Fig. 2.

Internal initiation of Nf DNA replication. The formation of the 4 TP-dNMP complexes was assayed and analyzed. Standard initiation reactions were carried out with 0.1 μM each of the indicated labeled dNTP (A, C, G or T) under the conditions specified in Materials and Methods, in the presence of 60 nM Nf DNA polymerase, 120 nM Nf TP and 5.5 μM the indicated oligonucleotide as template. Reactions were started by adding 1 mM MnCl₂ and, after incubation for 10 min at 10 °C, the reactions were stopped, processed, and analyzed by SDS/PAGE and autoradiography. The six 3′-terminal nucleotides of each ssDNA are also shown. The nucleotide changed in each template is indicated. For each template, the relative efficiency of the initiation reaction with each of the 4 possible substrates is indicated, considering as 100% the incorporation level of the preferred dNTP.