Abstract
Initiation of adenovirus DNA replication is strongly enhanced by two cellular transcription factors, NFI and Oct-1, which bind to the auxiliary origin and tether the viral precursor terminal protein-DNA polymerase (pTP.pol) complex to the core origin. NFI acts through a direct contact with the DNA polymerase, but the mode of action of Oct 1 is unknown. Employing glutathione S-transferase-POU pull-down assays and protein affinity chromatography, we have established that the POU domain contacts pTP rather than pol. The POU homeodomain is responsible for this interaction. The protein-protein contacts lead to increased binding of pTP-pol to the core origin, which is caused by a reduced off-rate. The enhanced formation of a pTP.pol.POU complex on the origin correlates with stimulation of replication. Using an immobilized replication system, we have studied the kinetics of dissociation of the Oct-1 POU domain during replication. In contrast to NFI, which dissociates very early in initiation, Oct-1 dissociates only when the binding site is rendered single-stranded upon translocation of the replication fork. Our data indicate that NFI and Oct-1 enhance initiation synergistically by touching different targets in the preinitiation complex and dissociate independently after initiation.
Highlights
Employing glutathione S-transferase-POU-specific domain (POUs) pull-down assays and protein affinity chromatography, we have established that the POU domain contacts precursor terminal protein (pTP) rather than pol
The main proteins required for efficient replication are encoded by the virus itself and are expressed early in infection. These are the DNA polymerase; the precursor terminal protein, which is required for correct initiation; and the DNA-binding protein (DBP), which is essential for elongation
Some of the pTP ran through the column, a significant amount was retained on the GST-POU column and eluted at approximately 225 mM NaCl
Summary
Employing glutathione S-transferase-POU pull-down assays and protein affinity chromatography, we have established that the POU domain contacts pTP rather than pol. In vitro Oct-1 stimulates replication 3–7-fold, depending on the pTP1⁄7pol concentration and a DNA independent interaction between the pTP1⁄7pol complex and the POU homeodomain was observed [16]. NFI and the POU Domain Bind Different Subunits in the pTP1⁄7pol Complex—Because we could not exclude the presence of inhibitors in the crude extracts, we purified pTP and pol separately, as well as the pTP1⁄7pol complex, and studied binding in a pull-down assay employing GST-POU or GST-NFI.
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