Protein n, a primosomal DNA replication protein of Escherichia coli: Purification and characterization

Protein n, essential in forming the primosome for the in vitro conversion of ∅X174 single-stranded (SS) DNA to the duplex replicative form (RF), has been purified about 5000-fold to near homogeneity from E. coli. Protein n is heat- and acid-resistant and N-ethyl-maleimide-sensitive. It appears to be a dimer of 12,000 (±2000)-dalton polypeptides. About 80 molecules of protein n are present/cell. Protein n binding to ∅X SS DNA depends on the presence of single-strand binding protein (SSB). This requirement for SSB reflects a direct interaction of protein n and SSD. About 30 protein n monomers can be bound to an SSB-coated circle. However, in forming the primosome on an SSB-coated ∅X circle, an input of only 2-3 protein n monomers is required and 1 monomer bound/circle. Retention of this low level of protein n on SSB-coated ∅X SS DNA is dependent upon protein n', a DNA-dependent ATPase (dATPase) that guides primosome assembly. This single protein n monomer is retained in the assembled primosome, which is conserved on the completed parental RF and participates in the next stage of the replicative cycle, production of progeny RF.