Abstract
All DNA polymerases misincorporate ribonucleotides despite their preference for deoxyribonucleotides, and analysis of cultured cells indicates that mammalian mitochondrial DNA (mtDNA) tolerates such replication errors. However, it is not clear to what extent misincorporation occurs in tissues, or whether this plays a role in human disease. Here, we show that mtDNA of solid tissues contains many more embedded ribonucleotides than that of cultured cells, consistent with the high ratio of ribonucleotide to deoxynucleotide triphosphates in tissues, and that riboadenosines account for three-quarters of them. The pattern of embedded ribonucleotides changes in a mouse model of Mpv17 deficiency, which displays a marked increase in rGMPs in mtDNA. However, while the mitochondrial dGTP is low in the Mpv17−/− liver, the brain shows no change in the overall dGTP pool, leading us to suggest that Mpv17 determines the local concentration or quality of dGTP. Embedded rGMPs are expected to distort the mtDNA and impede its replication, and elevated rGMP incorporation is associated with early-onset mtDNA depletion in liver and late-onset multiple deletions in brain of Mpv17−/− mice. These findings suggest aberrant ribonucleotide incorporation is a primary mtDNA abnormality that can result in pathology.
Highlights
Mammalian mitochondrial DNA is a small covalently closed circular molecule of ∼16 kb, encoding 13 essential components of the oxidative phosphorylation system (OXPHOS)
Ribonucleotide incorporation in mitochondrial DNA (mtDNA) is substantially higher in solid tissues than cultured cells
The assays confirmed the presence of numerous rNMPs scattered throughout the molecules (Figure 1, Supplementary Figure S1), and showed that the fragmentation was more marked for the mtDNA of solid tissues than for proliferating cells (Figure 1), indicating that ribosubstitution of mtDNA is considerably more prevalent in vivo, in post-mitotic tissues
Summary
Mammalian mitochondrial DNA (mtDNA) is a small covalently closed circular molecule of ∼16 kb, encoding 13 essential components of the oxidative phosphorylation system (OXPHOS). OXPHOS provides the bulk of the cell’s energy in the form of ATP, a reduction in the amount (depletion) or quality of the mtDNA (ranging from point mutations to multiple deletions) can cause an energy crisis and human pathologies [1].
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