
7
O
9
10
][R * I t
I
The basic properties (the molecular weight, thermal stability, sensitivity to camptothecin, ATP and novobiocin and immunological properties) were the same for the purified enzymes isolated from actively growing plasmodia and developed spherules which remained 72 h in the starvation medium. Discussion
Due to the large evolutionary distance between
Physarum and higher eukaryotes the first step of this work was the characterization of the purified Physarum enzyme. It has revealed that Physarum topoisomerase i
TABLE i
shares several properties with enzymes from higher eukaryotes. The molecular weight, ability to relax both positively and negatively supercoiled substrates, sensitivity to inhibitors and drugs are similar to other type I topoisomerases. Some minor properties of the Physarum topoisomerase seem to be specific of lower eukaryotes. These are: diminished thermostability [33,34] and the pH optimum, which is slightly shifted towards alkaline values [33]. In these respects the enzyme from Physarum nuclei resembles more the mitochondrial than the nuclear Lopoisomerases from higher eukaryotes [29]. Some unique structural features of Physarum enzyme were revealed by antibodies against human topoisomerase which did not recognize Physarum topoisomerase. However, summing up, we found no significant feature of Physarum topoisomerase ! which would allow to expect that the enzyme acted differently than those from higher eukaryotes. We found that the activity of topoisomerase ! decreased 2-fold in the course of Physarum spherulation. We did not observe any difference in basic properties between enzymes isolated from actively growing plasmodia and mature spherules. Such differences concerning sensitivity to ATP and campthotecin have been reported for different forms of topoisomerase I [35]. Thus, we think that the decrease of topoisomerase ! activity observed during spherulation of Physarum does not result from appearance of new or modified form of enzyme but rather reflects a diminished level of topoi-
Actiui O' of wpoisomerase I during sphertdation
somerase.
The activity of Physarum topoisomerase I was seriously affected by the purification procedure, especially these steps which required high ionic strength conditions. Thus, in order to monitor the activity during spherulation we determined it at an early step of purification, immediately after removal of chromosomal DNA, i.e., in the PEG extract. To be sure that the relaxation assay did not reveal any other activity than that of topoisomerase I "~'eperformed each assay both in the absence and in the presence of camptothecin (10 /~g/ml). To monitor possible changes in the activity of topoisomerase I we assayed the activity in samples prepared from PEG extract by successive 2-fold dilutions. Thus, we were able to detect any 2-fold difference in enzyme activities between particular samples. A 2-fold decrease was observed for matured spherules present in cultures starved for 48 and 72 h (Table 1). On the other hand, the incorporation of [J4C]thymidine and [~H]uridine dropped approx. 10-fold and d-fold, respectively, after 48 h of starvation (Table I).
Starvation
Control plasmodia 24h
Activity of
Incorporation (¢~ of control) *
topoisomerase ! (U/rag DNA.105)
114Clthymidine
[-~H]uridine
10.8+2.4 (n = 8) 7.6+1.9 (n 3) 5.1 + 1.8 (n=3) 4.5-]-1.2 (. = 3)
100
100
24
46
12
26
I0
23
=
48 h 72 h
* Measured as cpm per mg DNA.
A time-course of the decrease of topoisomerase ! activity during spherulation was correlated with that of transcription, replication and also with morphological changes observed microscopically. However, the decrease of the enzyme's activity (2-fold) was lower than a d; 3p of DNA (10-fold) and RNA (4-fold) synthesis. In ,he latter case a difference is even more pronounced if one considers the activity of topoisomerase I and rRNA synthesis because a decrease of transcription during spberulation of Physarum concerns mainly rRNA [16]. A basic involvement of topoisomerase 1 in transcription of rRNA genes is suggested by a concentration of
40 majority of eukaryotic topoisomerase 1 in the nucleolus [36]. The conclusion which can be drawn from the work on spherulation is that although the Physarum topoisomerase activity is regulated in concert with RNA and DNA synthesis a relative excess of the enzyme over genome's activities remains in dormant spherules. The activity stored is of about 5 - l 0 s U/rag DNA and is comparable with activity of topoisomerase I in actively transcribing tissues of higher eukaryotes [9]. It seems possible that a relatively high level of topoisomerase 1 activity in spherules facilitates genome's activities during early stages of germination. The latter process may start immediately at any point of spherulation due to reversibility of Pkysarum differentiation. Acknowledgements
This work was supported by the Polish Academy of Sciences within the project 3.13. We thank Dr. M. Chorzelska for sera from scleroderma-positive patients. R~e~nces I Gellert, M. {1981) Annu. Rev. Biochem. 50, 879-910. 2 Wang, J.C. (1985) Annu. Rev. Biochem. 54, 665-697. 30sheroff, N. {1989) Pharmac. Ther. 41, 223-241. 4 Bonven, BJ., Gocke, E. and Westergaard, O. (1985) Cell 41. 541-551. 5 Stewart, A.F. and Sehutz, G. (1987) Cell 50, 1109-1117. 6 Gilmour, D.S. and Elgin, S.C.R. {1987) Mol. Cell. Biol. 7, 141-148. 7 Ness, P.J., Koller, T. and Thoma. F. (1988) J. Mol. Biol. 200. 127-139. 8 Liu, L.F. and Wang, J.C. {1987) Proc. Natl. Acad. Sci. USA 84. 7024-7027. 9 Roca, J. and Mezquita, C. (1989) EMBO J. 8, 1855-1860. 10 Tricoli, J.V., Sahai. B.M., McCormick, P.J.. Jarlinski, S.J.. Bertram, J.S. and Kowalski, D. (1985) Exp. Cell Res. 158, 1-14. 11 Rainwater, R. and Mann, K. (1990) J. Virol. 64, 918-921. 12 Hsiang, Y.-H., Liu, L.F.. Hochster, H. and Potmesil. M. {1988) Proc. Am. Assoc. Cancer Res. 29, 172. 13 Potmesil, M., Hsiang, Y.-H., Liu, L.F., Bank, B., Grossberg, H.,
Kirschenbaum. S., Forlenza, T.J.. Penzinger, A., Kanganis, D., Knowles, D.. Traganos, F .and Silber, R. (1988) Cancer Res. 48, 3537-3543. 14 Heck, M. and Earnshaw, W.C. {1988) in Self-Assembling Architectare (Varner, J.E., ed.), pp. 243-263, Alan R. Liss, N e w York. 15 Dove, W.F., Dee, J., Hatano, S., HauglL F.B. and WohlfarthBottermann, K.E. {1986) in The Molecular Biology of Physarum Polycephalum, Plenum Press. N e w York and London. 16 Sauer, H.W., Babcock, K,L. and Rusch, H.P. (1970) Wilhelm Roux' Archly Entwicklungsmech. Org, 165, 110-124. 17 Davies, L.G., Dibner, M.D. and Baney, J.F, (1986) in Basic Methods in Molecular Biology, Elsevier,N e w York. 18 Daniel, J.W. and Baldwin, H.H. (1964) in Methods in Cell Physi, ology (Prescott, D M., ed,), pp. 9-41, Academic Press, N e w York. 19 Jockusch, B.M. and Walker, I.O. (1974) Eur. J. Biochem. 48, 417-425. 20 Liu, L.F. (1983) Methods Enzymol. tOO, 133-137. 21 Champoux, J.J, and McConaughy, B.L. (1976) Biochemistry 15, 4638-4642. 22 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 23 Burton, K. (1956) Biochem. J. 62, 315-322. 24 Maniatis, T., Frisch, E.F. and Sambrook, J. {1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N e w York. 25 Share, M., Pulleyblank, D.E. and Vinogra,d J. {1977) Nucleic Acid Res. 4, 1183-1205. 26 Laemmli, U.K. (1970) Nature 227, 680-685. 27 Smith, W.L. and Rollins, T.E. (1982) Methods Enzymol. 86, 213-222. 28 Fisher, H., Erdmann, S. and Holler, E. (1989) Biochemistry 28, 5219-5226. 29 Lazarus, G.M., Heiarich, J.P., Kelly, W.G., Sehmitz, S.A, and Castor& F.J. (1987) Biochemistry 26, 6195-6203. 30 Hsiang, Y.H., Hertzberg, R., Hecht, S. and Liu, L.F. (1985) J. Biol. Chem. 260, 14873-14878. 31 Gellert, M., O'Dea, M.H., Itoh. T. and Tomizawa, J.-l. (1976) Proc. Natl. Acad. Sei. U S A 73, 4474-4479. 32 Maul, G.G., French, B.T., Van Venrooij, W.J. and Jimenez, S.A. {1986) Proc. Natl. Acad. Sei. U S A 83, 5145-5149. 33 Badaracco, G., Plevani, P., Ruyechan, W.T. and Chang, LM.S. :1983) J. Biol. Chem. 258, 2022-2026, 3"* Rowe, T.C., Rusche, J.R., Brougham. M.J. and Holloman, W.K. (1981) J. Biol. Chem. 256, 10354-10361. 35 Liu, S.. Hwang, B., Liu, Z. and Cheng, Y. {1989) Cancer Res. 49, 1366-1370. 36 Muller, M.T., Pfund, W.F.. Mehta. V.B. and Trask, D.K. (1985) E M B O J. 4, 1237-1243.
36
BBAEXP 92184
Topoisomerase I in actively growing plasmodia and during differentiation of the slime mold Physarumpolycephalum K r z y s z t o f Staron, B a r b a r a K o w a l s k a - L o t h , R o b e r t M. C z e r w i n s k i , J o a n n a B a n d o r o w s k a and Joanna Guberska Department of Biochemistry, Warsaw University, Warszawa (Poland) (Received 15 May 1990)
Key words: DNA topoisomerase !; Enzyme purification; Spherulation; (P, polycephalum)
A type ! topoisomerase has been purified from nuclei of a slime mold Physarumpolycephalum and its activity was tested during spherulation. The final preparation contained a single polypeptide of about 100 kDa. Basic properties of Physarum tupoisomerase ! (substrate specificity, ionic requirement, sensitivity to inhibiturs) were similar to those of tupoisomerases from higher eukaryotes. Specific features of Pkvsarum enzyme were that it was rapidly inactivated at 45 °C and did not react with antibodies against human tupoisomerase !. The activity of topoisomerase ! in developed dormant spherules decreased approx. 2-fold, as compared with a 4-fold decrease of RNA and a 10-fold decrease of DNA synthesis. Basic properties of the enzyme remained unchanged during spherulation. Introduction Topoisomerases catalyse interconversions between topoisomers of circular or looped DNA and thus allow DNA replication, transcription and recombination (for reviews see Refs. 1-3). Of the two distinct classes of topoisomerases type I and type II enzymes act by a transient breaking of either single or double DNA strands, respectively. In eukaryotic cells topoisomerase I is preferentially associated with transcriptionally active chromatin [4-7]. It suggests that the main role of the enzyme is to relax the torsional stress resulting from a movement of the RNA polymerase complex [8]. A possible regulatory link between RNA synthesis and the activity of topoisomerase 1 remains obscure, altough it has been shown that the activity of topoisomerase I decreases precisely in parallel with transcriptional activity during differentiation of chicken spermatids [9]. On the other hand, a slight increase of topoisomerase I activity has been also observed in some cells stimulated to proliferation [10], infected with virus [11] or in several types of human cancer [12,13]. It suggests a link between topoisomerase I activity and DNA replication. Abbreviations: PEG, poly(ethyleneglycol); HAP, hydroxyapatite; PMSF, phenyimethylsulfonyl fluoride; DMSO, dimethylsulfoxide; NEM, N-cthylmaleimide. Correspondence: K. Staron, Department of Biochemistry, Warsaw University, AI. Zwirki i Wigury 93, 02-089 Warszawa, Poland.
However, in numerous other cells the activity of topoisomerase I has been found to be independent of proliferative capacity [14]. In this paper we present a study on topoisomerase I from Physarum polycephalum. Under unfavorable conditions (e.g., starvation, elevated temperature) Physarum plasmodia undergo a simple differentiation which leads to dormant spherules [15]. This is accompanied by the overall drop in RNA synthesis and halting of DNA replication [16]. We report here the purification and characterization of Physarum topoisomerase I and the pattern of changes of its activity during spherulation. Materials and Methods
pBR 322 pBR322 was purified from Escherichia coil host, HB 101, as described [17]. Positively supercoiled pBR322 was prepared by addition of ethidium bromide to the previously relaxed plasmid DNA to the concentration of 30/~g/ml.
Culture Microplasmodia of P. polycephalum (strain M3CIV) were grown in shaken cultures at 23°C in a semi-defined medium [18]. The cultures used for spherulation were grown in a culture medium for 48 h and were then transferred into starvation medium [18] and shaken up to 72 h. Formation of spherules was controlled microscopically.
37 Microplasmodia or spherules were labelled for 1 h at 23°C in the presence of 5 # C i / m l of [14C]thymidine (spec. act. 0.48 Ci/mmol) or [3H]uridine (spec. act. 25 Ci/mmol).
Purification of topoisomerase Nuclei were prepared from microplasmodia according to the detergent method [19]. Spherules were disrupted by grinding in liquid nitrogen. The nuclei were lysed in 1 M NaCI and treated with 6~ (w/v) PEG, according to Liu [20]. The PEG supernatant was purified in the following steps: (1) 1/20 volume of HAP was added and the suspension was stirred for 10 rain to adsorb the enzyme. HAP was washed 5-times with 1 mM potassium phosphate/10~ (v/v) glycerol/1 M NaCI/10 mM mercaptoethanol/50 mM Tris-HCI (pH 7.5) and the enzyme was eluted with the same medium but containing 200 mM potassium phosphate; (2) The preparation was dialysed against 5 mM KCI/10~ (v/v) glycerol/1 mM E D T A / 1 0 mM mercaptoethanol/25 mM Tris-HC! (pH 7.5) (column buffer). MgCI 2 was added to 5 mM and the preparation was chromatographed on CM-Sephadex column using KCI gradient in the column buffer ranging from 5 to 200 mM KCI. Topoisomerase ehited between 90-110 mM KCI; (3) Active fractions were combined, dialysed against the column buffer containing 5 mM MgCI 2 and loaded on DEAE-Sephadex column equilibrated in the same buffer. The column was developed with KC! gradient ranging from 5 to 200 mM KCI. The enzyme was eluted in the 50 mM KCI fraction; and (4) The final preparation was dialysed against 50~ (v/v) glycerol/10 mM mercaptoethanol/0.1 mM E D T A / 7 0 mM potassium phosphate (pH 7.0). The preparation was stored below -20°C. PMSF was present in all media at the concentration of 1 raM. Topoisomerase I from rabbit liver was isolated according to Champoux and McConaughy [21].
Assays Topoisomerase I assay measured the extent of relaxation of supercoiled pBR322 and was performed as described [20] except that the incubation was at 25 °C. 1 unit of activity relaxed 505 of the substrate DNA in 30 rain. lnbibitors, salts or buffers were mixed with the enzyme in the assay cocktail in the absence of substrate and incubated for 10 min at 25 o C. The exception was ethidium bromide which was added directly to the substrate. Camptothecin (lactone form) was dissolved in DMSO. For temperature dependence experiments the samples were incubated for 5 min at required temperatures. Then an equal volume of pBR322 was added to initiate the reaction which was performed at 25 ° C. Protein was determined by the method of Bradford [22]. DNA in nuclei was determined by the method of
Burton [23] after removal of RNA by alkaline hydrolysis.
Electrophoresis DNA electrophoresis was performed on 0.75~ agarose in 2 mM EDTA/40 mM Tris-acetate (pH 7.8), according to Maniatis et al. [24] or on 1.2~ agarose containing 7 5 / t g / m l chloroquine as described by Shure et ai. [25]. DNA bands were visualised by staining in 5 #M ethidium bromide and photographed under ultraviolet illumination. Proteins were analysed on 7.5~ SDS-polyacrylamide gels according to Laemmli [26].
A ntisera Sera containing antibodies to Scl 70 antigen were obtained from scleroderma-positive patients. IgG fraction was purified on Protein A-Sepharose according to Smith and Rollins [27]. Results
Purification procedure The major problem during the purification of P.
polycephalum topoisomerase I is a charged polymer, presumably polymalate [28], which is present in the extract of lysed nuclei, if not removed at the first stages of the procedure, it complexed the majority of proteins and affected further purification. We found that the polymer isolated from culture medium did not adsorb on HAP even at low concentration of phosphlte. Therefore, we used HAP as the first stage of the purification. The high ionic strength used (1 M NaCI) prevented the polymer from binding to the enzyme. The further purification protocol included CM- and DEAE-Sephadex chromatography. The final preparation migrated in SDS electrophoresis as a single band of the molecular weight of about 100 kDa with traces of low molecular ~eight material (Fig. 1). It was active in the relaxation assay under conditions specific for topoisomerase I (Fig. 2). The average activity of the final preparation was about 3.5 • l0 T U / r a g protein.
Basic properties of Physarum topoisomerase Physarum topoisomerase I shares several properties with type I enzymes from higher eukaryotes. They are listed below: (a) Ionic requirement and pH optimum. A slight stimulation of the activity occurred at 50-100 mM KC! and 5-10 mM MgCI 2. Higher concentrations of both KC! and MgCI 2 inhibited the enzyme. The enzyme was active above pH 6 and below pH 9 with the optimum at pH 8, which means, however, that it shifted slightly into the alkaline region as compared with mammalian topoisomerases [29]: (b) lnhibitors. The activity of the enzyme was inhibited by 1-5 mM NEM, 1-5 mM ATP and 20~ DMSO. It was also inhibited by
38 CH
HAP
DEAE
M
1
2
3
4
$
~;
7
IR,II'
-
e
~
-
94,000
67,000
Fig. 3. Effect of temperature cat Pkysarum (lanes 2-4) and rabbit (lanes 5-7) topoisomerases. Samples were preincubated 5 m/n at followingtemperatures: 2, 5 - 35°C: 3, 6 - 40°C; 4, 7 - 45°C and the reaction was performed for 30 rain at 25°C. Lane I - control pBR322. Indicationsas on Fig. 2.
Fig. l. SDS-polyacrylamidegel eleetrophoresis of preparations of Physarum topoisomerase ! at various stages of purification. HAP, hydroxyapat/te; CM, CM-Sephadex; DEAE, DEAE-Sephadex fractions; M, markersof the molecularweight.
concentration of about 1 m g / m l . The above values concern 50% inhibition of the activity; (c) Substrate specificity. The enzyme relaxed negative supercoiis a s well as positive ones introduced into relaxed plasmic D N A by the addition of ethidium bromide; a n d (d) Limited proteolysis. Limited proteolysis led to the active polypeptide of the molecular weight of 70 kDa.
D N A at about 1 /~g of double-stranded or 0.1 /zg of single-stranded DNA per 1 /tg of pBR322 DNA. The enzyme was inhibited by a topoisomerase i-specific alkaloid camptothecin [30] at the concentration of 2-10 /tg/ml. On the other hand, topoisomerase If-specific drug novoliocin [31] inhibited the enzyme only at the
Specific features of Physarum topoisomerase 1 Two features of Physarum topoisomerase were different from those of enzymes from higher eukaryotes:
43,000
1
2
II
thermal stabifity and immunological properties. Physarum topoisomerase is a heat-sensitive enzyme. It was completely inactivated after 5 min incubation at 4 5 ° C . Under the same conditions the enzyme from rabbit liver remained fully active (Fig. 3). Topoisomerase ! from Physarum was not recognized by antibodies against human enzyme. It has been shown that human topoisomerase l is responsible for immunological properties of Scl 70 antigen which is recognized by antibodies present in the sera from scleroderma-positive patients [32]. lgG isolated from such a serum inhibited the reaction catalysed by rabbit topoisomerase when 15 /Lg/ml or more lgG was used (Fig. 4). The activity of Physarum topoisomerase was not affected by similar concentrations of anti-Scl 70 antibodies (Fig. 4). Neither the rabbit nor the Physarum topoisomerase was inhibited by IgG isolated from control sera.
IR Fig. 2. Relaxation of topolsomers of pBR.~.2 by DEAE-Sephadex fractionof PhysarumtOlmhomerase.Lane l: ControlpBR322; lane 2: enzyme-treated plasmid. Electrophoresiswas performed in the presence of 75/~g/ml of chiomquine to allow for distinction between relaxed (la) and nicked (!1) form of the plasmid, i, supereoiledform of the plasm/d.
Activity of topoisomerase ! during spherulation Microscopic examination of Physarum cultured in the starvation medium revealed that the transition from plasmodia to spherules occurred between 24 and 48 h. Therefore, we tested the activity of topoisomerase I at both points and additionally after 72 h of starvation.
39 1
2
3
4
S
6
7
O
9
10
][R * I t
I
The basic properties (the molecular weight, thermal stability, sensitivity to camptothecin, ATP and novobiocin and immunological properties) were the same for the purified enzymes isolated from actively growing plasmodia and developed spherules which remained 72 h in the starvation medium. Discussion
Due to the large evolutionary distance between
Physarum and higher eukaryotes the first step of this work was the characterization of the purified Physarum enzyme. It has revealed that Physarum topoisomerase i
TABLE i
shares several properties with enzymes from higher eukaryotes. The molecular weight, ability to relax both positively and negatively supercoiled substrates, sensitivity to inhibitors and drugs are similar to other type I topoisomerases. Some minor properties of the Physarum topoisomerase seem to be specific of lower eukaryotes. These are: diminished thermostability [33,34] and the pH optimum, which is slightly shifted towards alkaline values [33]. In these respects the enzyme from Physarum nuclei resembles more the mitochondrial than the nuclear Lopoisomerases from higher eukaryotes [29]. Some unique structural features of Physarum enzyme were revealed by antibodies against human topoisomerase which did not recognize Physarum topoisomerase. However, summing up, we found no significant feature of Physarum topoisomerase ! which would allow to expect that the enzyme acted differently than those from higher eukaryotes. We found that the activity of topoisomerase ! decreased 2-fold in the course of Physarum spherulation. We did not observe any difference in basic properties between enzymes isolated from actively growing plasmodia and mature spherules. Such differences concerning sensitivity to ATP and campthotecin have been reported for different forms of topoisomerase I [35]. Thus, we think that the decrease of topoisomerase ! activity observed during spherulation of Physarum does not result from appearance of new or modified form of enzyme but rather reflects a diminished level of topoi-
Actiui O' of wpoisomerase I during sphertdation
somerase.
The activity of Physarum topoisomerase I was seriously affected by the purification procedure, especially these steps which required high ionic strength conditions. Thus, in order to monitor the activity during spherulation we determined it at an early step of purification, immediately after removal of chromosomal DNA, i.e., in the PEG extract. To be sure that the relaxation assay did not reveal any other activity than that of topoisomerase I "~'eperformed each assay both in the absence and in the presence of camptothecin (10 /~g/ml). To monitor possible changes in the activity of topoisomerase I we assayed the activity in samples prepared from PEG extract by successive 2-fold dilutions. Thus, we were able to detect any 2-fold difference in enzyme activities between particular samples. A 2-fold decrease was observed for matured spherules present in cultures starved for 48 and 72 h (Table 1). On the other hand, the incorporation of [J4C]thymidine and [~H]uridine dropped approx. 10-fold and d-fold, respectively, after 48 h of starvation (Table I).
Starvation
Control plasmodia 24h
Activity of
Incorporation (¢~ of control) *
topoisomerase ! (U/rag DNA.105)
114Clthymidine
[-~H]uridine
10.8+2.4 (n = 8) 7.6+1.9 (n 3) 5.1 + 1.8 (n=3) 4.5-]-1.2 (. = 3)
100
100
24
46
12
26
I0
23
=
48 h 72 h
* Measured as cpm per mg DNA.
A time-course of the decrease of topoisomerase ! activity during spherulation was correlated with that of transcription, replication and also with morphological changes observed microscopically. However, the decrease of the enzyme's activity (2-fold) was lower than a d; 3p of DNA (10-fold) and RNA (4-fold) synthesis. In ,he latter case a difference is even more pronounced if one considers the activity of topoisomerase I and rRNA synthesis because a decrease of transcription during spberulation of Physarum concerns mainly rRNA [16]. A basic involvement of topoisomerase 1 in transcription of rRNA genes is suggested by a concentration of
40 majority of eukaryotic topoisomerase 1 in the nucleolus [36]. The conclusion which can be drawn from the work on spherulation is that although the Physarum topoisomerase activity is regulated in concert with RNA and DNA synthesis a relative excess of the enzyme over genome's activities remains in dormant spherules. The activity stored is of about 5 - l 0 s U/rag DNA and is comparable with activity of topoisomerase I in actively transcribing tissues of higher eukaryotes [9]. It seems possible that a relatively high level of topoisomerase 1 activity in spherules facilitates genome's activities during early stages of germination. The latter process may start immediately at any point of spherulation due to reversibility of Pkysarum differentiation. Acknowledgements
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