Deleterious mitochondrial DNA point mutations are overrepresented in Drosophila expressing a proofreading-defective DNA polymerase γ

Abstract

Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited syndromes and the accumulation of somatic mtDNA mutations is implicated in aging and common diseases. However, the mechanisms that influence the frequency and pathogenicity of mtDNA mutations are poorly understood. To address this matter, we created a Drosophila mtDNA mutator strain expressing a proofreading-deficient form of the mitochondrial DNA polymerase. Mutator flies have a dramatically increased somatic mtDNA mutation frequency that correlates with the dosage of the proofreading-deficient polymerase. Mutator flies also exhibit mitochondrial dysfunction, shortened lifespan, a progressive locomotor deficit, and loss of dopaminergic neurons. Surprisingly, the frequency of nonsynonymous, pathogenic, and conserved-site mutations in mutator flies exceeded predictions of a neutral mutational model, indicating the existence of a positive selection mechanism that favors deleterious mtDNA variants. We propose from these findings that deleterious mtDNA mutations are overrepresented because they selectively evade quality control surveillance or because they are amplified through compensatory mitochondrial biogenesis.

Inactivation of the mitochondrial protease Afg3l2 results in severely diminished respiratory chain activity and widespread defects in mitochondrial gene expression

“…For locomotor analyses, flies were anesthetized by brief exposure to CO 2 and, allowed to recover for 1 day before the experiment. Climbing behavior was assessed using the Rapid Iterative Negative Geotaxis (RING) assay on day 1, according to a previously published protocol [ 47 ]. Briefly, plastic vials containing 10–15 flies each were loaded onto the RING apparatus.…”

Drosophila as a Model System to Investigate the Effects of Mitochondrial Variation on Innate Immunity

“…Mutations in POLG have been shown to cause large scale deletions and various other mutations to the mtDNA due to replication and/or repair machinery malfunctions and these have been connected to many mitochondrial diseases such as Alper's syndrome, parkinsonism and multiple other neurodegenerative disorders (23). POLG was mutated in Drosophila to create a proofreading-deficient form resulting to drastically increased somatic mtDNA mutation frequency and mitochondrial dysfunction, which manifested as a shortened lifespan, a progressive locomotor deficit and a loss of dopaminergic neurons (24).…”

The DNA polymerases of Drosophila melanogaster

“…Mutations in human POLG and POLG2 are associated with several diseases including mtDNA depletion syndromes, such as Alpers syndrome, and mtDNA deletion disorders, such as progressive external ophthalmoplegia [121]. Significantly, several studies in the last decade have manipulated the orthologous D. melanogaster genes to provide important insights into the etiology of, and possible therapies for, these diseases [108][109][110][111][112][113]122]. For example, two studies generated flies expressing exonuclease-or polymerase-deficient versions of PolG1 in order to investigate how defects in these two different activities of the enzyme may contribute to pathophysiology [112,113].…”

“…Significantly, several studies in the last decade have manipulated the orthologous D. melanogaster genes to provide important insights into the etiology of, and possible therapies for, these diseases [108][109][110][111][112][113]122]. For example, two studies generated flies expressing exonuclease-or polymerase-deficient versions of PolG1 in order to investigate how defects in these two different activities of the enzyme may contribute to pathophysiology [112,113]. Two other investigations studied the effects of mutations or knock-down of D. melanogaster PolG1 or PolG2 specifically in the nervous system to probe how mitochondrial dysfunction may lead to neuropathies and neurodegenerative disorders in humans [108,110].…”

“…Indeed, the genetic approach, often combined with biochemical assays, has been key to the several landmark discoveries about DNA polymerases that were made through research using D. melanogaster. These include the first characterization of mutations in PolG subunits [107,117], which ultimately led to a genetic dissection of the roles of the exonuclease and polymerase domains of PolG1 [112,113]; the function of PolQ within the MMEJ repair pathway [88][89][90]; and the role of PolD3 in facilitating the nuclear import of PolD1 [58]. Several of the mechanisms elucidated by these studies provided precedent for other organisms, particularly so in the case of PolQ.…”