Date of Award
Doctor of Philosophy (PhD)
Frank J. Castora
Mark S. Elliot
Christopher J. Osgood
Roger D. Nolan
Topoisomerases are DNA-modifying enzymes found in prokaryotes, eukaryotes, viruses and organelles such as chloroplast and mitochondria. Information about these enzymes in eukaryotic systems is mostly limited to nuclear enzymes, although our laboratory has been characterizing the biochemical and biophysical properties of the mammalian mitochondrial topoisomerases. We have determined the polarity of the attachment of mitochondrial topoisomerase I to its substrate DNA. To study the substrate preference and kinetic parameters of mitochondrial topoisomerase I, selected regions of mammalian mitochondrial DNA (mtDNA) were inserted into pGEM plasmid vectors following a series of modification and optimization experiments of currently available methods for PCR-cloning. These mtDNA containing recombinant plasmids were used in a kinetic analysis of the highly purified enzyme. 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. Two other recombinant plasmids, containing non-mitochondrial DNA inserts (pZT-800 and pZT-400) served as control substrates. Southern hybridization using probes specific for either control or mtDNA-containing plasmids 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 μΜ, 25.2 +/- 1.0 μM and 17.0 +/- 0.8 μΜ respectively, while those for control plasmids were 57.5 +/- 2.1 μΜ and 56.3 +/- 2.3 μΜ. pZT-Cytb was not preferentially relaxed compared to the control plasmid (Km= 53.4 +/- 2.0 μΜ vs.56.3 +/- 2.3 μΜ 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.
"Investigation of the Substrate Recognition Characteristics and Kinetics of Mammalian Mitochondrial DNA Topoisomerase I"
(1995). Doctor of Philosophy (PhD), dissertation, , Old Dominion University, DOI: 10.25777/sfsc-rb54