Initiation of replication of the Escherichia coli chromosomal origin reconstituted with purified enzymes.

A mixture of purified proteins has replaced a crude enzyme fraction capable of efficient replication of oriC-containing plasmids. The reconstituted enzyme system contains proteins which provide initiation, replication, and specificity functions required for dnaA-dependent replication specific for an oriC template. Replication can be separated into successive stages of RNA synthesis and DNA replication. Isolation of an intermediate no longer requiring RNA polymerase action requires the presence of dnaA protein, DNA gyrase, dnaB protein and dnaC protein. Intermediate formation likely involves binding of dnaA protein to a 9-bp sequence present 4 times as inverted repeats within the chromosomal origin sequence.