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

1982; Elsevier BV; Volume: 257; Issue: 22 Linguagem: Inglês

10.1016/s0021-9258(18)33515-4

1083-351X

J. Kobori, A Kornberg,

RNA and protein synthesis mechanisms

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'-0-(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 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 $X174 single-stranded DNA to the duplex replicative form.Tritium-labeled dnaC protein, absent from an isolated prepriming complex intermediate, was nevertheless bound to the $X replicative form DNA synthesized in vitro.These results suggest that stable inclusion of dnaC protein in the priming complex bound to DNA requires a completely assembled primosome.