The Escherichia coli dnaC gene product. III. Properties of the dnaB-dnaC protein complex.

Abstract

The Escherichia coli dnaB and dnaC proteins form a tight complex in the presence of ATP (Wickner, S., and Hurwitz, J., (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 921-925). The complexed dnaC protein is resistant to inhibition by the sulfhydryl reagent, N-ethylmaleimide. This protection is not observed when ATP is substituted by AMP, ADP, adenyl 5'-yl imidodiphosphate, or adenosine-5'-O-(3-thiotriphosphate); dATP provides partial protection. A sedimentation coefficient of 15.2 S determined by glycerol gradient sedimentation and a Stokes radius of 64 A determined by gel filtration suggests a molecular weight in the range of 400,000. The complex isolated by DEAE-cellulose chromatography contains six dnaC protein monomers of 29,000 daltons contains six dnaC protein monomers of 29,000 daltons contains six dnaC protein monomers of 29,000 daltons per dnaB protein hexamer (300,000 daltons) consistent with a calculated weight of 474,000. The isolated dnaB-dnaC protein complex functions in vitro in the replication of phage phi X174 single-stranded DNA to the duplex replicative form. Tritium-labeled dnaC protein, absent from an isolated prepriming com-dnaC protein, absent from an isolated prepriming complex intermediate, was nevertheless bound to the phiX replicative form DNA synthesized in vitro. These results suggest that stable inclusion od dnaC protein in the priming complex bound to DNA requires a completely assembled primosome.