Lethal Interaction of Nuclear and Mitochondrial Genotypes in Drosophila melanogaster

Drosophila melanogaster, like most animal species, displays considerable genetic variation in both nuclear and mitochondrial DNA (mtDNA). Here we tested whether any of four natural mtDNA variants was able to modify the effect of the phenotypically mild, nuclear tko(25t) mutation, affecting mitochondrial protein synthesis. When combined with tko(25t), the mtDNA from wild strain KSA2 produced pupal lethality, accompanied by the presence of melanotic nodules in L3 larvae. KSA2 mtDNA, which carries a substitution at a conserved residue of cytochrome b that is predicted to be involved in subunit interactions within respiratory complex III, conferred drastically decreased respiratory capacity and complex III activity in the tko(25t) but not a wild-type nuclear background. The complex III inhibitor antimycin A was able to phenocopy effects of the tko(25t) mutation in the KSA2 mtDNA background. This is the first report of a lethal, nuclear-mitochondrial interaction within a metazoan species, representing a paradigm for understanding genetic interactions between nuclear and mitochondrial genotype relevant to human health and disease.Peer reviewe

Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase

Pathological conditions impairing functions of mitochondria often lead to compensatory upregulation of the mitochondrial DNA (mtDNA) replisome machinery, and the replicative DNA helicase appears to be a key factor in regulating mtDNA copy number. Moreover, mtDNA helicase mutations have been associated with structural rearrangements of themitochondrial genome. To evaluate the effects of elevated levels of the mtDNA helicase on the integrity and replication of the mitochondrial genome, we overexpressed the helicase in Drosophila melanogaster Schneider cells and analyzed the mtDNA by two-dimensional neutral agarose gel electrophoresis and electron microscopy. We found that elevation of mtDNA helicase levels increases the quantity of replication intermediates and alleviates pausing at the replication slow zones. Though we did not observe a concomitant alteration in mtDNA copy number, we observed deletions specific to the segment of repeated elements in the immediate vicinity of the origin of replication, and an accumulation of species characteristic of replication fork stalling. We also found elevated levels of RNA that are retained in the replication intermediates. Together, our results suggest that upregulation of mtDNA helicase promotes the process of mtDNA replication but also results in genome destabilization.Peer reviewe

A cytoplasmic suppressor of a nuclear mutation affecting mitochondrial functions in Drosophila

Phenotypes relevant to oxidative phosphorylation (OXPHOS) in eukaryotes are jointly determined by nuclear and mitochondrial DNA (mtDNA). Thus, in humans, the variable clinical presentations of mitochondrial disease patients bearing the same primary mutation, whether in nuclear or mitochondrial DNA, have been attributed to putative genetic determinants carried in the other genome, though their identity and the molecular mechanism(s) by which they might act remain elusive. Here we demonstrate cytoplasmic suppression of the mitochondrial disease-like phenotype of the Drosophila melanogaster nuclear mutant tko25t, which includes developmental delay, seizure sensitivity, and defective male courtship. The tko25t strain carries a mutation in a mitoribosomal protein gene, causing OXPHOS deficiency due to defective intramitochondrial protein synthesis. Phenotypic suppression was associated with increased mtDNA copy number and increased mitochondrial biogenesis, as measured by the expression levels of porin voltage dependent anion channel and Spargel (PGC1α). Ubiquitous overexpression of Spargel in tko25t flies phenocopied the suppressor, identifying it as a key mechanistic target thereof. Suppressor-strain mtDNAs differed from related nonsuppressor strain mtDNAs by several coding-region polymorphisms and by length and sequence variation in the noncoding region (NCR), in which the origin of mtDNA replication is located. Cytoplasm from four of five originally Wolbachia-infected strains showed the same suppressor effect, whereas that from neither of two uninfected strains did so, suggesting that the stress of chronic Wolbachia infection may provide evolutionary selection for improved mitochondrial fitness under metabolic stress. Our findings provide a paradigm for understanding the role of mtDNA genotype in human disease

Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of hte human mitochondrial replicase, Pol gamma

Peer reviewe

A cytoplasmic suppressor of a nuclear mutation affecting mitochondrial functions in Drosophila

Phenotypes relevant to oxidative phosphorylation (OXPHOS) in eukaryotes are jointly determined by nuclear and mitochondrial DNA (mtDNA). Thus, in humans, the variable clinical presentations of mitochondrial disease patients bearing the same primary mutation, whether in nuclear or mitochondrial DNA, have been attributed to putative genetic determinants carried in the other genome, though their identity and the molecular mechanism(s) by which they might act remain elusive. Here we demonstrate cytoplasmic suppression of the mitochondrial disease-like phenotype of the Drosophila melanogaster nuclear mutant tko25t, which includes developmental delay, seizure sensitivity, and defective male courtship. The tko25t strain carries a mutation in a mitoribosomal protein gene, causing OXPHOS deficiency due to defective intramitochondrial protein synthesis. Phenotypic suppression was associated with increased mtDNA copy number and increased mitochondrial biogenesis, as measured by the expression levels of porin voltage dependent anion channel and Spargel (PGC1α). Ubiquitous overexpression of Spargel in tko25t flies phenocopied the suppressor, identifying it as a key mechanistic target thereof. Suppressor-strain mtDNAs differed from related nonsuppressor strain mtDNAs by several coding-region polymorphisms and by length and sequence variation in the noncoding region (NCR), in which the origin of mtDNA replication is located. Cytoplasm from four of five originally Wolbachia-infected strains showed the same suppressor effect, whereas that from neither of two uninfected strains did so, suggesting that the stress of chronic Wolbachia infection may provide evolutionary selection for improved mitochondrial fitness under metabolic stress. Our findings provide a paradigm for understanding the role of mtDNA genotype in human disease

Mg2+-Assisted Catalysis by B. Stearothermophilus TrpRS Is Promoted by Allosteric Effects

Mn2+-assisted catalysis by B. stearothermophilus TrpRS parallels that in polymerases and reduces specificity in amino acid activation. As predicted by non-equilibrium molecular dynamics simulations, multi-variant thermodynamic cycles with [ATP]-dependent Michaelis-Menten kinetics and Mn2+ for Mg2+ substitution demonstrate energetic coupling of ATP affinities to the metal; to lysines K111 and K192, which interact via the PPi leaving group; and to K195, which couples differently to the metal via the α–phosphate. However, net coupling to the metal opposes catalysis in both ground (Km) and transition (kcat) states. The 105-fold rate acceleration by Mg2+-protein interactions therefore requires additional favorable protein-metal couplings. Examples include longer-range, i.e., allosteric, interactions previously illustrated by the remote F37I mutation, which both reduces kcat and enhances catalytic assist by Mn2+, relative to that by Mg2+. These data support a model linking metal-assisted phosphoryl transfer catalysis to domain movement, and hence to free-energy transduction in a broad range of enzymes

Modeling Pathogenic Mutations of Human Twinkle in Drosophila Suggests an Apoptosis Role in Response to Mitochondrial Defects

The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1

In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an similar to 90% drop in K-cat with a decreased K-M, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were cooverexpressed in S2 cells, and functional interactions between them in vitro were minor.Peer reviewe

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems