Disease causing mutation (P178L) in mitochondrial transcription factor A results in impaired mitochondrial transcription initiation.

Mitochondrial transcription factor A (TFAM) is essential for the maintenance, expression, and packaging of mitochondrial DNA (mtDNA). Recently, a pathogenic homozygous variant in TFAM (P178L) has been associated with a severe mtDNA depletion syndrome leading to neonatal liver failure and early death. We have performed a biochemical characterization of the TFAM variant P178L in order to understand the molecular basis for the pathogenicity of this mutation. We observe no effects on DNA binding, and compaction of DNA is only mildly affected by the P178L amino acid change. Instead, the mutation severely impairs mtDNA transcription initiation at the mitochondrial heavy and light strand promoters. Molecular modeling suggests that the P178L mutation affects promoter sequence recognition and the interaction between TFAM and the tether helix of POLRMT, thus explaining transcription initiation deficiency.

DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion.

Mutations in POLG, encoding POLγA, the catalytic subunit of the mitochondrial DNA polymerase, cause a spectrum of disorders characterized by mtDNA instability. However, the molecular pathogenesis of POLG-related diseases is poorly understood and efficient treatments are missing. Here, we generate the PolgA449T/A449T mouse model, which reproduces the A467T change, the most common human recessive mutation of POLG. We show that the mouse A449T mutation impairs DNA binding and mtDNA synthesis activities of POLγ, leading to a stalling phenotype. Most importantly, the A449T mutation also strongly impairs interactions with POLγB, the accessory subunit of the POLγ holoenzyme. This allows the free POLγA to become a substrate for LONP1 protease degradation, leading to dramatically reduced levels of POLγA in A449T mouse tissues. Therefore, in addition to its role as a processivity factor, POLγB acts to stabilize POLγA and to prevent LONP1-dependent degradation. Notably, we validated this mechanism for other disease-associated mutations affecting the interaction between the two POLγ subunits. We suggest that targeting POLγA turnover can be exploited as a target for the development of future therapies.

Structural basis for adPEO-causing mutations in the mitochondrial TWINKLE helicase.

TWINKLE is the helicase involved in replication and maintenance of mitochondrial DNA (mtDNA) in mammalian cells. Structurally, TWINKLE is closely related to the bacteriophage T7 gp4 protein and comprises a helicase and primase domain joined by a flexible linker region. Mutations in and around this linker region are responsible for autosomal dominant progressive external ophthalmoplegia (adPEO), a neuromuscular disorder associated with deletions in mtDNA. The underlying molecular basis of adPEO-causing mutations remains unclear, but defects in TWINKLE oligomerization are thought to play a major role. In this study, we have characterized these disease variants by single-particle electron microscopy and can link the diminished activities of the TWINKLE variants to altered oligomeric properties. Our results suggest that the mutations can be divided into those that (i) destroy the flexibility of the linker region, (ii) inhibit ring closure and (iii) change the number of subunits within a helicase ring. Furthermore, we demonstrate that wild-type TWINKLE undergoes large-scale conformational changes upon nucleoside triphosphate binding and that this ability is lost in the disease-causing variants. This represents a substantial advancement in the understanding of the molecular basis of adPEO and related pathologies and may aid in the development of future therapeutic strategies.