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Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA
Published on: November 14, 2017
Short, single-stranded oligonucleotides mediate targeted nucleotide conversion using extracts from isolated liver
Betsy T Kren1, Phillip Y Wong, Clifford J Steer
1Department of Medicine, University of Minnesota Medical School, Minneapolis, MN 55455, USA. krenx001@tc.umn.edu
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.
Insights
Mitochondria possess enzymes for correcting single-point mutations using single-stranded oligonucleotides (ONs). This DNA repair mechanism differs from that used by chimeric ONs and may offer new treatments for mitochondrial diseases.
Area of Science:
- Mitochondrial Biology
- Molecular Genetics
- DNA Repair Mechanisms
Background:
- Human diseases often stem from single-point mutations in mitochondrial DNA.
- Previous work showed rat liver mitochondria can modify DNA using chimeric oligonucleotides (ONs).
- Nuclear and mitochondrial DNA repair pathways involve different factors and mechanisms.
Purpose of the Study:
- To investigate if mitochondria have the enzymatic machinery for single-stranded (SS) oligonucleotide-mediated DNA alterations.
- To determine if SS ONs can correct point mutations in mitochondrial DNA.
Main Methods:
- In vitro DNA repair assays using mutagenized plasmids.
- A bacterial read-out system to detect nucleotide modifications.
- Protein extracts from purified rat liver mitochondria and nuclei were utilized.
Main Results:
- Mitochondrial and nuclear extracts showed similar levels of site-specific nucleotide modifications with SS ONs.
- Quiescent liver extracts yielded higher conversion rates than regenerating liver extracts.
- SS ON-mediated correction appears to be a single-strand, strand-transfer process.
Conclusions:
- Mitochondria possess factors necessary for correcting single-point mutations using SS ONs.
- These factors differ from those required for chimeric ON-mediated DNA repair.
- Unmodified SS ONs may offer a novel therapeutic strategy for mitochondrial diseases.
