Type I DNA-topoisomerase activity in mitochondria
--Tarasenko, VI; Konstantinov, YM

The type I DNA-topoisomerase (DNA-TI) plays a key role in control of the topological state of the DNA molecule to regulate its supercoiling, introducing a break into one of its chains. Such change is important for replication and transcription. Data have been obtained on involvement of the type I DNA-TI in regulation of genetic information expression by changing the topological state of regulatory sites of particular genes (Caserta et al., J. Cell Biochem. 55:93-97, 1994). The present communication reports characteristics of the type I DNA-TI found in the genetic system of mitochondria from hybrid VIR42MV. The activity of the enzyme was mostly revealed in the solubilized fraction at 0.5% concentrations of Triton X-100. The DNA-topoisomerase activity was not found in intact mitochondria suspensions and in postmitochondrial supernatant, thus supporting intra-mitochondrial localization of the enzyme studied.

One of the characteristic properties of the type I DNA-topoisomerases is a dependence of the enzyme activity on Mg²⁺ ions. The presence of magnesium is necessary for topoisomerases of procaryotes to work, while the activity of eucaryotic nuclear topoisomerases is not absolutely dependent on Mg²⁺ ions though is greatly stimulated. However, the mechanism of the effect of Mg²⁺ ions on the mitochondrial enzymes is not clear. Figure 1 presents data on dependence of the mitochondrial topoisomerase activity on the presence of magnesium and potassium ions in the reaction medium. The topoisomerase activity was found only in the presence of Mg²⁺ ions. Potassium ions had some inhibitory effect. The maximum activity was observed at 10-20 mM MgCl₂ (the data are not given). These results are in agreement with those of studies of type I TI from wheat mitochondria (Echeverria et al., Plant Mol. Biol. 6:417-427, 1986) and Chenopodium album (Meisner et al., Biochem. Intern. 27:1119-1125, 1992), also demonstrating an absolute dependence of the enzymes on Mg²⁺ ions. Since the interaction between the type I TI and the DNA molecule is actually electrostatic the ionic strength of the solution is important for the enzyme to exhibit its activity. Figure 2 shows the effect of KCl concentration on DNA relaxation by the topoisomerase. The maximum activity of the enzyme was observed at 50 mM KCl concentration. A further increase in the ionic strength of the solution had an inhibitory effect. Such dependence is typical of the type I plant topoisomerases. The effect of some inhibitors of the DNA-topoisomerase activity on mitochondrial DNA-TI has been studied in a particular set of experiments (Fig. 3). EDTA and ethidium bromide, which are characteristic inhibitors of procaryotic TI type I as well as type II TI, failed to affect the activity of the mitochondrial enzyme. Only very high concentrations of EDTA resulted in reduction of the activity (Fig. 3, lane 4). On the other hand, an addition of spermidine into the incubation medium caused certain decreases of the topoisomerase activity (Fig. 3, lanes 8-10). Such effect of this compound is considered to be characteristic of DNA-topoisomerases of a procaryotic origin in contrast to eucaryotic enzymes which are shown to be stimulated by polyamines (Sitailo, Biopolymers and Cell (Russia) 7:97-103, 1991). The enzyme studied can use as a substrate the positively supercoiled DNA of the pUC19 plasmid (the data are not given), which is a typical property of eucaryotic TI. We have also attempted to study the effect of non-ionic detergent, Triton X-100, on the activity of mtDNA-TI (Fig. 4). It is shown that plasmid DNA is relaxed easier in the presence of nonsolubilizing (0.02%) concentrations of Triton X-100 (Fig. 4, lane 4,5). It is generally accepted that Triton X-100 affects the conformational state of the DNA to separate locally the chains of the double helix of nucleic acid. Such conformational variation is likely to be responsible for the enhanced activity of the enzyme.

The data on properties of the mtDNA-TI type I are summarized in Table 1. The majority of the properties described are similar to those of eucaryotic nuclear type I topoisomerases. However, such properties of the enzyme as Mg²⁺ ion dependence and partial inhibition by polyamines are characteristic of DNA-topoisomerases I of procaryotes. As similar data were obtained for mitochondrial TI of wheat and Chenopodium album it is assumed that an independent form of the enzyme with properties intermediate between procaryotic and eucaryotic DNA-TI I exists in plant mitochondria. At present we are making efforts to purify the mtDNA-topoisomerase I to as high as a homogenic state as possible, in order to search for and isolate the gene encoding the enzyme.

Table 1. Properties of maize mitochondrial type I topoisomerase.
 

Temperature optimum	35 C
pH optimum	7.5
Relaxation of positive supercoils	Present
Influence of Mg2+	Absolute dependence
Optimum concentration of KCl	50 mM
Effect of inhibitors and detergents:
EDTA	Absence of inhibition
Ethidium bromide	Absence of inhibition
Spermidine	Partial inhibition
Triton X-100	Stimulation

Figure 1. The influence of Mg²⁺ and K⁺ cations on type I mtDNA topoisomerase activity: 1, MgCl₂ + KCl; 2, KCl; 3, MgCl₂; 4, control (without cations); 5, pUC19 plasmid DNA (without enzyme).

Figure 2. The dependence of type I mtDNA topoisomerase activity from ionic strength of KCl solution: 1, 0 mM; 2, 10 mM; 3, 30 mM; 4, 50 mM; 5, 70 mM; 6, 90 mM, 7, 120 mM; 8, 150 mM; 9, 180 mM; 10, 210 mM; 11, 250 mM; 12, 300 mM; 13, pUC19 plasmid DNA.

Figure 3. The influence of potential inhibitors on topoisomerase activity: 1, 2 mM EDTA; 2, 10 mM EDTA; 3, 50 mM EDTA; 4, 0.5 mkg/ml ethidium bromide; 5, 5 mkg/ml ethidium bromide; 6, 10 mkg/ml ethidium bromide; 7, 2 mM spermidine; 8, 5 mM spermidine; 9, 20 mM spermidine; 10, pUC19 plasmid DNA.

Figure 4. The effect of Triton X-100 on mitochondrial type I topoisomerase activity: 1, pUC19 plasmid DNA (without enzyme); 2, 2 units; 3, 0.2 units; 4, 2 units + 0.02% Triton X-100; 5, 0.2 units + 0.02% Triton X-100.

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