We have previously mapped and characterized the DNA-binding domain of the SV70 tumor (T) antigen. Our data demonstrate that a region mapping between residues 131 to 371 is important for replication origin-specific DNA binding. By protease digestion experiments, we have also shown that a """"""""core"""""""" region mapping from approximately 140 to 281 is tightly associated with the DNA. Genetic data from other labs indicate that the essential sequences within this core region map from 140 to about 260. Together, these findings define the minimal region of T antigen which is needed to bind to the replication origin. As part of this proposed work, we plan to identify the sequences within the core element that are essential for the proper structure of the DNA- binding region and for making contacts with the DNA. This will be done by testing the activity of mutants with specific amino acid substitutions. Beside DNA-binding, the SV40 T antigen has several other activities that are involved in virus DNA replication. These include an ATPase, a single-strand DNA-binding activity, a helicase and an origin-specific DNA unwinding activity. Although the origin DNA-binding and ATPase domains have been carefully mapped, there is presently very little information about the regions that are responsible for the other three activities. We plan to map these regions by examining the in vitro activity of various tryptic fragments. Finally, in order to understand how the multiple functional domains of this protein communicate and interact with one another during viral DNA replication, we will generate various deletion mutants and test the effect of the deletions on T antigen function.
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