Regulation of Trypanosome DNA Replication

Ray, Dan

Abstract

The kinetoplastid flagellates represent a unique opportunity for investigating eukaryotic DNA replication mechanisms and their coordination with the cell cycle. These parasitic protozoa are distinguished by the presence of a single mitochondrion which contains approximately 5,000 copies of a plasmid-like DNA (minicircles) catenated into an enormous network together with 25-50 copies of the mitochondrial DNA (maxicircles). The kinetoplast DNA (kDNA) accounts for approximately 25% of the cellular DNA and, unlike mammalian mitochndrial DNA, replicates coordinately with the nuclear S phase. The long-term goal of this proposal is to determine the biochemical and enzymatic mechanisms os replication of the kDNA minicircles in the trypanosomatid Crithidia fasciculata and to identify features of the mechanism coordinating minicircle replication with the cell cycle. Kinetoplast-associated proteins involved in initiation and possible pre-initiation steps in minicircle replication will be purified and used to supplement crude kinetoplast extracts in order to develop a minicircle- dependent DNA replication system. The enzymes involved in RNA-primed initiation at minicircle replication origins will be purified and used to determine the mechanism and the sites of initiation. Antibodies against purified replication proteins will be used to localize individual proteins within the kinetoplast and to determine the possible association of additional replication proteins with the two topoisomerase-containing structures located, in previous studies, at the periphery of the kDNA network. The gene encoding the kinetoplast-associated topoisomerase will be cloned and sequenced and the expression of RNA and protein products of the gene will be determined at various times in the cell cycle. Protein- protein interactions of the topoisomerase will be sought and, if found, exploited to identify and purify possible associated proteins of the replication apparatus. This proposal addresses fundamental questions in modern cell biology and therefore is relevant to an understanding of both normal and abnormal growth and development of cells and tissues. In addition, unique features of the kinetoplast DNA replication mechanism may reveal appropriate targets for the development of new chemotherapeutic drugs for the treatment of trypanosomiasis and leishmaniasis.