MECHANISMS OF SIMIAN-VIRUS-40 T-ANTIGEN ACTIVATION BY PHOSPHORYLATIONOF THREONINE-124

Previous studies have shown that phosphorylation of simian virus 40 (S V40) T antigen at threonine 124 enhances the binding of T antigen to t he SV40 core origin of replication and the unwinding of the core origi n DNA via hexamer-hexamer interactions, Here, we report that threonine 124 phosphorylation enhances the interaction of T-antigen amino acids 1 to 259 and 89 to 259 with the core origin of replication, Phosphory lation, therefore, activates the minimal DNA binding domain of T antig en even in the absence of domains required for hexamer formation. Acti vation is mediated by only one of three DNA binding elements in the mi nimal DNA binding domain of T antigen, This element, including amino a cids 167, 215, and 219, enhances binding to the unique arrangement of four pentanucleotides in the core origin but not to other pentanucleot ide arrangements found in ancillary regions of the SV40 origin of repl ication, Interestingly, the same four pentanucleotides in the core ori gin are necessary and sufficient for phosphorylation-enhanced DNA bind ing, Further, we show that phosphorylation of threonine 124 promotes t he assembly of high-order complexes of the minimal DNA binding domain of T antigen with core origin DNA, We propose that phosphorylation ind uces conformational shifts in the minimal DNA binding domain of T anti gen and thereby enhances interactions among T-antigen subunits oriente d by core origin pentanucleotides, Similar subunit interactions would enhance both assembly of full-length T antigen into binary hexamer com plexes and origin unwinding.