Yeast Cells Expressing the Human Mitochondrial DNA Polymerase Reveal Correlations between Polymerase Fidelity and Human Disease Progression

Abstract

Mutations in the human mitochondrial polymerase (Pol‐γ) are associated with various mitochondrial disorders. To correlate biochemically quantifiable defects resulting from point mutations in Pol‐γ with their physiological consequences, we created ‘humanized’ yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol‐γ. In spite of differences in the replication and repair mechanism, the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and the yeast helicase. We examined the effects of four disease‐related point mutations (S305R, H932Y, Y951N and Y955C) and an exonuclease‐deficient mutant (D198A/E200A). In haploid cells, each mutant results in mtDNA depletion, increased mutation frequency leading to reductions in mtDNA content and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild‐type Pol‐γ suppresses mutation‐associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content and depolarized mitochondrial membranes. The severity of the Pol‐γ mutant phenotype in heterozygous diploid humanized yeast correlates with the age of disease onset and the severity of symptoms observed in humans. Grant Funding Source: Supported by NIH GM044613

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

“…This method was also validated by Southern-blot analysis (Baruffini et al, 2006 ). Petite mutants can also be examined by confocal fluorescence microscopy following 40,60-diamidino-2-phenylindole hydrochloride (DAPI) staining of cytoplasm, which in yeast also stains mtDNA (as in Stuart et al, 2006 ; Qian et al, 2014 ). In both rho − cells and mit − cells stained with DAPI, several small spots can be observed under the surface of the cells, whereas these spots are absent in rho 0 due to the lack of mtDNA.…”

“…To examine the quantity and integrity of the mitochondrial genomes in petite mutants, quantitative PCR (qPCR) methods were used (as in Stuart et al, 2006 ; Qian et al, 2014 ). The copy number of mitochondrial genomes was determined by qPCR of short mitochondrial targets: no detectable PCR products indicate the absence of mtDNA.…”

Polymorphisms in DNA polymerase γ affect the mtDNA stability and the NRTI-induced mitochondrial toxicity in Saccharomyces cerevisiae

“…A limitation to the use of yeast MIP1 to study the effects of mutations is that only conserved or semi-conserved residues can be studied. During the preparation of this manuscript, Qian and co-authors created a yeast model system in which both human Pol γ subunit genes, cloned under the yeast MIP1 promoter and in frame with the MIP1 fragment encoding the mitochondrial targeting signal, complement the absence of MIP1 , indicating that human Pol γ can replicate yeast mtDNA ( Qian et al, 2014 ). Interestingly, a comparison between the effects of four human mutations which have been studied both in the human POLG and in the MIP1 gene showed very similar results concerning mtDNA stability, mtDNA point mutability and dominance/recessivity in the two systems, indicating that the use of yeast MIP1 has a good predictive ability for conserved and semi-conserved residues.…”

“…The Saccharomyces cerevisiae DNA polymerase γ, encoded by the MIP1 gene, shows 43% similarity with the human Pol γ catalytic subunit. Thanks to this similarity, yeast has been largely used to validate the role of human putative pathological mutations, to understand biochemical consequences associated with these mutations, to find molecules able to rescue their detrimental effects and to study the pharmacogenetics of drugs such as valproate and stavudine ( Baile and Claypool, 2013; Baruffini and Lodi, 2010; Baruffini et al, 2006, 2007, 2011; Qian et al, 2014; Spinazzola et al, 2009; Stewart et al, 2010; Stricker et al, 2009; Stuart et al, 2006; Stumpf and Copeland, 2013; Stumpf et al, 2010; Szczepanowska and Foury, 2010 ). Yeast is a suitable model organism for the study of the effects of Pol γ mutations on mtDNA stability, thanks to its ability to survive in the absence of a functional mitochondrial genome.…”

On the scope and limitations of baker’s yeast as a model organism for studying human tissue-specific pathways

“…For the majority of disease-genes with known yeast orthologs, heterologous expression of the mammalian gene is functional in yeast and can compensate for the loss-of-function phenotype in yeast deletion strains(1). This approach has already been used to construct humanized yeast model cells to study cancers(39), apoptosis-related diseases(49), mitochondrial disorders(50), and neurodegenerative diseases(43). Perhaps one of the more encouraging examples is the very recent discovery of a new compound, N-aryl benzimidazole (NAB), which strongly protects cells from α -synuclein toxicity in the humanized yeast model of Parkinson’s disease(51).…”