Hypothesis

The rate of mitochondrial DNA polymerase γ (Pol γ)–mediated replication errors sets the pace of organismal aging by altering mitochondrial metabolite fluxes that rewire nuclear epigenetic landscapes, rather than by causing wholesale bioenergetic failure.

Mechanistic Basis

Pol γ lacks proofreading activity in many tissues, leading to a bias toward G→A and T→C transitions on the light strand [1]. These mutations accumulate at regulatory regions such as the origin of light‑strand replication, where even low heteroplasmy disrupts glucose metabolism [2]. We propose that the primary consequence is not oxidative damage but a shift in the balance of TCA‑cycle intermediates—particularly an increase in succinate and a decrease in NAD⁺—that is exported to the cytosol. Elevated succinate inhibits α‑ketoglutarate‑dependent dioxygenases, causing histone and DNA hypermethylation, while reduced NAD⁺ diminishes sirtuin activity, together tightening chromatin and suppressing stress‑response genes. This retrograde signaling creates a feed‑forward loop: nuclear epigenetic changes further down‑regulate Pol γ subunits, increasing error rates.

Testable Predictions

1. Enhancing Pol γ fidelity (e.g., by overexpressing its exonuclease domain or administering a small‑molecule stabilizer) will lower heteroplasmy at the origin of light‑strand replication without changing overall ROS levels.
2. Animals with higher Pol γ fidelity will show younger epigenetic clocks (DNA methylation age) and improved transcriptome signatures of youth, even when mitochondrial mass and ATP production remain unchanged.
3. Conversely, inducing a Pol γ error‑prone state will accelerate epigenetic aging and shorten lifespan, an effect that is not rescued by mitochondrial antioxidants such as MitoQ.

Experimental Design

• Generate a knock‑in mouse line expressing a Pol γ variant with enhanced 3′→5′ exonuclease activity (Pol γ^exo+). Include a littermate control expressing wild‑type Pol γ.
• Measure heteroplasmy levels at known hotspot sites using duplex sequencing at 6, 12, and 18 months.
• Quantify mitochondrial ROS (MitoSOX), NAD⁺/NADH ratio, and succinate concentrations in liver, muscle, and brain.
• Perform whole‑blood bisulfite sequencing to estimate epigenetic age and RNA‑seq for stress‑response pathways.
• Monitor frailty index, grip strength, and survival.
• As a pharmacological arm, treat wild‑type mice with a Pol γ‑stabilizing compound (identified via in silico screening) and repeat the above metrics.
• Include a MitoQ treatment group to test the independence from ROS scavenging.

Potential Outcomes

If Pol γ^exo+ mice display reduced heteroplasmy, normalized metabolite ratios, delayed epigenetic aging, and extended healthspan despite unchanged ROS, the hypothesis is supported. If altering Pol γ fidelity fails to affect epigenetic age or longevity, or if benefits are abolished by MitoQ, the hypothesis is falsified, pointing to oxidative damage as the dominant driver.