Molecular Characterization Of The Mitochondrial Dna Polymerase

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

Mitochondrial diseases are devastating disorders for which there is no cure and no proven treatment. About 1 in 2000 individuals are at risk of developing a mitochondrial disease sometime in their lifetime. Half of those affected are children who show symptoms before age 5 and approximately 80% of these will die before age 20. The human suffering imposed by mitochondrial and metabolic diseases is enormous, yet much work is needed to understand the genetic and environmental causes of these diseases. Mitochondrial genetic diseases are characterized by alterations in the mitochondrial genome, as point mutations, deletions, rearrangements, or depletion of the mitochondrial DNA (mtDNA). The mutation rate of the mitochondrial genome is 10-20 times greater than of nuclear DNA, and mtDNA is more prone to oxidative damage than is nuclear DNA. Mutations in human mtDNA cause premature aging, severe neuromuscular pathologies and maternally inherited metabolic diseases, and influence apoptosis. The primary goal of this project is to understand the contribution of the replication apparatus in the production and prevention of mutations in mtDNA. Since the genetic stability of mitochondrial DNA depends on the accuracy of DNA polymerase gamma (pol gamma), we have focused this project on understanding the role of the human pol gamma in mtDNA mutagenesis. Human mitochondrial DNA is replicated by the heterodimeric DNA polymerase gamma encoded by POLG and POLG2, in concert with the Twinkle helicase (TWNK) and the single stranded DNA binding protein (SSBP1) To date over 300 pathogenic mutations in POLG that cause a wide spectrum of disease including Progressive external ophthalmoplegia (PEO), parkinsonism, premature menopause, Alpers syndrome, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) or sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO). TAF1A, a gene encoding a TATA-box binding protein involved in ribosomal RNA synthesis, is a candidate gene for pediatric cardiomyopathy as biallelic TAF1A variants were reported in two families with affected individuals. Here, we report a third family with two siblings who presented with infantile restrictive cardiomyopathy and carried biallelic missense variants in TAF1A (NM_001201536.1:c.1021G>A p.(Gly341Arg) and c.781A>C p.(Thr261Pro)). Additional shared clinical features in the siblings included feeding intolerance, congenital leukoencephalopathy, ventriculomegaly and concern for primary immunodeficiency. The first-born sibling passed away at 6 months of age due to complications of hemophagocytic lymphohistiocytosis (HLH) whereas the second sibling underwent cardiac transplantation at 1 year of age and is currently well. We compare the clinical and molecular features of all the TAF1A associated cardiomyopathy cases. Our study adds evidence for the gene-disease association of TAF1A with autosomal recessive pediatric cardiomyopathy. In 2024 we published the first structure of the mitochondrial single-stranded DNA binding protein bound to single stranded DNA. The mitochondrial single-stranded DNA binding protein, mtSSB or SSBP1, binds to ssDNA to prevent secondary structures of DNA which could impede downstream replication or repair processes. Clinical mutations in the SSBP1 gene have been linked to a range of mitochondrial disorders affecting nearly all organs and systems. Yet, the molecular determinants governing the interaction between mtSSB and ssDNA have remained elusive. Similarly, the structural interaction between mtSSB and other replisome components, such as the mitochondrial DNA polymerase, Pol, has been minimally explored. Here, we determined a 1.9 Å X-ray crystallography structure of the human mtSSB bound to ssDNA. This structure uncovered two distinct DNA binding sites, a low- and a high-affinity site, confirmed through site-directed mutagenesis. The high-affinity binding site encompasses the clinically relevant residue, R38 and the highly conserved DNA base stacking residue, W84. Employing cryo-electron microscopy, we confirmed the tetrameric assembly in solution and capture its interaction with Pol. Finally, we derived a model depicting modes of ssDNA wrapping around mtSSB and a region within Pol that mtSSB binds. We also published a review on DNA repair pathways in the mitochondria. Despite the importance of maintaining mtDNA genomic integrity, fewer DNA repair pathways exist in the mitochondria than in the nucleus. However, mitochondria have numerous pathways that allow for the removal and degradation of DNA damage that may prevent accumulation of mutations. We reviewed the DNA repair pathways present in the mitochondria, sources of mtDNA mutations, and discussed the passive role that mtDNA mutagenesis may play in cancer progression.