The Escherichia coli dnaC gene product. II. Purification, physical properties, and role in replication

Abstract

The Escherichia coli dnaC protein, purified to homogeneity from overproducing plasmid strains, is a polypeptide of 31,000 daltons (determined on a denaturing gel). The native molecular weight as calculated from the sedimentation coefficient of 2.75 S and Stokes radius of 24.5 A is 29,000. dnaC protein is N-ethylmaleimide sensitive (Wickner, S., Berkower, L., Wright, M., and Hurwitz J. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 2369-2373), and has 3 sulfhydryl groups as determined with [14C]p-chloromercuribenzoate. The activity was assayed by complementation of a mutant dnaC extract or by reconstitution of a purified protein system which converts phi X174 single-stranded DNA to the duplex replicative form. In this conversion the dnaC protein is required during the initial prepriming stage of phi X174 DNA replication. Antiserum against dnaC protein specifically inhibits this stage but not the subsequent priming and elongation steps carried out by primase and the PolIII holoenzyme. Requirement for dnaC protein was also manifested in the in vitro replication of a plasmid DNA containing the E. coli origin of replication (oriC) by complementation of a mutant extract and specific inhibition by dnaC antiserum.