Mammalian mitochondrial DNA topoisomerase I preferentially relaxes supercoils in plasmids containing specific mitochondrial DNA sequences.
Selected regions of mammalian mitochondrial DNA (mtDNA) were inserted into pGEM plasmid vectors and used as substrates in a kinetic analysis of the highly purified bovine mitochondrial type I topoisomerase. Recombinant plasmids containing the bovine mtDNA heavy and light strand origins of replication (pZT-Hori and pZT-Lori, respectively), a major transcription termination region (pZT-Term) and a portion of cytochrome b gene (pZT-Cytb) were prepared. Southern hybridization using probes specific for either control or mtDNA-containing plasmid indicated a relative preference by the mitochondrial topoisomerase I to relax supercoils in pZT-Hori and pZT-Term. Quantitative determination of kinetic parameters derived from double-reciprocal Lineweaver-Burk plots showed that recombinant plasmids containing the heavy and light strand origins and the transcription termination region were preferentially relaxed by the mitochondrial enzyme with Km values 2.3- to 3.3-fold lower than controls. The Km values for pZT-Hori, pZT-Lori and pZT-Term were 21.0 +/- 0.9 microM, 25.2 +/- 1.0 microM and 17.0 +/- 0.8 microM, respectively, while those for control plasmids were 57.5 +/- 2.1 microM and 56.3 +/- 2.3 microM. pZT-Cytb was not preferentially relaxed compared to the control plasmid (Km = 53.4 +/- 2.0 microM vs. 56.3 +/- 2.3 microM, respectively) indicating that mitochondrial topoisomerase I preferentially interacts with certain mtDNA sequences but not others. Identical experiments with the purified nuclear enzyme did not differentiate between control or mtDNA containing plasmids.
Department of Biochemistry, Eastern Virginia Medical School, Norfolk 23507-1696, USA.
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D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D002417:Cattle; D004264:DNA Topoisomerases, Type I / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000032:analysis*; D005822:Genetic Vectors; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D013379:Substrate Specificity
D004272:DNA, Mitochondrial; D004264:DNA Topoisomerases, Type I