The presence of rNTPs decreases the speed of mitochondrial DNA replication

Abstract

Ribonucleotides (rNMPs) are frequently incorporated during replication or repair by DNA polymerases and failure to remove them leads to instability of nuclear DNA (nDNA). Conversely, rNMPs appear to be relatively well-tolerated in mitochondrial DNA (mtDNA), although the mechanisms behind the tolerance remain unclear. We here show that the human mitochondrial DNA polymerase gamma (Pol γ) bypasses single rNMPs with an unprecedentedly high fidelity and efficiency. In addition, Pol γ exhibits a strikingly low frequency of rNMP incorporation, a property, which we find is independent of its exonuclease activity. However, the physiological levels of free rNTPs partially inhibit DNA synthesis by Pol γ and render the polymerase more sensitive to imbalanced dNTP pools. The characteristics of Pol γ reported here could have implications for forms of mtDNA depletion syndrome (MDS) that are associated with imbalanced cellular dNTP pools. Our results show that at the rNTP/dNTP ratios that are expected to prevail in such disease states, Pol γ enters a polymerase/exonuclease idling mode that leads to mtDNA replication stalling. This could ultimately lead to mtDNA depletion and, consequently, to mitochondrial disease phenotypes such as those observed in MDS.

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.…”

“…As with nDNA duplication, single rNMPs are also incorporated during replication of mtDNA by Pol ɣ. Biochemical studies indicate that yeast Pol ɣ (Mip1) has an rNMP incorporation rate that is comparable to its nuclear counterparts (1 rNMP per 640 nucleotides in a long‐template assay where Pol ε and Pol δ insert 1 rNMP per 640 and 720 nucleotides, respectively) . Human Pol ɣ exhibits high discrimination against rNTPs and inserts only one rNMP per 2300 nucleotides in a similar assay . The persistent rNMPs found in mtDNA are therefore not due to unusually poor discrimination against rNTPs by the mtDNA polymerase.…”

“…One central reason might be the reverse transcriptase activity of Pol ɣ that allows it to efficiently replicate DNA templates containing incorporated rNMPs. Accordingly, in vitro analyses have shown that Pol ɣ bypasses single rNMPs embedded in a DNA template without any major negative effects on the processivity or fidelity of replication . In contrast, multiple consecutive rNMPs greatly reduce the efficiency of rNMP bypass by Pol ɣ.…”

“…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.…”