title: Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome.
abstract: The mitochondrial genome exists in numerous structural conformations, complicating the study of mitochondrial DNA (mtDNA) metabolism. Here, we describe the development of 2D intact mtDNA agarose gel electrophoresis (2D-IMAGE) for the separation and detection of approximately two-dozen distinct topoisomers. Although the major topoisomers were well conserved across many cell and tissue types, unique differences in certain cells and tissues were also observed. RNase treatment revealed that partially hybridized RNAs associated primarily with covalently closed circular DNA, consistent with this structure being the template for transcription. Circular structures composed of RNA:DNA hybrids contained only heavy-strand DNA sequences, implicating them as lagging-strand replication intermediates. During recovery from replicative arrest, 2D-IMAGE showed changes in both template selection and replication products. These studies suggest that discrete topoisomers are associated with specific mtDNA-directed processes. Because of the increased resolution, 2D-IMAGE has the potential to identify novel mtDNA intermediates involved in replication or transcription, or pathology including oxidative linearization, deletions or depletion.
authors.affiliation: Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street VET220E, Philadelphia, PA 19104, USA.
authors.forename: Jill E
authors.initials: JE
authors.lastname: Kolesar
authors.forename: Catherine Y
authors.initials: CY
authors.lastname: Wang
authors.forename: Yumiko V
authors.initials: YV
authors.lastname: Taguchi
authors.forename: Shih-Hsuan
authors.initials: SH
authors.lastname: Chou
authors.forename: Brett A
authors.initials: BA
authors.lastname: Kaufman
mesh_terms: D000818:Animals; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects; D026942:DNA Topoisomerases / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000032:analysis / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004996:Ethidium / Q000494:pharmacology; D054629:Genome, Mitochondrial; D006801:Humans; D051379:Mice; D012313:RNA / Q000737:chemistry
chemical_list: D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D012313:RNA; D026942:DNA Topoisomerases; D004996:Ethidium