KINETOPLAST DNA ASSOCIATED PROTEINS IN KINETOPLASTID PROTOZOA

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Members of the order Kinetoplastida, which include trypanosomes and leishmania, share one of the most unusual mitochondrial DNA structures found in nature. The single mitochondria present in these organisms contains a relatively large proportion of the cellular DNA (kinetoplast DNA, kDNA) organized in a disk-shaped structure and made up of a population of two types of topologically linked circular molecules. In spite of the recent advances made in our understanding of the mechanism of replication of the major DNA component of the networks, some outstanding questions about the structure and function of kDNA networks still remain unanswered. For example, it is not clear what mitochondrial function the network structure serves, nor how it is partitioned at the end of replication. The maintenance and possibly the function of kDNA may depend on some proteins components of the networks. Our preliminary studies with Trypanosoma cruzi indicate that several proteins remain associated with kDNA networks after exposure of trypanosome cells to a mild detergent in presence of 2M NaCl. Rabbit antibodies raised against these T.cruzi kDNA-protein complexes recognize some protein(s) that appear to be bound to the supercoiled form of T.cruzi minicircle DNA. More interestingly, using both immunofluorescence and immunoelectron microscopy, we have observed that the same antibody preparation cross-reacts with kinetoplast DNA present in related kinetoplastid protozoa. Based on these observations, the present research proposal is designed to unravel some basic features about the structure and function of kinetoplast DNA networks, by investigating the function of some of the proteins associated with kDNA. We propose first, to identify and characterize proteins bound to T.cruzi minicircles; second, to identify common kDNA-associated proteins (KAPs) present in kDNA network preparations from other kinetoplastid protozoa; and third, to isolate and clone the genes encoding for selected KAPs. By understanding the structure and function of kDNA-associated proteins we may learn a great deal about how kDNA networks are maintained and propagated. This information may also provide us with a more rational approach toward the complete eradication of these parasites, some of which are the causative agents of several important human diseases.