Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired

Misincorporation of ribonucleotides into DNA during genome replication has recently become recognized as a significant source of genomic instability. The frequency of ribonucleotides in the genome is determined by dNTP/rNTP ratios, the ability of the DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove misincorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by imbalancing cellular dNTP pools. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for repair of misincorporated ribonucleotides. Mapping ribonucleotides in DNA from yeast that have unbalanced dNTP pools with HydEn-seq (5´end sequencing of genomic fragments generated by alkaline hydrolysis). The genomic DNA of these cells were also digested with the ristriction enzyme PmeI to allow for an estimation of the abosulte number of mitochondrial ribonucleotides in these lines.