Kinetoplast DNA is the remarkable mitochondrial DNA in trypanosomes and related parasitic protozoa. These parasites cause majojr human diseases. The DNA is organized in the form of networks of thousands of interlocked minicircles and a few maxicircles. One network is present in the cell's single mitochondrion. Maxicircles are the functional equivalent of the mitochondrial DNA in other eukaryotes; the function of minicircles is not yet known. The long term objectives of this research are to understand the replication, structure, and, ultimately, the function of kinetoplast DNA. Minicircles replicate as free circles after release from the network by a topoisomerase. The progeny reattach to the network. Up to now, very little is known about the molecular mechanisms involved in the replication process. Several kinds of molecules have been identified in free minicircle preparations (e.g. Cairns forms and covalently-closed, nicked, gapped, and single-strand minicircles). The structures of these molecules and their role in replication will be studied in detail using EM and biochemical techniques. Hopefully, this information will lead to an understanding of the replication pathway of kinetoplast minicircles. Maxicircles replicate as network-bound rolling circles. After completion, the branch is cleaved off. It then recircularizes and reattaches to the network. The maxicircle origin will be mapped and the structure of the linear intermediate will be studied. As for kinetoplast structure, studies will be made on the packing of the network in its highly organized compact form inside the mitochondrion. Proteins, which may stabilize this structures, will be isolated and characterized. Also, the role of the minicircle """"""""bent helix"""""""" in stabilizing this structure will be evaluated. The bent helix causes unusual physical properties in minicircle restriction fragments, and further studies will also be conducted to evaluate what kind of sequences cause helix bending. These studies will contribute to an understanding of biochemical structures and pathways which are unique to these parasites. This knowledge may lead to new modes of chemotherapy.

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National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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Johns Hopkins University
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