Plasmodium Recombination of Machinery

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Malaria parasites, worldwide are responsible for 300-500 million new infections and 1-2 million deaths each year. An effective vaccine, however, remains elusive, partly due to antigenic diversity and the immune evasion strategies of the parasite. Recombination mechanisms are intimately linked with antigenic variation, a phenomenon of utmost significance for vaccine development against protozoan parasites like the malaria parasite. The long term objective of the proposed studies is to investigate mechanism(s) of genetic rearrangements associated with phenomenon like antigenic variation. An underlying tenet of the work proposed is that understanding the recombination mechanisms in Plasmodium will provide improved opportunities for the development of therapies. In other eukaryotes, homologous recombination (HR) plays a major role in chromosomal rearrangements, and Rad51 and Dmc1 proteins, the eukaryotic counterparts of bacterial RecA recombinase, are central molecules involved in HR during mitosis and meiosis. In eukaryotes, additional proteins like RPA and Rad54 functionally cooperate with Rad51 and mediate HR and the repair of damaged chromosomes. The hypothesis underlying proposed studies is that the Rad51 and other interacting proteins, as mediators of HR, play critical role(s) during growth and development of the parasite and facilitate gene rearrangements in P. falciparum. Molecular identification of Rad51 and Dmc1 homologues in P. falciparum and recent characterization of enzymatic properties of recombinant PfRad51, such as DNA strand exchange and ATPase activities, have strongly indicated a conserved functional role for PfRad51 in these organisms. We will test our hypothesis using biochemical as well as genetic approaches. Studies in the revised specific aims 1 and 2 will lead to in vitro characterization of the proteins involved in HR and thus probe into the biochemical basis of recombination and gene rearrangements in the parasites. PfRad51 gene knockout studies in the revised specific aim 3 will directly evaluate importance of PfRad51 in the erythrocytic growth of the parasite and analysis of repertoire of var gene transcripts. Moreover, studies on Dmc1 disruption (revised specific aim 3) will evaluate the role of meiosis specific recombinase during malaria transmission. The results of this study should be important in unraveling the recombination machinery and molecular and genetic basis for recombination and genetic rearrangements in P. falciparum.