Short, single-stranded oligonucleotides mediate targeted nucleotide conversion using extracts from isolated liver mitochondria

Betsy T. Kren, Phillip Y. Wong, Clifford J Steer

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Site-specific single-nucleotide changes in chromosomal DNA of eukaryotic cells have been achieved using chimeric RNA/DNA oligonucleotides (ONs) and short single-stranded (SS) ONs. However, a variety of human diseases originate from single-point mutations in the genome of mitochondrial DNA. We previously demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate targeted single-nucleotide changes using chimeric ONs in vitro. However, different factor(s) and/or mechanism(s) appear to be involved in SS and RNA/DNA ON mediated DNA repair. Because mitochondria are deficient in certain factors involved in nuclear DNA repair pathways, we investigated whether mitochondria possess the enzymatic machinery for SS ON mediated DNA alterations. Using in vitro DNA repair assays based on mutagenized plasmids and a bacterial read-out system, SS ONs were designed to correct the point mutations in the genes encoded by the different plasmids. In this system, protein extracts from purified rat liver mitochondria and nuclei catalyzed similar levels of site-specific nucleotide modifications using SS ONs. Interestingly, extracts isolated from quiescent liver mediated significantly higher conversion rates than those isolated from regenerating liver. The results suggest that mitochondria contain the factors necessary for correction of single-point mutations by SS ONs. In addition, at least some are different than those required for DNA repair by RNA/DNA ONs. Moreover, correction with SS ONs appears to occur one strand at a time suggesting that repair of the DNA substrate involves strand transfer. The ability of unmodified SS ONs to mediate targeted alteration of the mitochondrial genome may provide a new tactic for treatment of certain mitochondrial-based diseases.

Original languageEnglish (US)
Pages (from-to)531-546
Number of pages16
JournalDNA Repair
Volume2
Issue number5
DOIs
StatePublished - May 13 2003

Keywords

  • Cell cycle
  • Cell-free extracts
  • Gapped plasmid
  • Gene/mismatch repair
  • Primer extension
  • Reporter gene constructs

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