title: Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase.
abstract: We used engineered zinc finger peptides (ZFPs) to bind selectively to predetermined sequences in human mtDNA. Surprisingly, we found that engineered ZFPs cannot be reliably routed to mitochondria by using only conventional mitochondrial targeting sequences. We here show that addition of a nuclear export signal allows zinc finger chimeric enzymes to be imported into human mitochondria. The selective binding of mitochondria-specific ZFPs to mtDNA was exemplified by targeting the T8993G mutation, which causes two mitochondrial diseases, neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and also maternally inherited Leigh's syndrome. To develop a system that allows the monitoring of site-specific alteration of mtDNA we combined a ZFP with the easily assayed DNA-modifying activity of hDNMT3a methylase. Expression of the mutation-specific chimeric methylase resulted in the selective methylation of cytosines adjacent to the mutation site. This is a proof of principle that it is possible to target and alter mtDNA in a sequence-specific manner by using zinc finger technology.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom. mminczuk@mrc-lmb.cam.ac.uk
authors.forename: Michal
authors.initials: M
authors.lastname: Minczuk
authors.forename: Monika A
authors.initials: MA
authors.lastname: Papworth
authors.forename: Paulina
authors.initials: P
authors.lastname: Kolasinska
authors.forename: Michael P
authors.initials: MP
authors.lastname: Murphy
authors.forename: Aaron
authors.initials: A
authors.lastname: Klug
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D016335:Zinc Fingers
chemical_list: D004272:DNA, Mitochondrial; D011994:Recombinant Proteins; D015254:DNA Modification Methylases