Old Dominion University ODU Digital Commons Theses and Dissertations in Biomedical Sciences College of Sciences Summer 2000 Mechanistic Characterization of the Mitochondrial Type I DNA Topoisomerase and a Study of Genes Containing Type I DNA Topoisomerase-Related Domains Jaydee Dones Cabral Old Dominion University Follow this and additional works at: https://digitalcommons.odu.edu/biomedicalsciences_etds Part of the Biochemistry Commons Recommended Citation Cabral, Jaydee D.. "Mechanistic Characterization of the Mitochondrial Type I DNA Topoisomerase and a Study of Genes Containing Type I DNA Topoisomerase-Related Domains" (2000). Doctor of Philosophy (PhD), Dissertation, , Old Dominion University, DOI: 10.25777/fjx3-kb57 https://digitalcommons.odu.edu/biomedicalsciences_etds/11 This Dissertation is brought to you for free and open access by the College of Sciences at ODU Digital Commons. It has been accepted for inclusion in Theses and Dissertations in Biomedical Sciences by an authorized administrator of ODU Digital Commons. For more information, please contact [email protected]. MECHANISTIC CHARACTERIZATION OF THE MITOCHONDRIAL TYPE IDNA TOPOISOMERASE AND A STUDY OF GENES CONTAINING TYPE I DNA TOPOISOMERASE-RELATED DOMAINS by Jaydee Dones Cabral B.S. May 1994, College of William and Mary M.A August 1995, College of William and Mary A Dissertation Submitted to the Faculty of Old Dominion University and Eastern Virginia Medical School in Partial Fulfillment of the Requirement for the Degree of DOCTOR OF PHILOSOPHY BIOMEDICAL SCIENCES OLD DOMINION UNIVERSITY and EASTERN VIRGINIA MEDICAL SCHOOL August 2000 Approve^b Frank J. Cetera (Director) Mark S. Elliott (Member) William Wasilenko (Member) Howard u. wtnte (Memoer) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT MECHANISTIC CHARACTERIZATION OF THE MITOCHONDRIAL TYPE I DNA TOPOISOMERASE AND A STUDY OF GENES CONTAINING TYPE I DNA TOPOISOMERASE-RELATED DOMAINS Jaydee Dones Cabral Old Dominion University and Eastern Virginia Medical School 2000 Director Dr. Frank J. Castora 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 anti fungal 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-topoL, 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 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. antifungal/antimicrobial activity were examined using standard DNA relaxation assays. One of these drugs, designated Cl, 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 phyiogeny 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. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To my grandmother, Ligaya Morales, who taught me to value knowledge, and to my parents, Gil and Carmelita Cabral, who showed me, by example, the merits of hard work and perseverance. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS I extend many thanks to Dr. Castora for his guidance and support. I thank my dissertation committee, Dr. Elliott, Dr. Wasilenko, and Dr. White for their advice on my research and editing of my dissertation. I thank Dr. Rosenthal for her help with my research efforts. Thanks are also given to Barbara Conyers and Jeffrey Stephens, for their friendship. I thank my family for their love and support To my sister, Gigi, thanks for the photographs and slide work. And, thank you, Bret for your encouragement patience, and love. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Page LIST OF TABLES............................................................................................................. ix LIST OF FIGURES........................................................................................................... xi LIST OF GRAPHS..........................................................................................................xiii Chapter I. INTRODUCTION................................................................................................I BACKGROUND AND SIGNIFICANCE.................................................1 STATEMENT OF PROBLEM...............................................................10 II. MATERIALS AND METHODS......................................................................12 EQUIPMENT..........................................................................................12 MATERIALS..........................................................................................13 METHODS..............................................................................................13 Human Placental Tissue RNA Isolation ........................................13 Probe and Primer Design ...............................................................14 RT-PCR..........................................................................................17 Plaque PCR ....................................................................................19 pGEM Vector Cloning ...................................................................19 Cycle Sequencing ...........................................................................22 Bioinformatics Programs .............................................................. 23 Northern Hybridization ................................................................. 25 Transcript Synthesis, Labeling, and Detection ............................. 27 cDNA Library Screen................................................................... 28 Fluorescence In Situ Hybridization .............................................. 30 3’ Rapid Amplification of cDNA Ends ......................................... 31 Type I DNA Topoisomerase Isolation from Calf Liver ................ 33 5’ and 3’ End Labeling of dsDNA ................................................ 33 Standard Topoisomerase Relaxation Assay .................................. 33 Cleavage Assay ............................................................................. 34 K+SDS‘ Precipitation .................................................................... 36 Drug Studies .................................................................................. 38 III. RESULTS........................................................................................................40 MECHANISTIC ANALYSIS................................................................. 41 Purification of the Mitochondrial Type I DNA Topoisomerase... 41 Cleavage Assay ............................................................................