IRKUTSK, RUSSIA
Institute of Plant Physiology and Biochemistry
MOSCOW, RUSSIA
V. A. Engelhardt Institute of Molecular Biology
Effects of different types of inhibitors on mitochondrial DNA-topoisomerase I
--Konstantinov, YM, Tarasenko, VI, Grokhovsky, SL, Sukhanova, AS, Zhuze, AL

Molecular biological and genetic evidence clearly indicates that topoisomerases are important, often essential, cellular enzymes involved in nearly all aspects of DNA structure and metabolism (for review see Berger, Biochim. Biophys. Acta 1400:3-18, 1998). We have previously described (MNL 71:39-40, 1997) some characteristics of type I DNA-topoisomerase (topo I) from maize mitochondria. The objective of the present study was to investigate the effects of different types of inhibitors on mitochondrial DNA-topoisomerase I activity.

The mitochondria were isolated from 3-day-old etiolated maize seedlings of hybrid VIR42 MV by a standard method of differential centrifugation. Mitochondrial protein was determined by the Lowry method. The purification of topo I from isolated mitochondria included the stages of organelle solubilization, ammonium sulfate fractionation, chromatography on a column with DEAE-Toyopearl, and chromatography on a column with ds-DNA-cellulose as previously described (Tarasenko and Konstantinov, Biopolymers and Cell 14:111-116, 1998). We have studied the effect of some inhibitors currently used to characterize type I DNA-topoisomerase activities from different sources (Figure 1).

It is well known that the topo I inhibitors belong to the ligands with powerful antimicrobial and antitumor activity. The best known of these agents is plant alkaloid camptothecin, which specifically affects the rejoining step of the topo I reaction and therefore stabilizes the covalent protein-DNA cleavable complex. Therefore, camptothecin selectively kills cells in the S phase of the cell cycle because of topo I dysfunction during DNA replication (Chen and Liu, Annu Rev Pharmacol Toxicol 34:191-218, 1994). In our experiments camptothecin does not have any influence on the mitochondrial topo I activity in concentrations of 10 uM to 1 mM. Therefore there is a striking difference in sensitivity to camptothecin between topo I of maize mitochondria and the analogous enzyme from mammalian organelles.

A number of DNA non-intercalating ligands are known to bind to the minor groove of DNA with AT specificity (Zimmer and Wahnert, Prog. Biophys. Mol. Biol. 47:31-112, 1986). These agents are able to inhibit the cleavage activity of eukaryotic topo I (Chen et al., Proc. Natl. Acad. Sci. USA 90:8131-8135, 1993). In this work we investigated the effect of such minor groove-binding agents on topo I activity from mitochondria. Among these types of topo I inhibitors we used such well-known ligands as Hoechst dye 33258, distamycin A, netropsin, and ligands from a new series of high sequence-specific dimeric derivatives of netropsin and distamycin A: bis-netropsins (bis-Nts) and bis-distamycins (bis-Dsts).

Bisbenzimidazole Hoechst 33258 is a cell permeable, adenine-thymine binding fluorescent dye used to stain DNA during evaluation of the cell cycle, induction of apoptosis by various ligands and cell viability by flow cytometry. It is known that this dye inhibits topo I activity in vitro, like camptothecin. In our experiments Hoechst 33258 is shown to cause a 50% decrease of mitochondrial topo I activity at a concentration of 5 uM. Distamycin A also inhibits topo I (a two-fold decrease of activity at 1.2 uM). It was unexpected, but bis-Dst had lower inhibition activity in comparison with the parent compound distamycin A and caused 50% inhibition of topo I only at 40 uM.

We have also studied the effect of such derivatives of netropsin as bis-Lys-Nt(->5<-) and Pt-bis-Nt which caused a two-fold decrease of topo I activity at concentrations of 15 uM and 3.75 uM, respectively.

We conclude that type I DNA-topoisomerase activity from maize mitochondria is rather sensitive to the action of inhibitors currently used for studying the catalytic mechanism of topo I reaction. These agents also have wide clinical significance due to their efficacy as antimicrobial and antitumor agents. We propose that type I DNA-topoisomerase from maize mitochondria may serve as a convenient model system in a search and testing of new ligands with the highest antitumor and antiviral activities.

Acknowledgements: We would like to thank Dr. N.V.Dorofeev for help with the figure preparation. Financial support from the INTAS (Project Number 98-0522) is acknowledged.

Figure 1. Chemical structures of type I DNA-topoisomerase inhibitors used in this work.
 


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