Date of Award
Doctor of Philosophy (PhD)
Chemistry and Biochemistry
Frank J. Castora
Richard P. Ciavarra
Mark S. Elliot
Laura K. Moen
Mammalian mitochondria contain a unique DNA topoisomerase I with characteristics of both nuclear and bacterial type I DNA topoisomerases. This enzyme, purified from bovine liver, requires cations as cofactors and possesses optimal activity at pH 7.0, 37°C and either 150 mM K+ or Na+ ions. Similar to nuclear enzymes and different from the bacterial enzyme, divalent ions such as Ca2+ or Mg2+ will stimulate but are not essential for the enzymatic activity.
Like the nuclear DNA topoisomerase I, the mitochondrial enzyme is capable of relaxing both negatively and positively supercoiled DNA. The relaxation of negatively supercoiled DNA is directly inhibited by camptothecin and indirectly by m- AMSA, a DNA intercalator. The relaxation of positively supercoiled DNA is also inhibited by camptothecin, however, with 10 to 20 fold less efficiency. Like all DNA topoisomerases, the mitochondrial enzyme forms a transient enzyme-substrate complex through a phosphotyrosyl covalent linkage. This linkage was found to be at the 5' terminus of the DNA break, a manner similar to the bacterial DNA topoisomerase I . Mitochondrial DNA topoisomerase I slightly cross-reacts with antibody raised against nuclear DNA topoisomerase I indicating the partial similarity of the structure of the mitochondrial and nuclear enzymes. Recognition of the DNA substrate was found to be nonrandom judging from the distinct cleavage patterns of DNA cleavage assay results. Analysis of the sequences around 79 sites cleaved by the nuclear and mitochondrial enzyme indicated no consensus sequences, though a preference of T (43%, P = 0.01) at the -1 position was found.
There were three sites showing a strong preference for cleavage by this mitochondrial DNA topoisomerase I. All three had a G at the -1 position. Furthermore, these sequences can form a cruciform structure with distinct features no other cleavage sequences analyzed so far possess. Mitochondrial DNA topoisomerase I can bind to both singlestranded and double-stranded DNA with a preference for the latter. This preference differs from both the nuclear enzyme, which has no preference for either forms of DNAs, and the bacterial enzyme, which prefers single stranded DNA. A DNA topoisomerase II has also been partially purified from calf thymus mitochondria. This enzyme unknots DNA in the presence of ATP. Two type II DNA topoisomerase-targeting antitumor drugs, m-AMSA and VM-26, are able to inhibit the unknotting activity at concentrations above 0.5 /uM.
"Studies on Type I and Type II DNA Topoisomerases From Mammalian Mitochondria"
(1993). Doctor of Philosophy (PhD), Dissertation, Chemistry and Biochemistry, Old Dominion University, DOI: 10.25777/hyxs-sx24