0020-711X/92$5.00+ 0.00 Copyright 0 1992PergamonPressLtd
Inc. J. Bioehem.Vol. 24, No. 8, PP. 1303-1306,1992 Printed in Great Britain. All rights reserved
EFFECTS OF FLUORODEOXYURIDINE AND NALIDIXIC ACID ON THE ACTIVITY OF TOPOISOMERASE I IN PLASMODIA OF PHYSARUM POLYCEPHALUM KRZYSZTOFSTARON,BARBARAKOWALSKA-LOTHand ROBERTM. CZERWI~SKI Institute of Biochemistry, Warsaw University, Al. Zwirki i Wigury 93, 02-089 Warszawa, Poland (Received 29 November 1991)
Abstract-l. A regulatory coupling between the rate of cellular transcription and the activity of topoisomerase I was investigated in plasmodia of Physarumpolycephalwn treated with fluorodeoxyuridine or nalidixic acid. 2. Fluorodeoxyuridine at concentrations above 40 pg/ml lowered both the incorporation of [‘Hluridine and the activity of topoisomerase I to 10% of corresponding control values. 3. Nalidixic acid, in the range of concentrations between 20-50 pg/ml did not inhibit the incorporation of [‘Hluridine but lowered the activity of topoisomerase I by about half. 4. It is suggested that a coupling between the level of transcription and the activity of topoisomerase I in Physarum plasmodia involves only about a half of the topoisomerase I activity and is limited to transcription occurring on ribosomal genes.
INTRODUCrION Eukaryotic topoisomerase I is involved in numerous cellular processes which result in the accumulation of a superhelical tension in double-stranded DNA (Wang, 1985). The role of topoisomerase I in transcription is suggested by several lines of evidence. The absence (Sri11 and Sternglanz, 1988) or inhibition (Schaak et al., 1990) of topoisomerase I reduces the level of transcription, especially that catalysed by RNA polymerase I. Topoisomerase I has been also found to be preferentially bound to transcriptionally active regions of chromatin (Bonven et al., 1985; Stewart and Schutz, 1987). Particularly high concentration of the enzyme has been found in the nucleolus (Muller et al., 1985), where a majority of cellular RNA is produced. The activity of topoisomerase I has been reported to change in the course of transition from resting to proliferating state of the cell (Rosenberg et al., 1976; Tricoli et al., 1985) which is usually accompanied by a significant change in the rate of RNA synthesis. Closely correlated changes of the activity of topoisomerase I and of the rate transcription have been reported for differentiating spermatocytes (Rota and Mezquita, 1989). The above suggests that the overall activity of topoisomerase I in the cell could be regulated by the rate of cellular transcription. On the other hand, the data from a number of experimental systems do not point to the existence of any coupling between transcription and the activity of the enzyme (Heck and Earnshaw, 1988; Romig and Richter, 1990). Thus, a question arises to what extent the
coupling mentioned above is a common phenomenon for different organisms and different stimuli which lead to changes in the rate of transcription. To address this question we employed a slime mold Physurum polycephulum in order to study the effects of different stimuli on the putative coupling. Physarum undergoes two types of a simple differentiation, one of which (spherulation) occurs without meiosis whereas the other (sporulation) leads to the production of gametes and thus resembles a differentiation of spermatocytes. We showed in the previous work (Staron et al., 1991) that a decrease of the rate of transcription which occurred in the course of spherulation was accompanied by a less pronounced decrease of the activity of topoisomerase I. Independently of a direct description of changes occurring in the course of spherulation and sporulation we examined the effects of drugs which changed the rate of transcription and (or) the activity of topoisomerase I when administered in oivo to actively growing plasmodia. The results of the studies on two of such drugs: fluorodeoxyuridine (FUdR) and naladixic acid are presented in this work. MATERIALS AND METHODS
Microplasmodia of P. polycephalum (strain M,CIV) were grown in shaken cultures at 23°C in a semidefined medium (Daniel and Baldwin, 1964). Freshly prepared solutions of FUdR (in water) or nalidixic acid (in 0.1 M KOH) were added to the medium before the inoculation. In the latter case an equivalent amount of 0.1 M HCl was added to prevent a change of pH of the medium.
KRZYSZTOF STARONet al.
Microplasmodia were labelled for 1 hr at 23°C in the presence of 5 pCi/ml of [3H]uridine (sp. act. 25 Ci/mmol) or 5 &i/ml of [‘Tlthymidine (sp. act. 0.48 Ci/mmol). Nuclei were prepared from microplasmodia according to the Triton method (Jockusch and Walker, 1974). Activity of topoisomerase I was determined in the lysed nuclei after removal of chromosomal DNA with poly(ethyleneglyco1) (Staron et al., 1991). The assays were performed according to Liu (1983) using pBR322 as a substrate. The activity was referred to the DNA content in the nuclei determined by OD,, measurement made in 1% SDS, IOmM EDTA, pH 7.5. RESULTS In order to check the effects of FUdR and nalidixic acid on transcription and the activity of topoisomerase I, drugs were added to the culture medium I5 hr before harvesting plasmodia and isolation of nuclei. Such a period ensured the completion of changes introduced by each drug but was too short for the advancement of processes leading to the formation of spherules. At the range of FUdR concentration between 4&100 pg/ml the activity of topoisomerase I in nuclei was lowered to about 10% of the control value (Fig. 1). This was accompanied by a similar decrease of the incorporation of [3H]uridine into RNA. However, the time-courses of both processes were not the same. The inhibition of the incorporation of [3H]uridine was observed immediately after addition of FUdR to the medium whereas the lowering of the activity of topoisomerase I was preceded by ca 3 hr lag period (Fig. 2). Nalidixic acid administered at the concentration of 20-45 pg/ml decreased the activity of topoisomerase I in nuclei to about 40-50% of the control value (Fig. 3). In contrast to FUdR nalidixic acid did not inhibit the incorporation of [3H]uridine into RNA.
10 12 14 16
time (h) Fig. 2. Time-course of the changes of topoisomerase I activity (0) and incorporation of [rH]uridine (0) upon treatment of plasmodia with FUdR. Plasmodia were cultured in the medium containing 80pg/ml of FUdR. Each curve combines results from two experiments.
Instead a stimulation of the incorporation was observed at low concentrations of the drug. A similar stimulation was found for the same concentration range for the incorporation of [i4C]thymidine into DNA, although in the latter case a slight inhibition of the incorporation occurred at higher concentrations of the drug. Concentrations of nalidixic acid of 50pg/ml and higher led to rapid inhibition of both uridine and thymidine incorporation and decreased the activity of topoisomerase I to zero level. This was accompanied by a death of plasmodia within 12-15 hr. Neither FUdR nor nalidixic acid affected the activity of topoisomerase I in vitro up to the concentrations of 700 and 125 @g/ml, respectively.
Fig. 1. Changes of the activity of topoisomerase I (0) and incorporation of [jH]uridine (0) upon treatment of plasmodia with FUdR. Plasmodia were cultured in the medium containing indicated concentration of FUdR for 15 hr. Each curve combines results from two experiments.
nalidixic acid concentration
Fig. 3. Changes of the activity of topoisomerase I (O), incorporation of [‘Hluridine (a) and [‘4C]thymidine (A) upon treatment of plasmodia with nalidixic acid. Plasmodia were cultured for 15 hr in the medium containing indicated concentration of nalidixic acid. Each curve combines results from two experiments.
Topoisomerase I in Physorum DISCUSSION
Our results showed that in Physarwn plasmodia treated with FUdR the decrease of the activity of topoisomerase I was correlated with the inhibition of transcription. Decrease of the rate of transcription preceded the decrease of the enzyme activity excluding a possibility that lowered activity of topoisomerase I was a reason of impaired transcription. On the other hand the opposite relationship remained possible. In the previous work we found a correlation between the rate of transcription and the activity of topoisomerase I in the course of spherulation (Staron et al., 1991). The processes occurring during spherulation and upon the action of FUdR are similar in that under both conditions the inhibition of transcription concerns first of all the rRNA synthesis (Sauer et al., 1970; Myers, 1981). Thus, it seems that in Physarum plasmodia there is a part of rRNA synthesis that is coupled with the activity of topoisomerase I. This conclusion is in agreement with the finding that topoisomerase I is highly concentrated in nucleolus (Muller et al., 1985). The coupling may also reflect a special status of Physarum rDNA which is not attached to the nuclear matrix (Vogt and Braun, 1976), where topoisomerase II is bound (Gasser et al., 1986). It seems that topoisomerase I is the only enzyme responsible for topology of extrachromosoma1 rDNA of Physarum. The halting of RNA synthesis during spherulation resulted in 50% inhibition of the activity of topoisomerase I (Staron et al., 1991) whereas this activity was almost completely abolished upon the action of FUdR. It is possible that the reason of this latter phenomenon is the fact that the action of FUdR is not limited to the inhibition of RNA synthesis (Myers, 1981) and other factors not coupled with transcription contribute to the observed overall decrease of the activity of topoisomerase I. This notion finds support in the results of experiments performed with nalidixic acid. In contrast to FUdR nalidixic acid applied to Physarum at concentrations below 50 @g/ml stimulated incorporation of both thymidine and uridine but inhibited by about a half the activity of topoisomerase I in nuclei. A stimulation of the incorporation of pyrimimidine nucleotides by 4-quinolones has been reported for human lymphocytes although, in contrast to Physarum, the effect was significantly more pronounced for deoxyribonucleosides than for uridine (Forsgren et al., 1987), and observed for quinolone deratives rather than nalidixic acid (Forsgren et al., 1986). The stimulation observed in lymphocytes, which occurred at concentration of quinolones comparable to those used by us for Physarum, has been found to be due to a block of pyrimidine metabolism (Forsgren et al., 1986, 1987). A direct effect of nalidixic acid on DNA and RNA synthesis, resulting probably from binding of the
drug to DNA (Shen and Pemet, 1985), has been observed for mammalian cells at concentrations higher than lOOpg/ml (Hussy et al., 1986). We also found a clear inhibition of the incorporation of [3H]uridine and [14C]thymidine above 50 pg/ml of nalidixic acid. Therefore we concluded that a decrease of the activity of Physarum topoisomerase I observed between 20 and 45 pg/ml of nalidixic acid was not a result of the inhibition of RNA synthesis. The effect of nalidixic acid shows that the activity of topoisomerase I is not a rate limiting step for transcription in Physarum plasmodia. Instead, this activity can be lowered by about a half without the concomitant decrease of the rate of transcription. This is in a good agreement with the part of topoisomerase I activity found as not coupled with transcription during spherulation (Staron et al., 1991). A general conclusion which can be drawn from the results presented in the previous (Staron et al., 1991) and the present work is that there is a coupling between the rate of transcription and the activity of topoisomerase I in Physarum which is however hmited to about half of the overall activity of the enzyme. We hypothesize that only topoisomerase I which acts on ribosomal genes is involved in the coupling. work was supported by a grant G-MEN/62/90 from the Ministry of Education.
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