NFI and Oct-1 stimulate Ad5 DNA replication. The in vitro replication assays were performed using Ad5 XhoI-digested TP-DNA as a template. As a result of replication two dsDNA fragments containing the origin (dsDNA B and C) and single-strands originated from the second and subsequent rounds of replication (ssDNA B and C) are generated. The D fragment (dsDNA D) is lacking an origin, but is nonspecifically labelled by polymerase. The first two lanes represent the basal level of replication in the absence of NFI and Oct-1. To stimulate replication 50, 75, 110, 165 and 250 ng of NFI (lanes 3–7 and 13–17) or 35, 53, 80, 120 and 180 ng of Oct-1 (lanes 8–12 and 13–17) were added. Lanes 13–17 represent replication in the presence of both NFI and Oct-1. The level of stimulation was determined by comparing the replication signal (the sum of the ds and ss B and C bands) in the presence of NFI and/or Oct-1 with the average basal signal. A second independent experiment gave similar results.