NUCLEOTIDE BINDING PROPERTIES OF SV40 LARGE T PROTEIN

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The long-term goal of this project is to determine the location and parameters of the nucleotide binding site(s) in SV40 large T antigen (T-Ag), and investigate the relationship between ATPase, adenylylation and other functions of T-Ag. Since there are several activities involving nucleotide associated with the protein, we will first determine whether there is one or more binding sites for ATP and the binding affinity(s). We have proposed a topological model for a consensus ATP binding domain in the protein, without the aid of crystallographic analyses, which includes the active sites for ATPase and adenylation. The research plan will focus on testing aspects of this newly proposed model both biochemically and genetically. At least two reactive ATP analogs will be used to probe the ATP binding site in T-Ag, the modifications will be analyzed for their specificity, and the modified residues will be mapped. Using the model to facilitate the targets for mutagenesis, the ATP binding site will also be probed genetically. Each of the mutant T-Ags will be tested for its ability to bind and hydrolyze ATP, and to be adenylylated. The results of these studies might support aspects of the proposed model or refute them, and the biochemical properties of the mutant T-Ags could reveal whether there is a relationship between nucleotide ATPase and adenylylation. With the relevant T-Ag mutants, further investigation of the biochemical and/or functional relationship between nucleotide binding activities and other associated functions of the protein will be investigated such as helicase, oligomerization, autoregulation of viral mRNA synthesis, oncogenic transformation and SV40 DNA replication. These investigations are intended to help in resolving the individual functions of T-Ag from one another and to help in understanding the details of T-Ag's interactions with the infected- cell.