THE ESCHERICHIA-COLI DNAC GENE-PRODUCT .3. PROPERTIES OF THE DNAB-DNAC PROTEIN COMPLEX

Abstract

The E. coli dnaB and dnaC proteins form a tight complex in the presence of ATP (Wickner, S., and Hurwitz, J., 1975). The complexed dnaC protein is resistant to inhibition by the SH 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 15.2S determined by glycerol gradient sedimentation and a Stokes radius of 64 .ANG. determined by gel filtration suggests a MW in the range of 400,000. The complex isolated by DEAE-cellulose chromatography contains 6 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 .vphi.X174 single-stranded DNA to the duplex replicative form. 3H-labeled dnaC protein, absent from an isolated prepriming complex intermediate, was nevertheless bound to the .vphi.X174 replicative form DNA synthesized in vitro. Stable inclusion of dnaC protein in the priming complex bound to DNA apparently requires a completely assembled primosome.