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

Abstract

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.

Ribonucleotides in mitochondrial DNA

“…The persistent rNMPs found in mtDNA are therefore not due to unusually poor discrimination against rNTPs by the mtDNA polymerase. As has been found for many other DNA polymerases, the exonuclease activity of Pol ɣ does not contribute to rNMP removal .…”

“…The rNMPs found in mature mtDNA are therefore not related to the lengthy RNA species that temporarily coat the displaced H-strand in the RITOLS model nor are they longer RNA primers left unremoved after replication initiation. 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.…”

“…Our two genomes show striking dissimilarity with regard to ribonucleotide contentwhile rNMPs are virtually absent from nDNA, mature mammalian mtDNA contains an incorporated rNMP approximately every 500-900 nucleotides depending on the source material [8,9]. In vitro, the human mtDNA polymerase incorporates an rNMP every 2000-2300 nucleotides, which is comparable or even lower than the rNMP incorporation frequency of nuclear DNA polymerases [8][9][10], although comparisons are complicated by variations in assays and reaction conditions. Rather than differential rNMP incorporation, the difference in rNMP content between our two genomes is therefore mainly ascribed to the absence of efficient rNMP removal mechanisms in the mitochondria.…”

Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization

“…Southern blotting followed by probe hybridization has been a steadfast tool to study restriction fragment length polymorphisms and detect rearrangements in immunoglobulin and T cell receptor genes (Current Protocols article: Brown, ). Southern blotting is one of the best‐described methods for analyzing human mtDNA maintenance (Berglund et al., ; Chen & Cheng, ; Hayashi et al., ; Holt et al., ; Kaukonen et al., ; Kornblum et al., ; Lamantea et al., ; Lehtinen et al., ; Luoma et al., ; Moraes et al., , ; Moretton et al., ; Peeva et al., ; Rocher et al., ; Ronchi et al., ; Schon et al., ; Shokolenko et al., ; Song et al., ; Tengan & Moraes, ; Wallace et al., ). Skeletal muscle mtDNA deletions are associated with the mitochondrial disease progressive external ophthalmoplegia (PEO) and traditionally have been detected via Southern blot analyses (Kaukonen et al., ; Lamantea et al., ; Moraes et al., ; Moslemi, Melberg, Holme, & Oldfors, ).…”

“…Studies utilizing Southern blotting have proven to be powerful tools to assess mtDNA maintenance in human cell culture and patient samples (Berglund et al., ; Chen & Cheng, ; Hayashi, Takemitsu, Goto, & Nonaka, ; Holt, Dunbar, & Jacobs, ; Kaukonen et al., ; Kornblum et al., ; Lamantea et al., ; Lehtinen et al., ; Luoma et al., ; Moraes et al., ; Moraes, Atencio, Oca‐Cossio, & Diaz, ; Moretton et al., ; Peeva et al., ; Rocher et al., ; Ronchi et al., ; Schon, Naini, & Shanske, ; Shokolenko et al., ; Song, Wheeler, & Mathews, ; Tengan & Moraes, ; Wallace et al., ) as well as in model organisms such as mice and yeast (Griffiths, Doudican, Shadel, & Doetsch, ; Hance, Ekstrand, & Trifunovic, ; Milenkovic et al., ; Trifunovic et al., ; Tyynismaa et al., , ; Young, Theriault, Li, & Court, ). Here we describe a straightforward Southern blotting and nonradioactive probe hybridization method to estimate the quantity of mtDNA in human genomic DNA samples.…”