Mammalian mitochondrial DNA topoisomerase I preferentially relaxes supercoils in plasmids containing specific mitochondrial DNA sequences

• Published in: Biochimica et biophysica acta 1995-12, Vol.1264 (3), p.377-387
• Main Authors: Topcu, Zeki, Castora, Frank J.
• Format: Article
• Language: English
• Subjects: Animals; Base Composition; Base Sequence; Cattle; DNA Topoisomerases, Type I - biosynthesis; DNA Topoisomerases, Type I - isolation & purification; DNA Topoisomerases, Type I - metabolism; DNA, Mitochondrial - analysis; Genetic Vectors; Kinetic analysis topoisomerase I; Kinetics; Mitochondria, Liver - enzymology; Mitochondrion; Molecular Sequence Data; Plasmids - genetics; Sequence preference; Substrate Specificity
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/0167-4781(95)00180-8

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 K m values for pZT-Hori, pZT-Lori and pZT-Term were 21.0 ± 0.9 μM, 25.2 ± 1.0 μM and 17.0 ± 0.8 μM, respectively, while those for control plasmids were 57.5 ± 2.1 μM and 56.3 ± 2.3 μM. pZT-Cytb was not preferentially relaxed compared to the control plasmid ( K m = 53.4 ± 2.0 μM vs. 56.3 ± 2.3 μM, 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.