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

<div><p>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. 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.</p></div

Identification of free 5´- ends at OriH and ribonucleotides at OriL.

<p>(A) Reads per million on H-strand (upper panel) and L-strand (lower panel) at OriH using 5´-End-seq, (B) Reads per million on L-strand at OriL using HydEn-seq (upper panel) and 5´-End-seq (lower panel).</p

Ribonucleotide incorporation in mtDNA from fibroblasts from patients with genetic defects in <i>TK2</i>, <i>DGUOK</i> or <i>MPV17</i>.

<p>(A) Hierarchical clustering of HydEn-seq libraries (undigested or digested with HincII). (B) Ribonucleotide content for H-strand (H) and L-strand (L) for patient-derived mutant cell lines. (C) Ribonucleotide incorporation frequency per 1,000 bases of its complementary nucleotides calculated on H-strand and L-strand for fibroblasts and patient-derived mutant lines. (D) Quantification of ribonucleotides in H-strand and L-strand per mtDNA molecule in fibroblasts and patient-derived mutant lines.</p