Fig. 5.

In vitro protein-primed initiation with chimerical TPs. (A) Reaction mixtures contained, in addition to φ29 or Nf DNA polymerase (120 nM), and either the wild-type or chimerical TPs (240 nM), 3.6 μM the indicated oligonucleotide as template and 0.1 μM [α-³²P]dATP as initiator nucleotide. Reactions were started by adding 1 mM MnCl₂ and, after incubation for 1 h at 30 °C, were stopped, processed and analyzed by SDS-PAGE and autoradiography (see Materials and Methods for details). (B) The reactions were carried out essentially as described in A, using 0.1 μM [α-³²P]dCTP as initiator nucleotide. Reactions were started by adding 1 mM MnCl₂ and incubated at 30 °C for 1 h or 30 min in the case of φ29 or Nf DNA polymerase, respectively. The reactions were stopped, processed and analyzed by SDS-PAGE and autoradiography (see Materials and Methods for details). The various templates, DNA polymerases and TP variants, and the mobility of the TP-dAMP or TP-dCMP complexes are indicated.