Structural Analysis of DNA Replication Machinery of P. falciparum

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DESCRIPTION (provided by applicant): Malaria is a leading cause of disease and death in the world. Over 300 million people are infected annually, causing 1-3 million deaths per year. In addition to causing incapacitating fever and chills, malarial infection is causally-associated with Burkitt's Lymphoma, which accounts for ~74 percent of childhood malignant disorders in endemic regions. Of the species that cause malaria, Plasmodium falciparum is the most virulent, and causes nearly all associated deaths. P. falciparum contains an essential organelle, termed the apicoplast, that when its functions or replication are inhibited kills parasites related to P. falciparum. I propose to investigate PfPrex, the putative replicative machinery of P. falciparum's apicoplast, as a promising drug target. PfPrex is evolutionarily well-conserved and has demonstrated primase, helicase, exonuclease and polymerase activities. It is proteolytically matured into several smaller domains in vivo, which is reminiscent of viral poly-protein strategies. Inhibition of these enzymatic activities, or of the maturation of PfPrex, will likely inhibit replication of the apicoplast, and in turn cause the death of the parasite. The goals of these proposed aims is to illuminate mechanisms of apicoplast function, and to identify essential elements of PfPrex as targets for novel drugs to treat malarial infection and prevent endemic Burkitt's Lymphoma. I propose to accomplish this by addressing the following two questions: (1) What is the three-dimensional structure of the primase domain of PfPrex? and (2) What forms of PfPrex function in vivo? First, I have cloned the functional primase domain of PfPrex from genomic DNA, over-expressed and purified this protein from E. coli, and begun to characterize it biochemically. I will now continue to examine PfPrex primase through in vitro assays, and determine its three-dimensional structure using x-ray crystallography. Second, using polyclonal antibodies that I have developed against PfPrex's TOPRIM domain, I will examine if proteolytic cleavage of PfPrex is required for its efficient function, as well as to identify the mature, functional forms of PfPrex in vivo throughout the asexual life cycle of the parasite. PUBLIC HEALTH RELEVANCE: Of the parasite species that cause malaria, Plasmodium falciparum causes nearly all associated deaths, and also is causally-associated with endemic Burkitt's Lymphoma. I intend to study a protein, called PfPrex, which this parasite likely needs to grow and survive. Using information gained about how PfPrex assembles, functions and is processed in the parasite, I will identify specific new drug targets to inhibit its functions, which will likely kill the parasite, treat malarial infection, and prevent endemic Burkitt's Lymphoma.