Oxidative DNA damage stalls the human mitochondrial replisome

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.

DNA damage related crosstalk between the nucleus and mitochondria

“…PrimPol, a DNA primase-polymerase, was identified in the mammalian mitochondria [117] and evidence for its ability to bypass DNA lesions such as 8-oxoG was presented [118]. A conflicting report suggests that while PrimPol is unable to promote DNA damage bypass at a replication fork, its interactions with proteins involved in mtDNA replication, such as the Twinkle helicase, may be important for mtDNA metabolism [119]. …”

Mechanistic and biological considerations of oxidatively damaged DNA for helicase-dependent pathways of nucleic acid metabolism

“…The Twinkle helicase operates at the mtDNA replication fork with the mitochondrial single-stranded binding protein (mtSSB) to unwind the parental duplex DNA circle allowing DNA synthesis by POLγ to proceed smoothly [203]. Recently, Stojkovic et al [204] directly tested in a reconstituted system with purified recombinant proteins and defined DNA substrates with representative oxidized lesions (8-oxoguanine or an abasic site) the efficiency of DNA synthesis by the human mitochondrial replisome which included Twinkle helicase. They found substantial stalling at sites of the base lesions by POLγ, even when TWINKLE and mtSSB were present.…”

“…They found substantial stalling at sites of the base lesions by POLγ, even when TWINKLE and mtSSB were present. PrimPol translesion polymerase, which was reported to localize to both nuclei and mitochondria [205], also failed to stimulate oxidative damage bypass by POLγ; however, Twinkle was able to stimulate PrimPol DNA synthesis on either damaged or undamaged DNA substrates at dNTP levels thought to be characteristic of cycling cells (2 µM dTTP, 1 µM dCTP, 2 µM dATP and 1 µM dGTP) [204]. …”

The role of mitochondria in cardiac development and protection

“…Although this type of modifications do not cause bulky lesions that one would expect to impair transcription and replication, oxidative damage seems to stall mtDNA replication both in vitro [97] and in vivo [92]. In addition, 8-oxo-dG's are read as dA's during replication and will result in G>A mutations as observed in the mutation pattern of Sod2 +/-mouse hearts [28].…”