Reconstitution of a nine-protein system that initiates bacteriophage λ DNA replication

We have established an in vitro system, composed of highly purified bacteriophage λ and Escherichia coli proteins, that specifically replicates supercoiled templates bearing the λ replication origin (oriλ). The complete system is composed of three groups of proteins: the virus-encoded initiator proteins (the λ O and P proteins), the E. coli replication fork propagation machinery (single-stranded DNA-binding protein, dnaB helicase, dnaG primase, DNA polymerase III holoenzyme, and DNA gyrase), and two bacterial heat shock proteins (dnaJ and dnaK proteins). DNA replication in this system is initiated at or near oriλ and proceeds unidirectionally rightwards through θ-structure intermediates, ultimately yielding a pair of intertwined daughter circles as the final product. In striking contrast to the situation in vivo and in crude in vitro systems, initiation of γ DNA replication in the purified protein system does not require 'transcriptional activation' of the origin region by E. coli RNA polymerase. We conclude that E. coli primase generates the primers for all leading and lagging strand DNA chains synthesized in this reconstituted λ replication system.