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

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 heavy- and 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 and genome stability. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may constitute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication Overall design: Mapping mitochondrial ribonucleotides in human cells (HeLa cells, healthy fibroblast cells as well as fibroblast cells from patients with mutations in TK2, MPV17 and DGUOK), via HydEn-seq (end sequencing of genomic fragments generated by alkaline hydrolysis). The genomic DNA of these cells were also digested with the ristriction enzyme HincII to allow for an estimation of the abosulte number of mitochondrial ribonucleotides in these lines.