Two regions of simian virus 40 T antigen determine cooperativity of double-hexamer assembly on the viral origin of DNA replication and promote hexamer interactions during bidirectional origin DNA unwinding

Phosphorylation of simian virus 40 large tumor (T) antigen on threonine 124 is essential for viral DNA replication. A mutant T antigen (T124A), in whi ch this threonine was replaced by alanine, has helicase activity, assembles double hexamers on viral-origin DNA, and locally distorts the origin DNA s tructure, but it cannot catalyze origin DNA unwinding. A class of T-antigen mutants with single-amino-acid substitutions in the DNA binding domain (cl ass 4) has remarkably similar properties, although these proteins are phosp horylated on threonine 124, as we show here. By comparing the DNA binding p roperties of the T124A and class 4 mutant proteins with those of the wild t ype, we demonstrate that mutant double hexamers bind to viral origin DNA wi th reduced cooperativity. We report that T124A T-antigen subunits impair th e ability of double hexamers containing the wild-type protein to unwind vir al origin DNA, suggesting that interactions between hexamers are also requi red for unwinding. Moreover, the T124A and class 4 mutant T antigens displa y dominant-negative inhibition of the viral DNA replication activity of the wild-type protein. We propose that interactions between hexamers, mediated through the DNA binding domain and the N-terminal phosphorylated region of T antigen, play a role in double-hexamer assembly and origin DNA unwinding . We speculate that one surface of the DNA binding domain in each subunit o f one hexamer may form a docking site that can interact with each subunit i n the other hexamer, either directly with the N-terminal phosphorylated reg ion or,vith another region that is regulated by phosphorylation.