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The study was performed on a seven- to eight-day- old suspension cell culture of carrot (Daucus carota) grown in the Murashige–Skoog medium containing 2%.
Doklady Biochemistry and Biophysics, Vol. 377, 2001, pp. 82–84. Translated from Doklady Akademii Nauk, Vol. 377, No. 2, 2001, pp. 263–265. Original Russian Text Copyright © 2001 by Konstantinov, Tarasenko, Rogozin.

BIOCHEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY

Redox Modulation of the Activity of DNA Topoisomerase I from Carrot (Daucus carota) Mitochondria Yu. M. Konstantinov, V. I. Tarasenko, and I. B. Rogozin Presented by Academician V.K. Shumnyi July 6, 2000 Received September 12, 2000

Siberian Institute of Plant Physiology and Biochemistry, Siberian Division, Russian Academy of Sciences, ul. Lermontova 132, Irkutsk, 664033 Russia

molecular-weight thiols was demonstrated for the first time. Highly conserved cysteine residues in the core domain of the topoisomerase I molecule, which were capable of formation of regulatory disulfide bonds, were localized with the use of computer simulation.

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The study was performed on a seven- to eight-dayold suspension cell culture of carrot (Daucus carota) grown in the Murashige–Skoog medium containing 2% sucrose [10]. Mitochondria were isolated as described previously [11] with some modifications. DNA topoisomerase I activity in the mitochondrial lysate was determined as described in [12] by evaluating the relaxation of negatively supercoiled plasmid DNA [8]. DNA of the plasmid pRT104 isolated by alkaline lysis was used as a substrate. Mitochondrial topoisomerase I exhibited a high sensitivity to such inhibitors as camptotecin, distamycin, and netropsin. Quantitative evaluation of the effect of redox agents on the activity of topoisomerase I was performed using the Sigmagel

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By now, the molecular mechanisms of redox regulation of gene expression at the transcription level have been studied in most detail in prokaryotes [1]. However, many aspects of redox regulation of gene activity in both nuclear and mitochondrial eukaryotic genomes are considerably less understood [2–5]. Earlier, we demonstrated [6, 7] that redox conditions have a pronounced effect on the intensity of genetic processes in mitochondria. We showed that oxidation caused by addition of potassium ferricyanide or oxidized glutathione to mitochondria led to activation of transcription and translation. Conversely, reduction caused by addition of sodium dithionite or reduced glutathione to mitochondria significantly suppressed these processes. However, the exact molecular mechanism of redox regulation of mitochondrial gene expression in vivo remains obscure. Earlier, we suggested that the mechanism of redox control of genetic functions in mitochondria consisted of two components, being based on the interaction of a specific redox sensor(s) and regulator(s) of redox response [6]. According to this hypothesis, the glutathione-mediated redox system can function as a redox sensor responding to changes in the redox state of the respiratory chain and redox potential of mitochondria as a whole. However, the transcription regulation factors involved in the response of mitochondria to the changes in redox conditions remain to be identified. Earlier, it was shown that sulfhydryl groups are important for expression of the activity of plant DNA topoisomerase I [8, 9]. With this in mind, we assumed that the molecule of this enzyme could contain regulatory redox-sensitive cysteine residues that provided the sensitivity of the enzyme to the changes in the redox state of glutathione and, possibly, some other lowmolecular-weight biothiols in the organelles. We studied the influence of redox conditions on the activity of DNA topoisomerase I from carrot. The possibility of redox modulation of the DNA-relaxing activity of topoisomerase I in plants mediated by low-

FII

FI

Fig. 1. Effect of redox agents on the relaxing activity of DNA topoisomerase I from carrot mitochondria. The concentrations of the redox agents in the incubation medium was 5 mM. FI and FII designate the supercoiled and relaxed forms of the plasmid DNA, respectively.

1607-6729/01/0304-0082$25.00 © 2001 MAIK “Nauka /Interperiodica”

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Fig. 2. Alignment of the amino acid sequences of DNA topoisomerase I from different plant species. Only regions containing highly conserved cysteine residues (shown in gray) are presented. (1) Daucus carota; (2) Nicotiana tabacum; (3) Arabidopsis thaliana; and (4) Pisum sativum.

software (PKWARE Inc., United States). Amino acid sequences of topoisomerase I from different plant species were taken from the PIR database. To reveal the possible influence of redox conditions on the activity of mitochondrial DNA topoisomerase I, we used chemical redox agents (i.e., potassium ferricyanide and sodium dithionite) and the physiological redox agent glutathione, which is known to participate in the redox regulation of expression of many genes [13]. The data obtained on the effects of redox conditions on DNA relaxing activity of mitochondrial topoisomerase I are shown in Fig. 1. As is seen from the figure, oxidation promoted by addition of potassium ferricyanide or oxidized glutathione (GSSG) caused a substantial decrease in the topoisomerase I activity in mitochondrial lysate, whereas reduction promoted by addition of sodium dithionite or reduced glutathione (GSH) stimulated the enzyme activity. Data summarized in the table show that GSSG inhibited the relaxing activity of topoisomerase I much more effectively than potassium ferricyanide, while GSH and sodium dithionite had a comparable activating effects. The effect of the glutathione redox system on the activity of mitochondrial topoisomerase I discovered in these experiments indicate that the thiol–disulfide exchange reactions in cysteine residues of topoisomerase I molecule are involved into modulation of its activity. This is consistent with the data obtained earlier that SH groups in the molecule of topoisomerase I from plant mitochondria play a key role in its catalytic activity [8, 9]. DOKLADY BIOCHEMISTRY AND BIOPHYSICS

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Analysis of amino acid sequences of type I topoisomerase of various plant species indicates the presence of six highly conserved cysteine residues (Fig. 2). They are located predominantly in the core domain of the molecule (Fig. 3). Only one of these residues is located in the C-terminal domain. The data on the effect of the oxidized and reduced forms of glutathione on the relaxing activity of carrot mitochondrial topoisomerase I suggest that a certain pair of cysteine residues in the enzyme molecule can undergo reversible oxidation to form a regulatory disulfide bond, thus modulating the enzyme activity. The results of computer analysis of the model three-dimensional structure of carrot topoisomerase I, which was created using the data on the crystal structure of human topoisomerase I, suggest that a disulfide bond is formed between Cys-651 (or -652) and Cys-771 (Fig. 3). In general, this hypothEffect of redox agents on the relaxing activity of DNA topoisomerase I from carrot mitochondria Activity of % (from the Experimental conditions topoisomerase I, control value) relative units Control Potassium ferricyanide GSSG Sodium dithionite GSH 2001

56.6 14 7.7 95.7 98

100 25 13.6 169 173

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KONSTANTINOV et al. N-terminal domain

Core domain

Linker domain

Cys Cys Cys Cys Cys 430 525 651 652 771

C-terminal domain

Cys 878

ACKNOWLEDGMENTS This study was supported by INTAS (project no. 97-0522). REFERENCES

*

*

Tyr 863

Fig. 3. The domain structure of the protein molecule of DNA topoisomerase I from carrot mitochondria. The scheme is created based on the data published in [15]. Cys and Tyr are the cysteine and tyrosine residues in the active site, respectively. The numbers indicate location of the amino acid residues. The asterisks indicate the sites whose cysteine residues can form disulfide bonds (as judged by the results of computer simulation).

esis is consistent with the current view that the function of eukaryotic topoisomerase I requires a conformational change in the protein molecule [14]. Judging by the results of spectroscopic studies of the complexes formed by recombinant human topoisomerase I and site-specific DNA substrates, it is most likely that this structural transformation of the enzyme is determined by the highly conserved core and C-terminal polypeptide domains. It should be noted that the discovered significant conformational transitions of topoisomerase I, which were observed during its binding to the DNA substrate and the cleavage [14], entailed changes in the configuration of disulfide bonds, along with other changes. Thus, we demonstrated for the first time the possibility of redox modulation of the activity of topoisomerase I from plant mitochondria. It is most likely that this property of the enzyme is accounted for by the presence of highly conserved redox-sensitive cysteine residues in the core domain of the protein molecule.

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DOKLADY BIOCHEMISTRY AND BIOPHYSICS

Vol. 377

2001