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DNA polymerase γ and disease: what we have learned from yeast.

ABSTRACT: Mip1 is the Saccharomyces cerevisiae DNA polymerase γ (Pol γ), which is responsible for the replication of mitochondrial DNA (mtDNA). It belongs to the family A of the DNA polymerases and it is orthologs to human POLGA. In humans, mutations in POLG(1) cause many mitochondrial pathologies, such as progressive external ophthalmoplegia (PEO), Alpers' syndrome, and ataxia-neuropathy syndrome, all of which present instability of mtDNA, which results in impaired mitochondrial function in several tissues with variable degrees of severity. In this review, we summarize the genetic and biochemical knowledge published on yeast mitochondrial DNA polymerase from 1989, when the MIP1 gene was first cloned, up until now. The role of yeast is particularly emphasized in (i) validating the pathological mutations found in human POLG and modeled in MIP1, (ii) determining the molecular defects caused by these mutations and (iii) finding the correlation between mutations/polymorphisms in POLGA and mtDNA toxicity induced by specific drugs. We also describe recent findings regarding the discovery of molecules able to rescue the phenotypic defects caused by pathological mutations in Mip1, and the construction of a model system in which the human Pol γ holoenzyme is expressed in yeast and complements the loss of Mip1.

SUBMITTER: Lodi T

PROVIDER: S-EPMC4362329 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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DNA polymerase γ and disease: what we have learned from yeast.

Lodi Tiziana T Dallabona Cristina C Nolli Cecilia C Goffrini Paola P Donnini Claudia C Baruffini Enrico E

Frontiers in genetics 20150317

Mip1 is the Saccharomyces cerevisiae DNA polymerase γ (Pol γ), which is responsible for the replication of mitochondrial DNA (mtDNA). It belongs to the family A of the DNA polymerases and it is orthologs to human POLGA. In humans, mutations in POLG(1) cause many mitochondrial pathologies, such as progressive external ophthalmoplegia (PEO), Alpers' syndrome, and ataxia-neuropathy syndrome, all of which present instability of mtDNA, which results in impaired mitochondrial function in several tissu ...[more]

PMID: 25852747

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Project description:Mutations in mitochondrial DNA (mtDNA) are an important cause of disease and perhaps aging in human. DNA polymerase gamma (pol γ), the unique replicase inside mitochondria, plays a key role in the fidelity of mtDNA replication through selection of the correct nucleotide and 3'-5' exonuclease proofreading. For the first time, we have isolated and characterized antimutator alleles in the yeast pol γ (Mip1). These mip1 mutations, localised in the 3'-5' exonuclease and polymerase domains, elicit a 2-15 fold decrease in the frequency of mtDNA point mutations in an msh1-1 strain which is partially deficient in mtDNA mismatch-repair. In vitro experiments show that in all mutants the balance between DNA synthesis and exonucleolysis is shifted towards excision when compared to wild-type, suggesting that in vivo more opportunity is given to the editing function for removing the replicative errors. This results in partial compensation for the mismatch-repair defects and a decrease in mtDNA point mutation rate. However, in all mutants but one the antimutator trait is lost in the wild-type MSH1 background. Accordingly, the polymerases of selected mutants show reduced oligonucleotide primed M13 ssDNA synthesis and to a lesser extent DNA binding affinity, suggesting that in mismatch-repair proficient cells efficient DNA synthesis is required to reach optimal accuracy. In contrast, the Mip1-A256T polymerase, which displays wild-type like DNA synthesis activity, increases mtDNA replication fidelity in both MSH1 and msh1-1 backgrounds. Altogether, our data show that accuracy of wild-type Mip1 is probably not optimal and can be improved by specific (often conservative) amino acid substitutions that define a pol γ area including a loop of the palm subdomain, two residues near the ExoII motif and an exonuclease helix-coil-helix module in close vicinity to the polymerase domain. These elements modulate in a subtle manner the balance between DNA polymerization and excision.

Project description:Mitochondria are arising as critical modulators of antiviral tolerance by releasing mtDNA/mtRNA fragments to cytoplasm upon infection, activating virus sensors and type-I interferon response. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here, we investigated mitochondrial recessive ataxia syndrome (MIRAS), caused by a common European founder mutation in DNA polymerase gamma (POLG1). The patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages-of-onset and symptoms, indicating unknown modifying factors contributing to disease manifestation. We report that the mitochondrial DNA (mtDNA) replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV, SARS-CoV-2) and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release to cytoplasm and slow interferon response in MIRAS allow an early replicative advantage for viruses, leading to augmented pro-inflammatory response, subacute loss of GABAergic neurons, liver inflammation and necrosis. A population databank of ~300,000 Finns demonstrates enrichment of immunodeficient traits in p.W748S carriers. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications to mitochondrial disease spectrum, including epilepsy, ataxia, and parkinsonism. In this metabolomic dataset: To investigate the in vivo response of MIRAS to viral infection, we generated a MIRAS-knock-in mouse model and subjected the mice to TBEV infection. These animals carry a homozygous knock-in mutation and the accompanying polymorphic variant homologous to the human ancestral MIRAS allele (p.W726S+E1121G in mice and p.W748S+E1143G in humans). We performed metabolomic analyses on the mouse brain (cerebral cortex) isolated from the control (wildtype) and MIRAS mice at 4 days post TBEV infection (n=5 mice per condition).

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DNA polymerase γ and disease: what we have learned from yeast.

Publications

DNA polymerase γ and disease: what we have learned from yeast.

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