Aberrant ribonucleotide incorporation and multiple deletions in mitochondrial DNA of the murine MPV17 disease model

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.

Parkinsonism and spastic paraplegia type 7: Expanding the spectrum of mitochondrial Parkinsonism

“…Instead, subjects with a complex phenotype, usually combining ataxia and PEO, had some of the highest levels of mtDNA among the SPG7 patients (250 per haploid nuclear DNA compared to 153 in pure spastic forms). Multiple deletions or depletion are alternative outcomes in a number of single‐gene mtDNA disorders, and in a mouse model of mtDNA disorder, multiple deletions and depletion have been reported in different tissues . The latter study led to the suggestion that mtDNA depletion is a consequence of slow DNA replication to prevent stalling and the formation of deletions and thus avoid the development of PEO.…”

Ribonucleotides in mitochondrial DNA

“…The estimated number of rNMPs per double-stranded mtDNA molecule ranges from 36 and 54 rNMPs in cultured HeLa and primary fibroblast cell lines, respectively [9], to 65 rNMPs in mouse liver [8]. In accordance with these numbers, Moss et al [65] reported a higher frequency of rNMPs in solid tissues than in cultured cell lines. The location of mtDNA rNMPs has been mapped using next-generation sequencing approaches, and they appear to be randomly distributed across the mammalian mitochondrial genome [9,65].…”

“…In accordance with these numbers, Moss et al . reported a higher frequency of rNMPs in solid tissues than in cultured cell lines. The location of mtDNA rNMPs has been mapped using next‐generation sequencing approaches, and they appear to be randomly distributed across the mammalian mitochondrial genome .…”

“… reported a higher frequency of rNMPs in solid tissues than in cultured cell lines. The location of mtDNA rNMPs has been mapped using next‐generation sequencing approaches, and they appear to be randomly distributed across the mammalian mitochondrial genome . There is no obvious strand bias in rNMP frequency between the two strands of mtDNA isolated from mouse tissues or from cultured human cells .…”

“…However, the correlation between rNTP/dNTP ratios and mtDNA rNMP frequency in mammals might not be as clear‐cut as in budding yeast. For example, although dNTP pools were only found to be altered in the liver of MPV17 knockout animals, additional tissues (the heart and brain) showed increased mtDNA rGMP incorporation that could not be explained by an increased rGTP pool . One possible explanation for why rNTP/dNTP ratios might not directly predict mtDNA rNMP incorporation is that the nucleotide levels measured from cellular or mitochondrial extracts might not accurately reflect the levels available to the mitochondrial replisome during mtDNA synthesis, for example, due to spatial and temporal variations in dNTP levels.…”