Replication initiated at the origin (oriC) of the E. coli chromosome reconstituted with purified enzymes

Abstract

A crude soluble enzyme system capable of authentic replication of a variety of oriC plasmids has been replaced by purified proteins constituting three functional classes: initiation proteins (RNA polymerase, dnaA protein, gyrase) that recognize the oriC sequence and presumably prime the leading strand of the replication fork; replication proteins (DNA polymerase III holoenzyme, single-strand binding protein, primosomal proteins) that sustain progress of the replication fork; and specificity proteins (topoisomerase I, RNAase H, protein HU) that suppress initiation of replication at sequences other than oriC, coated with dnaA protein. Protein HU and unidentified factors in crude enzyme fractions stimulate replication at one or more stages. Replication has been separated temporally and physically into successive stages of RNA synthesis and DNA synthesis.