FIG. 6.

Effect of temperature and of ATP on the E2-dependent recruitment of wild-type E1 or E1 (353 to 649) to the origin (ori). DNA-protein complexes were assembled with E2 and with either wild-type E1 (A) or E1 (353 to 649) (B) made by in vitro translation. Due to differences in their levels of expression in reticulocyte lysate in vitro, approximately twice as much E1 (353 to 649) protein was present in the assay shown in panel B than wild-type E1 (1 to 649) in the assay shown in panel A. The immunoprecipitation of complexes was done with the K72 polyclonal antibody. Binding reactions and immunoprecipitations were carried out at the indicated temperature (4, 23, or 37°C) and in the presence (+) or absence (−) of ATP (or ADP) and magnesium at concentrations of 5 and 3 mM, respectively. Detection of bound DNA was done as described in the legend to Fig. 2A. The autoradiograms in panels A and B were obtained after exposure of the gels for 3 and 15 h, respectively. (C) Graph generated by quantification of the data in panels A and B with a PhosphorImager. For each panel, the intensity of each ori band was normalized to the amount of ori probe loaded in lane 1. The amount of probe loaded in lane 1 was the same for both gels. The intensity of the ori band detected with wild-type E1 at 23°C in the absence of ATP-Mg was set arbitrarily at 100%.