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.
Highlights
The replication of DNA is a highly accurate process where free deoxyribonucleoside triphosphates are incorporated opposite their complementary base
Certain forms of mtDNA depletion syndrome (MDS) are caused by imbalances in the mitochondrial deoxyribonucleoside triphosphate pool, which have been shown to lead to altered levels of the ribonucleotides that are embedded in mitochondrial DNA (mtDNA)
We address the impact of these ribonucleoside monophosphates (rNMPs) on the mitochondrial DNA polymerase Pol γ at nucleotide concentrations that resemble those found inside a cell
Summary
The replication of DNA is a highly accurate process where free deoxyribonucleoside triphosphates (dNTPs) are incorporated opposite their complementary base. The presence of embedded ribonucleoside monophosphates (rNMPs) in DNA induces structural and chemical changes [3,4] that contribute to unwanted effects such as genome instability and replication stress [5,6] Due to their negative influence on DNA stability, rNMPs are actively removed from nuclear DNA (nDNA) by the ribonucleotide excision repair (RER) pathway that is initiated by cleavage at the incorporated rNMP by the enzyme RNase H2 [6,7]. Recent studies using fibroblast cell lines show that rNMPs are present at a frequency of approximately 54 rNMPs per 16 kb mammalian mtDNA molecule [10] It is currently unclear whether the rNMPs embedded in mtDNA have a functional significance or if they merely are relatively well-tolerated in the mitochondria and do not undergo the prompt removal observed in nDNA. Mutations in the gene coding for RNase H1, an endonuclease implicated in the removal of longer stretches of rNMPs, cause adultonset mitochondrial encephalomyopathy marked by multiple mtDNA deletions [15], underscoring the importance of at least a certain level of rNMP removal from mtDNA
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