Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase.

TitleSequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase.
Publication TypeJournal Article
Year of Publication2006
AuthorsMinczuk, MA, Papworth, MA, Kolasinska, P, Murphy, MP, Klug, A
JournalProc Natl Acad Sci U S A
Volume103
Issue52
Pagination19689-94
Date Published2006 Dec 26
ISSN0027-8424
KeywordsAmino Acid Sequence, Animals, Base Sequence, Cell Line, Cercopithecus aethiops, DNA Methylation, DNA Modification Methylases, DNA, Mitochondrial, Mitochondria, Models, Molecular, Mutation, Protein Binding, Protein Structure, Tertiary, Protein Transport, Recombinant Proteins, Substrate Specificity, Zinc Fingers
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.

DOI10.1073/pnas.0609502103
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
Citation Key10.1073/pnas.0609502103
PubMed ID17170133
PubMed Central IDPMC1750892
Grant ListMC_U105184324 / / Medical Research Council / United Kingdom
MC_U105663142 / / Medical Research Council / United Kingdom