Abstract
Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase γ holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage, and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism.
Highlights
Specialized lesion bypass DNA polymerases are recruited to synthesize past the oxidative DNA lesion and allow the replication fork to finish replication[18]
We used DNA templates comprising of synthetic oligonucleotides to determine the action of the various mitochondrial DNA (mtDNA) replication proteins upon encounter of oxidative DNA damage (8-oxo-G or abasic sites)
At “normal” dNTP concentrations, DNA polymerase γshows moderate stalling at 8-oxo-G sites, in particular when the DNA is coated with mitochondrial single-stranded DNA-binding protein (mtSSB) as is expected to be the case in vivo (Fig. 1B)
Summary
Specialized lesion bypass DNA polymerases are recruited to synthesize past the oxidative DNA lesion and allow the replication fork to finish replication[18]. The recently discovered PrimPol enzyme has been proposed to participate in restarting replication during the nuclear DNA damage response[21] and in both nuclear and mitochondrial translesion synthesis (TLS)[19,22,23]. Several groups have used recombinant PrimPol to characterize the properties of this enzyme[19,22,23,24,25,26] These studies have shown that PrimPol’s primase activity can use both NTPs and dNTPs to synthesize primers de novo. The interaction of PrimPol with the components of the mitochondrial DNA replisome has not been studied, with the exception of a recent report that showed in vivo interaction between PrimPol and mtSSB27. We found PrimPol to be ineffective in mediating the bypass of AP or 8-oxo-G damage by the mtDNA replisome at “normal” dNTP concentrations. We present that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis, suggesting PrimPol plays an important role in mtDNA metabolism
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