Oxidative damage diminishes mitochondrial DNA polymerase replication fidelity

Area of Science:

• Mitochondrial biology
• Molecular genetics
• Oxidative stress research

Background:

• Mitochondrial DNA (mtDNA) accumulates mutations at a higher rate than nuclear DNA due to its proximity to reactive oxygen species (ROS).
• Emerging evidence indicates that mtDNA mutations may arise from DNA replication errors rather than direct oxidative damage.

Purpose of the Study:

• To investigate how the mitochondrial DNA replicase, DNA polymerase gamma (Pol γ), contributes to elevated mutation rates under oxidative conditions.
• To elucidate the impact of Pol γ oxidation on its polymerase and proofreading activities.

Main Methods:

• Studied the effect of oxidation on the polymerase (pol) and proofreading exonuclease (exo) activities of purified Pol γ.
• Utilized mass spectrometry to identify sites of oxidation on Pol γ.
• Assessed the fidelity of oxidized and unoxidized Pol γ during DNA replication in vitro.

Main Results:

• The exonuclease (exo) domain of Pol γ is significantly more sensitive to oxidation than the polymerase (pol) domain.
• Oxidized Pol γ exhibits a reduced exonuclease activity, leading to an editing-deficient state.
• Oxidized Pol γ shows a 20-fold increase in replication error rate compared to the unoxidized enzyme.
• Mass spectrometry identified the Pol γ exo domain as a primary site for oxidation, with altered active site charge affecting DNA binding.

Conclusions:

• Oxidation of mitochondrial DNA polymerase gamma (Pol γ) impairs its proofreading exonuclease activity.
• This impairment leads to increased DNA replication errors and contributes to the high mutation frequency observed in mitochondrial DNA under oxidative stress.
• Oxidative stress indirectly elevates mtDNA mutation rates by damaging the mitochondrial replicase.