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.