Enzymatic replication of the origin of the Escherichia coli chromosome.

Abstract

The physiologically relevant features of an enzyme system that replicates plasmids bearing the origin of the E. coli chromosome (oriC) were studied. The system depends completely on low levels of exogenously furnished supercoiled oriC plasmids, uses only those plasmids that contain the intact oriC region of .apprx. 245 base pairs, and initiates replication within or near the oriC sequence and proceeds bidirectionally. Replication in this system proceeds linearly, after a 5-min lag, for 30-40 min to produce as much as a 40% increase over the input DNA, depends on RNA polymerase and gyrase as indicated by total inhibition by rifampicin and nalidixate, depends on replication proteins (e.g., dnaB protein and single-stranded DNA binding protein) as judged by specific antibody inhibitions and operates independently from protein synthesis. Replication in this system also depended on dnaA activity, as suggested by the inactivity of enzyme fractions from each of 2 dnaA temperature-sensitive mutant strains, and complementation (with a 15-fold overproduction of complementing activity) by a fraction from a strain containing the dnaA gene cloned in a multicopy plasmid. Resolution and analysis of factors that control the initiation of a chromosome cycle should become accessible through this enzyme system.