Flagellated protozoa of the Order Kinetoplastida contain a single mitochondrion termed a kinetoplast. Two distinct populations of double stranded circular DNA, the maxicircles and the minicircles, are localized within the kinetoplast as a single highly catenated network. These organisms are generally parasitic and are responsible for a variety of human and animal diseases including leishmaniasis, African sleeping sickness and Chagas disease. We will determine the biochemical mechanisms involved in the replication of the DNA minicircles of the trypanosomatid Crithidia fasciculata. These studies should provide insight into eukaryotic replication mechanisms in general and may reveal unique features of kinetoplast replication that can be targeted in the development of chemotherapeutic agents. We will isolate and characterize minicircle replication intermediates synthesized in isolated kinetoplasts and will develop a model of the mechanics of the replication and partitioning of minicircles. Replication of minicircles in soluble extracts will be investigated as a means of identifying specific enzymatic steps in the replication process and developing assays for the purification of the individual enzymes required for minicircle duplication. Replication products produced in vitro will be characterized by physical and biochemical methods in order to elucidate the various enzymatic reactions involved in the overall replication mechanisms. Antibodies prepared against specific components of the replication machinery will be used to investigate the requirements for individual components in different stages of replication, to determine their cellular localization and to provide reagents for rapid immunoaffinity purification of the various proteins.