Mitochondria were prepared from 3—day—old etiolated seedlings of maize (Zea mays L. hybrid VIR 46 MV) by the standard method of differential centrifugation. RNA synthesis was measured in mitochondria according to the method of Wilson et al. (Eur. J. Biochem. 242, 81-85, 1996) with the use of [3H]-UTP (specific radioactivity was 1332 TBq mol-1). Reactions were started by the addition of mitochondria and performed at 30 C. Protein was determined by the Lowry method. All kinetic data were obtained from at least 3 independent experiments. Statistical analysis was performed using Students paired t-test.
Figure 2 shows that the specific inhibitor of DNA topoisomerases I, camptothecin, caused substantial repression of RNA synthesis in isolated mitochondria in a dose-dependent manner. Bisbenzimidazoles Hoechst 33258 and Hoechst 33342 belong to the minor groove-binding compounds which are known to bind to the minor groove of DNA with A+T specificity and to cause widening of the minor grooves (Neidle et al., 1987, Biochem. J. 243:1-13). Hoechst 33342 has enhanced membrane permeability in comparison with Hoechst 33258 (Chen et al., 1993, Proc. Natl. Acad. Sci. USA 90:8131-8135). Figure 3 shows the influence of the bisbenzimidazoles at various concentrations on the transcriptional activity of mitochondria. It can be seen that both inhibitors of topoisomerase I repressed the activity of mitochondrial RNA synthesis in a dose-dependent manner, while Hoechst 33342 under, concentrations of 1 and 10 µM was more efficient as repressor of mitochondrial transcriptional activity in comparison with Hoechst 33258.
It is well known that DNA topoisomerases are important, often essential, cellular enzymes involved in nearly all aspects of DNA structure and metabolism (Berger, 1998, Biochim. Biophys. Acta 1400:3-18). The data obtained show clearly that DNA topoisomerase I is involved in transcription of mitochondrial genome under in vitro conditions. We suggest that an in organello RNA synthesis system of isolated maize mitochondria may serve as an additional model system for probing and studying anti-DNA topoisomerase I activities.
Financial support from the INTAS (Project
Number 97-0522) is acknowledged.
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