Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomes, parasitic protozoa that cause major human disease. The major subject of our study is Trypanosoma brucei that causes African sleeping sickness; kDNA is an unusual structure, consisting of DNA circles that are catenated into a giant network. The network contains several thousand minicircles and a few dozen maxicircles. The subject of this proposal is the replication of kDNA. Major steps in kDNA synthesis include the sequential release of minicircles from the network by a topoisomerase to form free minicircles, replication of the free minicircles via theta structures, reattachment of the progeny minicircles to the network, and division of the double-size network into two daughter networks. The major emphasis of this proposal is to discover new proteins involved in kDNA replication. Because of the unusual structure of kDNA and because it is essential for parasite viability, these proteins could be targets for anti-trypanosome chemotherapy. One approach for discovering new kDNA replication proteins, in Specific Aim 1, is to screen an RNA interference library for cells with depleted kDNA, and preliminary data show that this approach is feasible. A second approach, in Specific Aim 2, is based on proteomics, in which mitochondrial DNA binding proteins are identified by tandem mass spectrometry. Again, preliminary data indicate that this strategy is also feasible. The proposed experiments in Specific Aim 3 are to characterize and determine the function of proteins discovered in Aims 1 and 2. Characterization will involve study of enzymatic properties and intracellular localization. Functional analysis will depend heavily on gene silencing by RNAi and an analysis of changes in structure of kDNA networks and free minicircle replication intermediates. [unreadable] [unreadable]