title: Differential phenotypes of active site and human autosomal dominant progressive external ophthalmoplegia mutations in Drosophila mitochondrial DNA helicase expressed in Schneider cells.
abstract: We report the cloning and molecular analysis of Drosophila mitochondrial DNA helicase (d-mtDNA helicase) homologous to human TWINKLE, which encodes one of the genes responsible for autosomal dominant progressive external ophthalmoplegia. An RNA interference construct was designed that reduces expression of d-mtDNA helicase to an undetectable level in Schneider cells. RNA interference knockdown of d-mtDNA helicase decreases the copy number of mitochondrial DNA (mtDNA) approximately 5-fold. In a corollary manner, overexpression of d-mtDNA helicase increases mtDNA levels 1.4-fold. Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype. Overexpression of mutants analogous to human autosomal dominant progressive external ophthalmoplegia mutations shows differential effects. Overexpression of I334T and A442P mutants yields a dominant negative effect as for the active site mutants. In contrast, overexpression of A326T, R341Q, and W441C mutants results in increased mtDNA copy number, as observed with wild-type overexpression. Our dominant negative analysis of d-mtDNA helicase in cultured cells provides a tractable model for understanding human autosomal dominant progressive external ophthalmoplegia mutations.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Matsushima
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824. Electronic address: lskaguni@msu.edu.
authors.forename: Laurie S
authors.initials: LS
authors.lastname: Kaguni
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D004265:DNA Helicases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics*; D005799:Genes, Dominant; D006801:Humans; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D017246:Ophthalmoplegia, Chronic Progressive External / Q000201:enzymology / Q000235:genetics*; D010641:Phenotype; D034622:RNA Interference
chemical_list: D004272:DNA, Mitochondrial; D029721:Drosophila Proteins; D004265:DNA Helicases