SV40 T Antigen Structure and Helicase Mechanisms

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SV40 large T antigen (LT) is a viral oncoprotein with diverse biological functions. It is involved in cellular transformation through regulating the activities of tumor suppressors. It also plays an important role in viral DNA replication by assembling around the origin into a double hexamer that function not only as a helicase to open up the origin and unwind the fork DNA, but also as a platform for recruiting the essential cellular replication proteins, such as RPA. Our goals are to understand the structural basis of LT functions in these diverse biological processes by studying LT structures in their various oligomeric and conformational states. Four specific aims are built upon our recent progress in the biochemistry/protein chemistry of LT and the crystallization of a LT fragment containing the larger C-terminal portion (residues 251-630). (i) Crystal structures of larger LT containing the N-terminal domains will be determined to learn how the different domains interact with each other. The structures will reveal the specific interactions for oligomerization that are important for DNA helicase activity. These structures will also provide insight into the mechanisms of how LT regulates tumor suppressors through protein-protein interactions in cell transformation. (ii) The mode of interactions between LT and DNA at the origin and at the replication fork will be studied by determining the structures of the complexes containing LT fragments and the corresponding DNA substrates. (iii) The structures of LT in the presence and absence of ATP and its analogs will be determined in order to understand the mechanisms of the ATP induced conformational switch. These structures will elucidate the structural basis for the ATP-driven DNA-unwinding process, and provide a guide for the studies of the structure/function relationship of LT through site-mutagenesis. (iv) Finally, the structure of the complex of LT with RPA will be determined to understand the molecular interactions between the two proteins, which will provide insight into how LT coordinates with RPA in the origin and fork DNA unwinding. This research will provide information useful for understanding the general molecular mechanisms of cell cycle control and DNA replication process in eukaryotes, both of which will be valuable for the potential development of anti- tumor virus strategies, and for human intervention of abnormal cell cycles that lead to cancer.