NUCLEOTIDE BINDING PROPERTIES OF SV40 LARGE T PROTEIN

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I intend to examine the nucleotide binding properties of SV40 large T protein and determine their relationship to T function. SV40 T is known to be involved in the initiation events for SV40 DNA replication, but even though we know a great deal about the complex formed by T and the SV40 DNA origin we cannot explain the actual mechanism of initiation. There are many modifications reported for T including phosphorylation, acetylation, ADP-ribosylation, and oligomerization, some of which have been found to affect the biochemical activity of T. From my research into the structure-function relationships of large T, I discovered that a nucleotidyl-protein complex was formed following incubation of partially purified SV40 T and ATP in the presence of magnesium. ATP was the preferred nucleotide, and Alpha32P-ATP, Alphathio35S-ATP and 3H-ATP all labeled SV40 Large T. In this grant proposal methods are outlined for determining the nature of the chemical linkage between T and nucleotide, and for identifying the amino acid involved in the linkage. The binding site on the protein can be mapped and compared with the present deletion map of SV40 large T with respect to functional domains. Then I shall pursue preliminary data which suggest that T may function as a nucleotidyl transferase. After forming the T-nucleotidyl complex using radioactive ATP, radioactive label can be released from the complex in the presence of Mg, pyrophosphate(PPi) and polydT. There is no significant release of label in the presence of Mg with a) monophosphate, b) polydT without PPi, or c) PPi and polydG, -dC, or -dA. This grant proposal outlines methods of substantiating this observation. If so, the interaction between T and host cell replication enzymes could constitute that of a primer or primase involved in bringing the first adenine nucleotide to the initiation site(s). Site directed mutagenesis involving the nucleotide binding site of T and reconstitution of mutant virus would allow analysis of the functional significance of this modification in vivo as well as production of mutant T for biochemical analysis. Tracing the putative nucleotide transfer reaction might help to resolve the phenomenon of T-induced initiation of DNA replication from the transformation function of SV40 T.