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) ϳ5-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.One of the important functions of mitochondria is the production of ATP by the oxidative phosphorylation pathway. Animal mitochondrial DNA (mtDNA) 2 encodes 13 polypeptides involved in oxidative phosphorylation, whereas all of the factors essential for mtDNA replication are encoded in the nuclear genome (1). In general, mutations in these factors result in both loss of mtDNA integrity via base substitution mutations, duplications, and deletions and mtDNA depletion (2).Autosomal dominant progressive external ophthalmoplegia (adPEO) is a human mitochondrial disorder associated with the presence of multiple deletions of mtDNA (2-5). The disease is manifest in adulthood, typically at 20 -40 years of age; symptoms include muscle weakness, wasting, exercise intolerance, ataxia, hearing loss, cardiomyopathy, and peripheral neuropathy (2). Most of the adPEO families carry heterozygous mutations in one of three genes: ANT1 (the adenine nucleotide translocator 1), POLG (mitochondrial DNA polymerase), and TWINKLE (mtDNA helicase) (2, 6 -8).TWINKLE protein shares high homology with the bacteriophage T7 gene 4 protein (T7 gp4), which contains both helicase and primase catalytic activities located in its carboxyl-and amino-terminal halves, respectively (7). Like other DNA helicases of the DnaB family, T7 gp4 functions as a hexameric ring (9 -11). The amino acid sequence of the helicase domain of T7 gp4 is well conserved in TWINKLE, whereas that of the primase domain is not. TWINKLE exhibits 5Ј-3Ј DNA helicase activity on double-stranded DNA substrates and functions in reconstituted mtDNA replication forks in vitro (12, 13). It co-localizes with mtDNA in structures designated as mitochondrial nucleoids in vivo (7). Recently, TWINKLE was demonstrated to modu...