Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA

Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these.We find that ribonucleotides are distributed evenly between the heavyand light-strand of mtDNA.The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis.The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides.Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells.Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication.Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication. Author summaryHuman mitochondria contain a small double-stranded DNA genome (mtDNA) of only 16,569 base pairs (bp) that encodes 13 essential subunits of the oxidative phosphorylation system.Depletion of mtDNA and different types of mtDNA mutations cause mitochondrial disease, and are also implicated in biological ageing.For almost half a century it has been known that mtDNA contains ribonucleotides, but their identity and precise location are not known.The source of these ribonucleotides and their relevance for mitochondrial genome stability in healthy individuals and in patients with mitochondrial defects has not been addressed.We have used a combination of next-generation sequencing, and in vivo