Structure and function of the ACMV geminivirus replication initiator protein

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The replication-associated protein (Rep) of plant geminiviruses is a multifunctional protein necessary for initiation of viral replication by rolling circle replication and for interaction with various host cell factors. In addition, geminivirus Rep proteins possess ATPase/helicase function. African cassava mosaic virus Rep protein will be ectopically expressed in fission yeast (Schizosaccharomyces pombe) and purified from cell extracts for further analysis. By using electron microscopy (EM) and size exclusion chromatography (SEC) the formation of Rep protein oligomers in the presence of different cations as well as adenosine nucleotides and DNA will be assessed. These experiments are aimed to dissect if Rep complexes with different numbers of monomers might be involved for different functions of the Rep protein (e.g. endonucleolytic cleavage for initiation of replication, helicase). Based on these analyses, three dimensional structures of the functional Rep oligomers will be determined by electron microscopy and image processing. The structures will help to understand the different functions that are combined in the Rep protein on a molecular level.