The mitochondrial replicative DNA helicase (mtDNA helicase) unwinds double-stranded mitochondrial DNA (mtDNA) by using NTP hydrolysis at the mtDNA replication fork. Moreover, single-stranded DNA annealing activity and branch migration are identified in human mtDNA helicase, expanding its roles in mtDNA repair or recombination. The mtDNA helicase is also proposed to tether mitochondrial nucleoid to the mitochondrial inner membrane or to load itself on the closed DNA. Consequently, defects in the mtDNA helicase gene cause several mitochondrial diseases. I have pursued a comprehensive understanding of the structural and functional roles for both human and Drosophila mtDNA helicases and Drosophila Ind1, a putative interacting partner of the Drosophila mtDNA helicase, in mitochondrial DNA maintenance. In particular, research in this dissertation has focused mainly on the N-terminal primase-like domains, which have no primase activity in metazoan mtDNA helicases and contain a species-specific iron-sulfur cluster in Drosophila mtDNA helicase. Structural studies of human mtDNA helicase present several different oligomeric states and structural dynamicity of its full-length form and demonstrate that the zinc binding-like domain imparts the structural flexibility to the helicase. In addition, open-ring structures and one-arm extended structures are observed, supporting a self-loading mechanism (Chapter 2). Functional studies in chapter 3 suggest that the N-terminal domains of the mtDNA helicases function as a binding module that interacts with a metal cofactor, lipid, and single-stranded DNA. I confirm the presence of the iron-sulfur cluster in the N-terminal domain of Drosophila mtDNA helicase and also show that the N-terminal domain serves a role in membrane binding through electrostatic interactions with cardiolipin. In addition, the positively-charged region where pathogenic mutants are clustered in the RNA polymerase-like domain of human mtDNA helicase contributes to single-stranded DNA binding that is likely required to mtDNA helicase self-loading, translocation, and annealing (Chapter 3). Drosophila Ind1, as a putative iron-sulfur donor to Drosophila mtDNA helicase, has been characterized. Its mitochondrial localization, dimerization, and the membrane binding property driven by electrostatic and hydrophobic interactions were demonstrated. In particular, the membrane binding property of Drosophila Ind1 may facilitate the transfer of an iron-sulfur cluster to membrane-bound recipients: complex I and possibly Drosophila mtDNA helicase (Chapter 4). Although the putative interaction between the mtDNA helicase and the Ind1 was not detected under given conditions in this research, I hypothesize that the expected protein-protein interactions may occur under optimized conditions with an appropriate cofactor (ATP) and chaperone proteins, based on experimental results and comparison with other protein in its Mrp/ MinD family in chapter 4. Including the experimental plans to test this hypothesis, I designed the detailed plans that expand studies in this dissertation. The proposal is categorized into three unsolved questions: biological roles of the iron-sulfur cluster in Drosophila mtDNA helicase, effects of Dm Ind1 on oxidative phosphorylation and mtDNA replication, and new binding partners of the mtDNA helicase (Chapter 5). These approaches will contribute to the better understanding of the mtDNA helicase in various mechanisms to maintain mtDNA stability.
Read
