title: Yeast cells expressing the human mitochondrial DNA polymerase reveal correlations between polymerase fidelity and human disease progression.
abstract: Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. 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. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also 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 rapid mtDNA depletion, increased mutation frequency, 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 approximate age of disease onset and the severity of symptoms observed in humans.
authors.affiliation: From the Institute for Cellular and Molecular Biology.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Qian
authors.forename: Aashiq H
authors.initials: AH
authors.lastname: Kachroo
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Yellman
authors.forename: Edward M
authors.initials: EM
authors.lastname: Marcotte
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Johnson
mesh_terms: D019943:Amino Acid Substitution; D000074002:DNA Polymerase gamma; D004261:DNA Replication / Q000235:genetics; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D004272:DNA, Mitochondrial / Q000096:biosynthesis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D002549:Diffuse Cerebral Sclerosis of Schilder / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics; D051336:Mitochondrial Membranes / Q000378:metabolism; D020125:Mutation, Missense; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human
keywords: DNA Polymerase; DNA Replication; Human Mitochondria; Mitochondrial DNA; Nucleic Acid Enzymology; Yeast