Oxidative DNA damage stalls the human mitochondrial replisome

Area of Science:

• Mitochondrial biology
• DNA replication and repair
• Oxidative stress

Background:

• Oxidative stress causes cellular damage, including to DNA.
• Limited understanding exists on oxidative stress's impact on mitochondrial DNA (mtDNA) and its replication.
• mtDNA is vulnerable to oxidative damage due to proximity to reactive oxygen species production.

Purpose of the Study:

• To investigate how oxidative stress affects mtDNA replication using purified proteins.
• To determine the role of the mitochondrial translesion synthesis polymerase PrimPol in bypassing oxidative DNA damage.
• To analyze replication dynamics at varying deoxynucleotide triphosphate (dNTP) levels.

Main Methods:

• In vitro replication assays using purified human mtDNA replication proteins: DNA polymerase γ holoenzyme, mtSSB, Twinkle helicase, and PrimPol.
• Utilized DNA templates containing oxidative damage.
• Experiments conducted at dNTP concentrations mimicking cycling and non-dividing cells.

Main Results:

• The mtDNA replication machinery significantly stalled at oxidative damage sites, especially at lower dNTP concentrations found in non-dividing cells.
• PrimPol did not facilitate bypass of oxidative lesions, challenging its conventional translesion synthesis role in mitochondria.
• The Twinkle helicase was observed to stimulate PrimPol's DNA synthesis activity in vitro.

Conclusions:

• PrimPol's function in mitochondria may not involve conventional translesion synthesis for oxidative DNA damage.
• The interaction between Twinkle and PrimPol suggests an alternative, yet unidentified, role for PrimPol in mtDNA metabolism.
• Oxidative stress poses significant challenges to mtDNA replication, particularly under conditions of low dNTP availability.