Yeast cells expressing the human mitochondrial DNA polymerase reveal correlations between polymerase fidelity and human disease progression (573.1)

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