INITIATION OF DNA-REPLICATION BY SIMIAN-VIRUS-40 T-ANTIGEN IS INHIBITED BY THE P107 PROTEIN

The p107 protein is related to the Rb protein by a 60-kDa region of ho mology called the pocket domain that binds cellular proteins such as E 2F and cyclins A and E as well as the viral oncoproteins E1A, E7, and SV40 T antigen. The p107 and Rb proteins have both been implicated as negative regulators of cell growth. We have examined the effect of the unphosphorylated pocket domain of p107 on specific stages of the T an tigen-mediated replication of SV40 DNA in vitro. The pocket domain inh ibited replication by preventing the assembly of T antigen at the SV40 core origin DNA containing binding site II. However, both proteins fo rmed a ternary complex with DNA containing T antigen-binding site I. T he pocket domain of p107 did not inhibit the oligomerization of T anti gen in the absence of DNA, and the p107 derivative is bound to the int ermediates of this reaction. In the unwinding assay, once T antigen wa s preassembled as hexamers at the core origin, the pocket domain bound to and stabilized the complex, resulting in an increase in the yield of unwound product. Preformed T antigen hexamers complexed with the po cket domain bind to a synthetic replication fork, and this complex sup ported unwinding. Consistent with this, the p107 pocket domain had no effect on the helicase activity of T antigen in an assay using a parti al duplex substrate. However, a complex containing p107 and T antigen assembled at the core origin did not support SV40 DNA replication in H eLa cell crude extracts or in the monopolymerase reaction. This inhibi tion is due to the inability of the complex to bind to DNA polymerase alpha, which is required for the initiation of DNA synthesis. The data suggest pathways by which the pocket domain of p107 can negatively re gulate T antigen-mediated replication in vitro. The binding of T antig en to p107 is discussed with respect to its role in mitigating negativ e cell growth control, resulting in viral mediated transformation.