A Single-Molecule View of the Functional Helicase-Primase Complex of the Bacteriophage T4

Abstract

We have studied the assembly mechanisms and DNA unwinding activity of the bacteriophage T4 helicase-primase (primosome) complex using single molecule Fluorescence Resonance Energy Transfer (sm-FRET) techniques. These experiments employed surface-immobilized DNA model replication forks labeled in the duplex region with donor/acceptor (Cy3/Cy5) chromophore pairs, and the time-dependent SM-FRET signal was monitored during the DNA unwinding process. We used these approaches to investigate the subunit stoichiometry of the primosome and the assembly pathway required to form a functional and fully active primosome-DNA complex. Our results confirm that the gp41 hexameric helicase binds only weakly to the DNA fork junction, but that the addition of a single subunit of gp61 primase stabilizes the primosome complex at the fork junction, resulting in the formation of a fully active helicase with a gp41:gp61 subunit stoichiometry of 6:1. The functional primosome complex exhibited enhanced [GTP]-dependent processive activity, which was reflected in an increase in the number of sm-FRET conversion events. Moreover, we showed that the use of other assembly pathways resulted in incorrect subunit stoichiometries, the formation of metastable DNA-protein aggregates and a loss of helicase functionality. These single-molecule studies support the results of recent ensemble experiments and provide a direct ‘real time' visualization of the assembly pathway and unwinding activity of the functional T4 primosome complex.