IRKUTSK, RUSSIA

Institute of Plant Physiology and Biochemistry

Characterization of nuclear and mitochondrial DNA topoisomerases I --Konstantinov, YM, Tarasenko VI It is well known that along with DNA and RNA polymerases DNA topoisomerases are of key importance in the fundamental genetic processes such as replication, transcription, recombination and repair. Once deprived of topoisomerases the cell fails to make up for them and thus perishes. Besides the nucleus, topoisomerases are found in plant mitochondria and chloroplasts. No molecular biological studies of maize topoisomerases I of nuclear and mitochondrial localization have been made up to the present. Neither topoisomerase I gene structure, nor the features of their protein products, is known.

We have previously described (MNL 71:39-40,1997; MNL 73:39-41, 1999) some characteristics of mitochondrial type I DNA-topoisomerase including its sensitivity to different type inhibitors and redox conditions. The aim of the present work was to investigate some characteristics of nuclear topoisomerase I in comparison with the enzyme of mitochondrial localization.

Nuclei were prepared from 3-day-old etiolated maize seedlings of hybrid VIR42 MV generally as described earlier (Chiatante, Bryant, J. Exp. Bot. 45:959-965, 1994). The purification of topo I from isolated nuclei included the stages of organelle solubilization, ammonium sulfate fractionation, chromatography on the column with DEAE-Toyopearl, and chromatography on the column with single-stranded-DNA-cellulose. The mitochondria were isolated by a standard method of differential centrifugation. The method of topo I purification from mitochondria was the same as described earlier (MNL 73: 40-41, 1999). Protein was determined by the Lowry method.

We have previously reported (MNL 73: 39-40, 1999) about the redox modulation of mitochondrial topo I activity under different redox conditions. The study of the influence of redox conditions on the activity of nuclear topo I showed that this enzyme has similar sensitivity to the redox agents used as compared with mitochondrial enzyme. The addition of both oxidising and reducing agents caused changes in the activity of topo I from the nucleus. An activation of topo I was shown in the presence of reduced agents such as sodium dithionite or reduced glutathione and its significant repression following the addition of oxidised agents such as potassium ferricyanide or oxidised glutathione.

We have studied also the effects of different topo I inhibitors (camptothecin, distamycin A, netropsin, bis-netropsines, Hoechst33258, Hoechst33342) on the relaxation activity of this enzyme. There were no significant differences between nuclear and mitochondrial topo I in sensitivity to these inhibitors. However, nuclear topo I can be distinguished from its mitochondrial counterpart by its different affinity to single stranded-DNA-cellulose. In contrast to mitochondrial topo I, which binds only to double-stranded-DNA-cellulose, enzyme from nuclei readily binds to this nucleic acid resin. For plant nuclear and mitochondrial topo I, it is the only known difference between these enzymes, which might point to dissimilarities in their protein structure.

Financial support from the INTAS (Project Number 97-0522) is acknowledged.
 
 


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