Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis, aging and age-related diseases

Abstract

As regulators of bioenergetics in the cell and the primary source of endogenous reactive oxygen species (ROS), dysfunctional mitochondria have been implicated for decades in the process of aging and age-related diseases. Mitochondrial DNA (mtDNA) is replicated and repaired by nuclear-encoded mtDNA polymerase γ (Pol γ) and several other associated proteins, which compose the mtDNA replication machinery. Here, we review evidence that errors caused by this replication machinery and failure to repair these mtDNA errors results in mtDNA mutations. Clonal expansion of mtDNA mutations results in mitochondrial dysfunction, such as decreased electron transport chain (ETC) enzyme activity and impaired cellular respiration. We address the literature that mitochondrial dysfunction, in conjunction with altered mitochondrial dynamics, is a major driving force behind aging and age-related diseases. Additionally, interventions to improve mitochondrial function and attenuate the symptoms of aging are examined.

Keywords: Age-related diseases; Aging; DNA polymerase gamma; Mitochondrial DNA mutations; MtDNA replication; POLG.

Figure 1

The four complexes of the ETC and ATP synthase are located in the inner mitochondrial membrane. Electrons from NADH and FADH₂ are transferred from Complex I (NADH dehydrogenase) and Complex II (succinate dehydrogenase) to coenzyme Q (CoQ) before being transported to Complex III (cytochrome c reductase). From Complex III, cytochrome c (Cyt c) delivers the electrons to Complex IV (cytochrome c oxidase), which ultimately reduces oxygen to water. This flow of electrons down the ETC generates a proton electrochemical gradient, which is used by Complex V (ATP synthase) to synthesize ATP. ATP is then transported back into the intermembrane space by the ANT1-4 and beyond to the cytosol with a combination of ANT and VDAC. Protons can also enter the matrix through the uncoupling protein (UCP), which results in the uncoupling or separation of electron transport through the ETC from ATP synthesis. Premature electron leakage from Complexes I and II form ROS as superoxide (O₂^•−) which can be transformed into H₂O₂ or the hydroxyl radical (HO^•). Subunits of each of the five complexes of the ETC, with the exception of Complex II, are encoded by both mtDNA and nDNA as recounted in the table.

Figure 2

A schematic diagram of the mtDNA replication machinery. POLRMT forms the RNA primer (jagged red line) needed to initiate DNA replication in conjunction with TFAM (royal blue), TFB2M (gray). After initiation, POLRMT, TFAM and TFB2M separately leave the DNA and the RNA primer is degraded by RNase H1 (yellow). In a 5′ to 3′ direction, the Twinkle helicase (pink) unwinds dsDNA at the replication fork. The ssDNA is stabilized by mtSSB (light blue), while MGME1 (red) degrades ssDNA. MtLigIII (white) seals the mtDNA nick. Nascent DNA (solid red line) is synthesized by Pol γ (green). Topoisomerases (brown) relieve the torsional tension in the DNA caused by unwinding.

Figure 3

Clonal expansion and heteroplasmy. Individual mitochondria contain many copies of mtDNA. During replication, a mutation and/or deletion can occur. This damage accumulates by clonal expansion. When the fraction of mutated mtDNA relative to total mtDNA exceeds 60% depending on the tissue affected, dysfunction and disease are measurable. The mitochondria are represented as the brown ovals, green circles are healthy mtDNA and red circles are damaged mtDNA. The yellow lightening bolt represents damage occurring during replication and the blue circles represent the replication machinery.

Figure 4

Factors causal to aging. Spontaneous errors in the mitochondrial replication machinery cause mtDNA damage. Accumulations of this mtDNA damage occur via clonal expansion until tissue specific heteroplasmy is reached and mitochondrial dysfunction is measurable. Increased mitochondrial dysfunction along with decreased mitochondrial dynamics leads to aging and age-related diseases. Mitochondrial dysfunction may also cause increased apoptosis, which is associated with aging. At low levels, ROS triggers stress response and biogenesis pathways and increases mitochondrial dynamics, which are protective and prevent aging. Caloric restriction and exercise functions similarly. Lastly, high levels of ROS can cause damage to cellular components, such as DNA, RNA, protein and lipids, which may also contribute to aging.