Trypanosomes are protozoa which cause several types of disease. This proposal is for the study of a unique type of mitochondrial DNA found in trypanosomes (kinetoplast DNA) and its interaction with a new type II topoisomerase. Topoisomerases alter the topology of DNA by causing transient breaks in the phosphodiester chain and then resealing the chain. The structure of kinetoplast DNA is very unusual. The mechanism of action of this topoisomerase will be compared to those of previously described topoisomerases. It is known that certain drugs used to treat trypanosomal infections interact with this topoisomerase but not others, causing cleavage of DNA to occur but resealing not to. Synthetic DNA substrates will be prepared which mimic bent regions in the kinetoplast DNA, cross- over junctions, minicircles, and enzyme binding site sequences. These results will be compared to those obtained with natural DNA substrates. The information obtained will add to our knowledge of how DNA is packaged and processed in cells. %%% Trypanosomes are parasitic protozoa which cause sleeping sickness and Chagas' disease. Several drugs are available to treat these diseases. They apparently function by inhibiting an enzyme called topoisomerase II, which is found in trypanosomal mitochondria. This enzyme is involved in DNA replication, and after drug treatment the DNA is cleaved but not resealed. A variety of approaches using synthetic DNAs with structural features similar to naturally occurring DNA will be used to determine the mechanism of action of the enzyme and compare it to other topoisomerases.
