Structural Studies of the SV40 Large T Antigen

University Of Virginia Charlottesville, Charlottesville VA

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Abstract

The Simian Virus 40 (SV40) has served as a powerful tool in understanding the detailed events involved in the initiation and regulation of DNA replication. Interesting, only one protein, Large T antigen (T-ag), is necessary for the initiation viral replication. The SV40 T-ag is a multifunctional protein that serves to force the host cell into S- phase, initiate viral replication, and regulate its own expression at the level of transcription. This protein is known to bind to both the SV40 core origin DNA and to synthetic DNA replication forks. Studies in Dr. Nick Cozzarelli's group have shown that in the presence of synthetic replication forks and ADP, T-ag forms two distinct complexes with differing gel mobilities. One primary goal the proposed research will be to generate 3-D reconstructions of these complexes using both negative stain and cryo-EM techniques. The resulting reconstructions will aid In understanding the DNA binding and helicase activities of Large T-ag. Large T-ag is composed of several interrelated functional and structural domains. Amino acid sequence maps of these domains have been defined; however, the location of these domains in the 3-dimensional structure of the Large T-ag is unknown. The second primary goal of the proposed research is to generate numerous 3-D reconstructions of mutant and truncated forms of the SV40 T-ag and of T-ag complexed with a variety of cellular proteins. These reconstructions will be used to develop a detailed 3-D map and structural models for the domain organization of T-ag that will help identify interactions that are necessary for Large T-ag function.