Sequential early stages in the in vitro initiation of replication at the origin of the Escherichia coli chromosome

Complexes previously identified in the reconstitution of stages in the initiation of replication of plasmids (oriC) bearing the origin of the Escherichia coli chromosome have been examined further. These are: (i) an ATP complex of dnaA protein, (ii) an initial complex of ATP·dnaA protein with oriC DNA, (iii) an open complex in which a portion of the oriC duplex has been opened by dnaA protein action, (iv) a prepriming complex of the open complex with dnaB, dnaC, and HU proteins, and (v) a complex with a small bubble opened at oriC by dnaB helicase action and by coating with single strand-binding protein (SSB). Helicase and gyrase actions can enlarge the bubble; coupling to priming and replication propagates bidirectional fork movement. Formation and stability of these complexes are profoundly affected by ATP, Mg²⁺, and temperature, as well as the levels of the participating proteins, including HU and SSB. As examples, the open complex is stable to isolation at a temperature near 38°C but not at 24°C; the prepriming complex requires an elevated temperature and high ATP levels for its formation, but is maintained at a low temperature and is destabilized by Mg²⁺. These successive steps, subject to a variety of controls, are designed to open the supercoiled duplex for priming and bidirectional replication.