title: Mitochondrial DNA replication proceeds via a 'bootlace' mechanism involving the incorporation of processed transcripts.
abstract: The observation that long tracts of RNA are associated with replicating molecules of mitochondrial DNA (mtDNA) suggests that the mitochondrial genome of mammals is copied by an unorthodox mechanism. Here we show that these RNA-containing species are present in living cells and tissue, based on interstrand cross-linking. Using DNA synthesis in organello, we demonstrate that isolated mitochondria incorporate radiolabeled RNA precursors, as well as DNA precursors, into replicating DNA molecules. RNA-containing replication intermediates are chased into mature mtDNA, to which they are thus in precursor-product relationship. While a DNA chain terminator rapidly blocks the labeling of mitochondrial replication intermediates, an RNA chain terminator does not. Furthermore, processed L-strand transcripts can be recovered from gel-extracted mtDNA replication intermediates. Therefore, instead of concurrent DNA and RNA synthesis, respectively, on the leading and lagging strands, preformed processed RNA is incorporated as a provisional lagging strand during mtDNA replication. These findings indicate that RITOLS is a physiological mechanism of mtDNA replication, and that it involves a 'bootlace' mechanism, in which processed transcripts are successively hybridized to the lagging-strand template, as the replication fork advances.
authors.affiliation: MRC-Mitochondrial Biology Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Aurelio
authors.initials: A
authors.lastname: Reyes
authors.forename: Lawrence
authors.initials: L
authors.lastname: Kazak
authors.forename: Stuart R
authors.initials: SR
authors.lastname: Wood
authors.forename: Takehiro
authors.initials: T
authors.lastname: Yasukawa
authors.forename: Howard T
authors.initials: HT
authors.lastname: Jacobs
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Holt
mesh_terms: D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D003854:Deoxyribonucleotides / Q000378:metabolism; D005363:Ficusin / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D012313:RNA / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D000077278:RNA, Mitochondrial
chemical_list: D003432:Cross-Linking Reagents; D004272:DNA, Mitochondrial; D003838:Deoxyadenine Nucleotides; D003854:Deoxyribonucleotides; D012322:RNA Precursors; D000077278:RNA, Mitochondrial; C011746:3'-deoxyadenosine 5'-triphosphate; D012313:RNA; D005363:Ficusin