Figure 2.
In vitro protein-primed initiation with chimerical TPs. Reaction mixtures contained, in addition to the indicated wild-type DNA polymerases (15 nM), and wild-type (30 nM) or chimerical (120 nM) TPs, 1.6 nM of the indicated TP-DNA. Reactions were started by adding 1 mM MnCl2. After incubation for 1 min (wild-type systems) or 10 min (with chimerical TPs) at 30°C, the reactions were stopped, processed and analysed by SDS–PAGE and autoradiography (see Materials and Methods section for details). The various TP-DNAs, DNA polymerases and TP variants, as well as the mobility of the TP-dAMP complexes are indicated. For clarity, the active complexes are depicted on top of the figure. ϕ29 DNA polymerase and TP are coloured in yellow, whereas GA-1 DNA polymerase and the homologous TP are in green. The different parts of chimerical TPs are coloured according to such a colour code.