Figure 6.

Specific recognition of replication origins by chimerical DNA polymerase. (A) TP-primed initiation assays were carried out in the presence of 1 mM MnCl₂, 1.6 nM of the indicated TP-DNA, 120 nM of chimerical DNA polymerase and 240 nM of the indicated TP. As control, 15 nM and 30 nM of wild-type ϕ29 DNA polymerase and TP, respectively, were used. After incubation for 10 min at 30°C, samples were stopped and processed as described in Materials and Methods section. (B) Effect of ϕ29 DBP on the activity of chimerical DNA polymerase. The assay was performed in the presence of 1 mM MnCl₂, 1.6 nM of ϕ29 TP-DNA as template, in the absence (−) or presence (+) of 35 µM of ϕ29 DBP. Under these conditions, 60 nM of chimerical DNA polymerase and 120 nM of the indicated TP were incubated for 20 min at 30°C. As control, 30 and 60 nM of wild-type ϕ29 DNA polymerase and TP, respectively, were incubated for 2 min at 30°C. Further processing of the samples was performed as described under Materials and Methods section.