Assuming mitochondrial DNA topoisomerase I (topo I) as one of the candidates for playing such a regulatory role, we have shown (MNL 73:39-40, 1999) the modulation of mitochondrial topo I activity under different redox conditions: an activation of DNA relaxation driven by topo I in the presence of reduced glutathione (GSH), and its significant repression following the addition of oxidised glutathione (GSSG). The aim of the present work was to verify our hypothesis that this redox modulation is realized at the level of DNA binding activity of enzyme.
Mitochondria were isolated from 4-day-old etiolated maize seedlings of hybrid VIR42 MV by a standard method of differential centrifugation. The method of topo I isolation was described earlier (MNL 73:40-41, 1999), with an additional stage of purification on non-denaturing PAGE. EMSA was carried out as described by Ikeda and Gray (Mol. Cell. Biol. 19:8113-8122, 1999), with minor modifications. Assays employed double stranded DNA probes containing regions recognized by eukaryotic topo I, as well as coxI and atp9 promoter fragments.
We have not found any DNA binding activity of purified topo I to DNA probes used in the standard assay conditions in the absence of redox agents. However, we have shown that the enzyme is able to form the stable complex with double stranded DNA in the presence of 5 mM GSH in the incubation mixture (Figure 1). In contrast, oxidised glutathione appeared not to be able to stimulate DNA binding activity of topo I. Furthermore, the presence of such redox agents as sodium dithionite and potassium ferricyanide has not led to induction of DNA binding (data not shown). Thus, data obtained point to a potential regulatory role played by the glutathione system in relation to mitochondrial topo I. This influence exists at the level of topo I DNA binding, which is the first stage of enzyme-DNA interaction. GSH might modulate topo I activity by favouring DNA-protein non-covalent complex stabilization.
It is not clear why we have not detected any DNA binding of topo I in the absence of GSH. We suppose that the complex formed between enzyme and DNA in such conditions is much less stable, and readily dissociates during the process of electrophoresis. It is also possible that the presence of GSH leads to changes in topo I affinity to one or another nucleotide sequence. Further investigations are needed to verify this hypothesis.
Financial support from the Russian Foundation for Basic Research (Grant No. 01-04-48162) is acknowledged.
1. The effect of reduced and oxidised glutathione on DNA binding activity
of mitochondrial topoisomerase I.
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