Figure 4.

Formation of the TP–dAMP complex catalyzed by wild-type or mutant Φ29 DNA polymerase in the presence or absence of Φ29 TP–DNA. The reactions were carried out as described in Materials and Methods, in the presence of 125 ng TP. The template-dependent reaction (top) was carried out in the presence of 10 mM MgCl₂, 0.5 µg TP–DNA and 5 ng wild-type or mutant Φ29 DNA polymerase. Incubation was for 4 min at 30°C. The template-independent reaction (bottom) was performed in the presence of 1 mM MnCl₂ and 80 ng mutant or wild-type Φ29 DNA polymerase. Incubation was for 120 min at 30°C. The samples were analyzed by SDS–PAGE and autoradiography. The band corresponds to the TP–dAMP initiation complex. Quantification was by densitometric analysis of the band corresponding to the labeled TP–dAMP complex, detected by autoradiography.