THE OCT-1 POU DOMAIN STIMULATES ADENOVIRUS DNA-REPLICATION BY A DIRECT INTERACTION BETWEEN THE VIRAL PRECURSOR TERMINAL PROTEIN-DNA POLYMERASE COMPLEX AND THE POU HOMEODOMAIN

The bipartite POU domain of transcription factor Oct-1 stimulates aden ovirus DNA replication through an interaction with the octamer sequenc e present in the auxiliary origin. Employing an immobilized lit vitro DNA replication system, we show that the POU domain enhances the forma tion of a pre-initiation complex composed of the viral precursor termi nal protein-DNA polymerase (pTP-pol) complex and the origin. To invest igate the mechanism of stimulation we have explored protein-protein in teractions between the POU domain and the pTP-pol complex. Such an int eraction could be detected using a GST-POU fusion protein bound to glu tathione-agarose beads. Binding was also observed with the POU homeodo main (POUHD), albeit weaker than with the intact POU domain, but not w ith the POU specific subdomain. Four point mutations localized in the POUHD were analyzed for pTP-pol binding. Two of these, E22A and E30A, bound pTP-pol equally as well as the wildtype, while the other two, Q2 4A and E29A, were able to bind 2- to 4-fold better. These mutations ar e localized in the same region where the HSV transactivator VP16 binds , but did not coincide with the VP16 contacts. A direct correlation be tween pTP-pol binding and stimulation of DNA replication lit vitro was observed for all mutants, suggesting that stimulation by the POU doma in is caused by an interaction with the viral pTP-pol complex.