title: Mapping topoisomerase sites in mitochondrial DNA with a poisonous mitochondrial topoisomerase I (Top1mt).
abstract: Mitochondrial topoisomerase I (Top1mt) is a type IB topoisomerase present in vertebrates and exclusively targeted to mitochondria. Top1mt relaxes mitochondrial DNA (mtDNA) supercoiling by introducing transient cleavage complexes wherein the broken DNA strand swivels around the intact strand. Top1mt cleavage complexes (Top1mtcc) can be stabilized in vitro by camptothecin (CPT). However, CPT does not trap Top1mtcc efficiently in cells and is highly cytotoxic due to nuclear Top1 targeting. To map Top1mtcc on mtDNA in vivo and to overcome the limitations of CPT, we designed two substitutions (T546A and N550H) in Top1mt to stabilize Top1mtcc. We refer to the double-mutant enzyme as Top1mt*. Using retroviral transduction and ChIP-on-chip assays with Top1mt* in Top1mt knock-out murine embryonic fibroblasts, we demonstrate that Top1mt* forms high levels of cleavage complexes preferentially in the noncoding regulatory region of mtDNA, accumulating especially at the heavy strand replication origin OH, in the ribosomal genes (12S and 16S) and at the light strand replication origin OL. Expression of Top1mt* also caused rapid mtDNA depletion without affecting mitochondria mass, suggesting the existence of specific mitochondrial pathways for the removal of damaged mtDNA.
authors.affiliation: From the Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Dalla Rosa
authors.affiliation: From the Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Shar-Yin N
authors.initials: SY
authors.lastname: Huang
authors.affiliation: From the Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Keli
authors.initials: K
authors.lastname: Agama
authors.affiliation: From the Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Salim
authors.initials: S
authors.lastname: Khiati
authors.affiliation: From the Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Hongliang
authors.initials: H
authors.lastname: Zhang
authors.affiliation: From the Laboratory of Molecular Pharmacology, Developmental Therapeutics Branch, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892 pommier@nih.gov.
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D000818:Animals; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid
chemical_list: D004272:DNA, Mitochondrial; D004264:DNA Topoisomerases, Type I
keywords: Chromatin Immunoprecipitation (ChiP); DNA Topoisomerase; DNA Topology; Mitochondrial DNA (mtDNA); Mitochondrial Metabolism; Top1mt; mtDNA Transcription