Figure 7.

Effect of Nf DBP on in vitro Nf TP-DNA replication. (A) Effect of Nf DBP on the formation of the TP-dAMP initiation complex and limited elongation. The assay was performed as described in Materials and Methods, using 10 mM MgCl₂, 0.5 μg of Nf TP-DNA, 10 ng of Nf DNA polymerase, 10 ng of Nf TP and the indicated concentration of the corresponding dNTP. Samples were incubated either in the absence (−) or presence of 10 μg of Nf DBP. After incubation for 10 min at 30°C, the initiation and transition products were detected by high resolution SDS–PAGE. Completely replicated molecules remain at the interphase with the stacking gel. The length of the different partially elongated products and the position corresponding to full-length Nf TP-DNA are indicated on the left. (B) Effect of Nf DBP on the truncated elongation of the TP-dAMP complex. The assay was performed as described in (A), using 2 mM MnCl₂ and increasing dATP concentrations in the presence of 10 μM dGTP and dTTP. When indicated, the reaction mixture contained 10 μM dCTP and/or 10 μg of Nf DBP. Samples were processed and analysed as described in (A).