Phosphorylation of simian virus 40 T antigen on Thr 124 selectively promotes double-hexamer formation on subfragments of the viral core origin

Cell cycle dependent phosphorylation of simian virus 40 (SV40) large tumor antigen (T-ag) on threonine 124 is essential for the initiation of viral DN A replication. A T-ag molecule containing a Thr-->Ala substitution at this position (T124A) was previously shown to bind to the SV40 core origin but t o be defective in DNA unwinding and initiation of DNA replication. However, exactly what step in the initiation process is defective as a result of th e T124A mutation has not been established. Therefore, to better understand the control of SV40 replication, we have reinvestigated the assembly of T12 4A molecules on the SV40 origin. Herein it is demonstrated that hexamer for mation is unaffected by the phosphorylation state of Thr 124. In contrast, T124A molecules are defective in double-hexamer assembly on subfragments of the core origin containing single assembly units. We also report that T124 A molecules are inhibitors of T-ag double hexamer formation. These and rela ted studies indicate that phosphorylation of T-ag on Thr 124 is a necessary step for completing the assembly of functional double hexamers on the SV40 origin. The implications of these studies for the cell cycle control of SV 40 DNA replication are discussed.