Date of Award

Summer 2000

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Frank J. Castora

Committee Member

Mark S. Elliot

Committee Member

William Wasilenko

Committee Member

Howard D. White

Abstract

The mitochondrial type I DNA topoisomerase (mt-topo I) serves an important function in the mitochondrion by relaxing mtDNA supercoils to allow for replication of the mitochondrial genome as well as for gene expression. The mt-topo I's role in essential processes, such as replication and transcription, makes it an ideal candidate as a target for antitumor or antifungal drugs. To gain further insight into mt-topo I mechanism, a cleavage assay and drug inhibition studies were performed. As well, a search for the mt-topo I gene or genes containing type I topoisomerase-related domains was conducted. To characterize the mt-topo I mechanism, the 5′ or 3′ binding of the mt-topo I to cleaved DNA was assessed by electrophoresis on an agarose gel after treatment with and without proteinase K (PK). The results of the cleavage assay revealed that the mt-topo I formed a covalent linkage to the 5′ end of cleaved DNA. In addition, drug inhibition studies were performed to aid in distinguishing between the mt-topo I and the nuclear type I topoisomerase (nu-topo I). In agreement with earlier work with the calf thymus mt-topo I, the calf liver mt-topo I was found to be slightly more sensitive to inhibition by dimethyl sulfoxide (DMSO) than was the nuclear enzyme. In an attempt to identify new drugs that might selectively inhibit the mt-topo I, a series of seven compounds with antifungal/antimicrobial activity were examined using standard DNA relaxation assays. One of these drugs, designated C1, was found to inhibit the nuclear topo I but not the mitochondrial enzyme and thus might serve as a useful tool to discriminate between these enzymes. To search for genes containing type I topoisomerase-related domains, reverse transcriptase polymerase chain reaction (RT-PCR) was performed to amplify conserved eukaryotic and prokaryotic topoisomerase I domains using primers that were homologous to these conserved regions. An amplified topo-related fragment, 575-1, which possessed a 57% similarity to a topo II signature, 56% similarity to a prokaryotic type I topo signature, and no similarity to the known nu-topo I, as indicated by prositescan searches, was used as a probe to screen a human cDNA library and in a northern hybridization. Fragment 575-1 detected a large 9 kb message which was three times the size of known type I topoisomerase messages. Due to the large message size, a further investigation to compare 575-1 to known eukaryote type I topoisomerase species was performed using the computational biology program, MEME. The program MEME finds conserved motifs in a group of DNA sequences. An RT-PCR positive control nu-topo I fragment, designated 575-6, was detected and included in the MEME run. 575-6, matched motif 1 out of 6 motifs identified by MEME. 575-1 contained none of six type I topoisomerase motifs identified. The program, Dnapars, revealed via an unrooted phylogeny tree that 575-1 was distantly related when compared to seven known eukaryotic type I topoisomerase species. In conclusion, the mt-topo I gene is fundamentally different from known eukaryotic type I topoisomerases. Degenerate primers developed from eukaryotic type I topoisomerase conserved domains were unable to amplify a region of the mt-topo I gene in RT-PCR reactions, although the known nu-topo I was detected.

Comments

Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/fjx3-kb57

ISBN

9780493079035

Included in

Biochemistry Commons

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