Figure 5.

Formation of TP-dAMP complex catalysed by Nf and GA-1 DNA polymerases. The assays were performed as described under Materials and Methods in the presence of 1 mM (for GA-1 and φ29 DNA polymerases) and 2 mM (for Nf DNA polymerase) MnCl₂, 5 ng of TP, 500 ng of TP-DNA, 0.1 μM [α-³²P]dATP (1 μCi), and the indicated amounts of DNA polymerase. After incubation at 30°C for the indicated times, samples were analysed by SDS–PAGE and autoradiography. The position of the TP-dAMP initiation complex is indicated. Quantification was by densitometry of the band corresponding to the labelled TP-dAMP complex, detected by autoradiography.