The SV40 T antigen specifically interacts with the sequences at the SV40 DNA replication origin to initiate virus DNA replication and to regulate early and late viral gene expression. To date this interaction is understood best at the DNA level and relatively little is known about the polypeptide regions or domains which are involved in the binding to the virus DNA. Likewise, there is little information about the general secondary structure of T antigen. We propose to examine DNA binding regions of T antigen by mapping polypeptide fragments that bind to the DNA in vitro and, secondly, by studying the association of the DNA with various previously characterized mutant T antigens. Information on secondary structure will be obtained by mapping internal disulfide bonds in the polypeptide chain. Together these experiments will provide us with data regarding the structural organization of this protein and will allow us to better understand the protein component of the T antigen - SV40 DNA interaction. T antigen is phosphorylated at multiple sites at the NH2- and COOH-terminal regions. We have previously determined that the protein is phosphorylated at the NH2-terminal region in a specific stepwise process which involves phosphorylation and dephosphorylation events. The function of this phosphorylation pathway is not known but the fact that it is highly regulated suggests that it is linked to the biochemistry of the protein in the cell. We plan to map the specific T antigen residues which become phosphorylated and dephosphorylated as the polypeptide matures in the cell. In addition, we propose to determine if there is a relationship between these phosphorylation states and DNA or ATP binding activity. Finally, because the subpopulation of T antigen which is present at the cell surface is important to the mechanism of tumor rejection and may also be involved in transformation, we propose to study the structure of this fraction of T antigen with respect to oligomerization and phosphorylation states.